Selection for cooperativity causes epistasis predominately between native contacts and enables epistasis-based structure reconstruction

Epistasis and cooperativity of folding both result from networks of energetic interactions in proteins. Epistasis results from energetic interactions among mutants, whereas cooperativity results from energetic interactions during folding that reduce the presence of intermediate states. The two concepts seem intuitively related, but it is unknown how they are related, particularly in terms of selection. To investigate their relationship, we simulated protein evolution under selection for cooperativity and separately under selection for epistasis. Strong selection for cooperativity created strong epistasis between contacts in the native structure but weakened epistasis between nonnative contacts. In contrast, selection for epistasis increased epistasis in both native and nonnative contacts and reduced cooperativity. Because epistasis can be used to predict protein structure only if it preferentially occurs in native contacts, this result indicates that selection for cooperativity may be key for predicting structure using epistasis. To evaluate this inference, we simulated the evolution of guanine nucleotide-binding protein (GB1) with and without cooperativity. With cooperativity, strong epistatic interactions clearly map out the native GB1 structure, while allowing the presence of intermediate states (low cooperativity) obscured the structure. This indicates that using epistasis measurements to reconstruct protein structure may be inappropriate for proteins with stable intermediates.

Two mutations have an epistatic interaction if their combined effect on a trait is not equal to the sum of their independent effects (1). The effect may be on fitness, function, or a physical property such as stability. Epistasis has been demonstrated many times experimentally. It has been found to impact the rate of adaptation (2), to constrain mutational trajectories leading to drug resistance (3, 4), and to impact yeast metabolism (5). It has been observed in the evolution of influenza (6, 7), between beneficial mutations in an evolving population of Escherichia coli (8), during the evolution of RNA viruses (9), and in the evolution of new enzyme activity (10, 11). Epistasis influences the amino acid preferences at different sites (12) and can have a substantial impact on protein evolution by restricting certain evolutionary pathways and by opening up new ones, resulting in sequences and functions that were not previously available (13). It has been suggested that epistasis is highly pervasive, affecting up to 90% of substitutions (14).Experimentally measured epistasis can be used to predict the three-dimensional (3D) native structure of a protein. For example, Olson et al. (15) measured the epistasis between the majority of possible residue pairs of the guanine nucleotide-binding protein (GB1), which was used by Rollins et al. (16) to predict the protein’s 3D structure. Such prediction methods assume that the majority of epistatic pairs are in contact in the native state, an assumption supported by experimental evidence (15). In the native state structure, the side chains of residues in contact interact, and so they no longer behave independently. This can result in nonadditivity in terms of protein properties such as stability. However, native contacts are not the only interactions that determine protein properties. Mutations in contacts present in intermediate states and unfolded state structures that alter the stability of those states relative to the native state will impact properties such as stability. It is therefore unclear why experimental evidence suggests that mostly native contacts interact epistatically.     

Structural and functional dissection of reovirus capsid folding and assembly by the prefoldin-TRiC/CCT chaperone network

Intracellular protein homeostasis is maintained by a network of chaperones that function to fold proteins into their native conformation. The eukaryotic TRiC chaperonin (TCP1-ring complex, also called CCT for cytosolic chaperonin containing TCP1) facilitates folding of a subset of proteins with folding constraints such as complex topologies. To better understand the mechanism of TRiC folding, we investigated the biogenesis of an obligate TRiC substrate, the reovirus σ3 capsid protein. We discovered that the σ3 protein interacts with a network of chaperones, including TRiC and prefoldin. Using a combination of cryoelectron microscopy, cross-linking mass spectrometry, and biochemical approaches, we establish functions for TRiC and prefoldin in folding σ3 and promoting its assembly into higher-order oligomers. These studies illuminate the molecular dynamics of σ3 folding and establish a biological function for TRiC in virus assembly. In addition, our findings provide structural and functional insight into the mechanism by which TRiC and prefoldin participate in the assembly of protein complexes.

Chaperones perform the essential function of folding proteins that cannot achieve a native conformation in an unassisted manner. The eukaryotic chaperonin TRiC (TCP1-ring complex, also called CCT for cytosolic chaperonin containing TCP1) mediates the adenosine triphosphate (ATP)-dependent folding of a subset (∼10%) of newly translated cytosolic proteins (1). TRiC substrates include the cytoskeletal proteins actin and tubulin (2, 3), tumor suppressor proteins (4, 5), and aggregation-prone proteins that accumulate in neurodegenerative diseases such as Huntington disease (6, 7). TRiC has a toroidal structure formed by two rings stacked back to back, each composed of eight paralogous subunits that form a barrel with a central cavity (8, 9). The central cavity functions as a chamber within which newly translated polypeptides are sequestered in a protected protein-folding environment (10, 11). ATP binding and hydrolysis trigger cyclic changes in the conformation of TRiC between an open form in the nucleotide-free state and a closed, folding-active conformation in the ATP-hydrolysis state (12, 13). Despite its quasisymmetry, there is a high degree of specialization within the eight subunits that form TRiC, with individual subunits differing in substrate-binding capacity (14), ATP-binding and hydrolytic activity (12, 15, 16), and surface hydrophobicity (8). The diversification of these subunits is thought to contribute to the capacity of TRiC to fold a wide array of substrates with complex topologies.TRiC functions in concert with other molecular chaperones, including Hsp70 (17) and prefoldin (PFD) (18), to direct protein-folding pathways. Other chaperones, including ribosome-associated chaperones such as Hsp70, may function more promiscuously and earlier in the folding process to stabilize unfolded or partially folded proteins in conformations that can either fold without further assistance or be recognized by more specialized chaperones (19). PFD, a jellyfish-structured TRiC cochaperone (20), can also bind nascent polypeptides (18, 21). Hsp70 and PFD may direct the transfer of certain substrates to TRiC (17, 18). PFD can contribute directly to TRiC-mediated protein folding (22), but mechanisms by which cochaperones cooperate with TRiC to fold individual substrates are poorly defined.As obligate intracellular pathogens, viruses replicate within host cells and use host chaperones to fold viral polypeptides. Proteins from diverse families of viruses have been identified as TRiC substrates (23–25). TRiC performs an essential folding function in the replication of mammalian orthoreoviruses (reoviruses) (26). Reoviruses are ubiquitous, infecting most mammals early in life, and have been linked to celiac disease in humans (27). Reoviruses have a proteinaceous outer capsid composed of μ1 and σ3, which coalesce in a 1:1 stoichiometric ratio to form heterohexamers. The σ3 component of the outer capsid is an obligate TRiC substrate (26). σ3 is aggregation prone (28) and exists in multiple oligomeric forms in the cell (26). The mechanism of TRiC/σ3 binding, folding, and release is unclear. In addition, the chaperones that cooperate with TRiC to fold and assemble σ3 into a complex with its binding partner, μ1, are unknown.In this study, we identify a network of host chaperones that interact with reovirus σ3, including Hsp70, Hsp90, PFD, and TRiC. We establish a function for PFD in preventing σ3 aggregation and enhancing σ3 transfer to TRiC. Cryoelectron microscopy (cryo-EM) and cross-linking mass spectrometry (XL-MS) resolve the structure of σ3 within the TRiC folding chamber, revealing TRiC/σ3 interaction interfaces and the orientation of the σ3/μ1 oligomerization domain within TRiC. Biochemical studies establish a function for TRiC in assembling folded σ3 into a complex with μ1 through a process that is enhanced by PFD. Functional assays demonstrate that TRiC folds σ3 into its biologically active and infectious conformation. Together, this work provides mechanistic insight into the structure and function of TRiC and PFD in the folding and assembly of a heterooligomeric protein complex.     

Allosteric cooperation in a de novo-designed two-domain protein

We describe the de novo design of an allosterically regulated protein, which comprises two tightly coupled domains. One domain is based on the DF (Due Ferri in Italian or two-iron in English) family of de novo proteins, which have a diiron cofactor that catalyzes a phenol oxidase reaction, while the second domain is based on PS1 (Porphyrin-binding Sequence), which binds a synthetic Zn-porphyrin (ZnP). The binding of ZnP to the original PS1 protein induces changes in structure and dynamics, which we expected to influence the catalytic rate of a fused DF domain when appropriately coupled. Both DF and PS1 are four-helix bundles, but they have distinct bundle architectures. To achieve tight coupling between the domains, they were connected by four helical linkers using a computational method to discover the most designable connections capable of spanning the two architectures. The resulting protein, DFP1 (Due Ferri Porphyrin), bound the two cofactors in the expected manner. The crystal structure of fully reconstituted DFP1 was also in excellent agreement with the design, and it showed the ZnP cofactor bound over 12 Å from the dimetal center. Next, a substrate-binding cleft leading to the diiron center was introduced into DFP1. The resulting protein acts as an allosterically modulated phenol oxidase. Its Michaelis–Menten parameters were strongly affected by the binding of ZnP, resulting in a fourfold tighter K_m and a 7-fold decrease in k_cat. These studies establish the feasibility of designing allosterically regulated catalytic proteins, entirely from scratch.

The emergence of life and the evolution of the three superkingdoms required the recombination of preexisting protein domains to perform ever-increasingly complex functions (1). The majority (∼90%) of multidomain proteins are made up of end-to-end linked domains; in the remaining ∼10% of cases, a domain insertion occurs, creating a continuous and a discontinuous domain (2, 3). In enzymes and small molecule binding proteins, the bilobed architecture facilitates the formation of active sites between individual domains; redox-active proteins often combine multiple domains to orient multiple cofactors for productive electron transfer; and allosterically regulated proteins combine binding domains with catalytic or signal-transducing domains (4). Therefore, the addition of a domain to an existing protein expands, alters, or modulates its functionality (5). Protein engineers have been inspired by this modularity to generate artificial multidomain proteins with improved properties or to create nanostuctured and sensor devices (6–10). Toward this end, different methodologies have been developed to fuse the different domains by: 1) introducing designed or naturally occurring peptide linkers (11); 2) superimposing and fusing one or two turns of terminal alpha helices of connecting helical proteins (12, 13); and 3) computationally designing new structural elements to interface the different domains in a fragment based approach (14). In each case, the domain architectures of the artificial multidomain proteins fell into either end-to-end or domain insertion topology. However, the de novo design and structure determination of allosterically regulated multidomain proteins (in which both domains are designed from scratch) have not been reported.Here, we describe the design of a protein that combines domains capable of binding ZnP, the Zn[5,10,15,20-tetrakis(trifluoromethyl)porphinato], and diiron cofactors into a single tightly coupled framework. While multicofactor proteins have been widely used to explore redox coupling (15–22), high-resolution structures of multicofactor proteins have not been described in the literature, limiting what can be learned and achieved in such systems. The present work differs in two fundamental manners from earlier work on the design of multicofactor proteins. First, the goal of the present study was to examine how the binding of redox-inert ZnP cofactor allosterically modulates the catalytic activity of a second diiron-binding domain. Second, the structure of the designed multicofactor protein was determined by X-ray crystallography. It is also noteworthy that the computational methods, adopted in this work, could be readily extended to the design of electronically coupled systems for light-triggered electron energy storage and utilization, particularly given the ability to design proteins that incorporate metal ion clusters (15, 21, 23–31).The diiron-binding component of our two-domain protein is based on the DF family of de novo proteins (32, 33), which have been optimized to catalyze various two and four-electron reactions, including ferroxidase, oxidase, and monooxygenase activities (34–40). The second domain is based on PS1, which binds the synthetic ZnP (41). PS1 has a well-structured hydrophobic core, which positions the porphyrin-binding domain for productive interaction with this cofactor. The structure of PS1 has been solved by NMR in both the apo-bound and ZnP-bound state. The structures are nearly identical in the hydrophobic core, but the apo-protein is more open and flexible near the opposite end of the bundle. This structural transition allows the protein to bind the cofactor in an alligator-like chomping motion. We reasoned that such transition might be used to regulate the properties of the DF diiron site in the neighboring domain. Both DF and PS1 are four-helix bundles, so we envisioned a coaxial arrangement to facilitate interdomain communication, as in bacterial signaling proteins (42). However, DF and PS1 have distinct bundle architectures with respect to their interhelical packing, helical offsets, and helical registers, which together presented challenges for structural design. To address these challenges, we extended previous fragment-based approaches (43–48) and designed artificial multidomain proteins with allosterically communicating sites.     

A conserved folding nucleus sculpts the free energy landscape of bacterial and archaeal orthologs from a divergent TIM barrel family

The amino acid sequences of proteins have evolved over billions of years, preserving their structures and functions while responding to evolutionary forces. Are there conserved sequence and structural elements that preserve the protein folding mechanisms? The functionally diverse and ancient (βα)_1–8 TIM barrel motif may answer this question. We mapped the complex six-state folding free energy surface of a ∼3.6 billion y old, bacterial indole-3-glycerol phosphate synthase (IGPS) TIM barrel enzyme by equilibrium and kinetic hydrogen–deuterium exchange mass spectrometry (HDX-MS). HDX-MS on the intact protein reported exchange in the native basin and the presence of two thermodynamically distinct on- and off-pathway intermediates in slow but dynamic equilibrium with each other. Proteolysis revealed protection in a small (α1β2) and a large cluster (β5α5β6α6β7) and that these clusters form cores of stability in I_a and I_bp. The strongest protection in both states resides in β4α4 with the highest density of branched aliphatic side chain contacts in the folded structure. Similar correlations were observed previously for an evolutionarily distinct archaeal IGPS, emphasizing a key role for hydrophobicity in stabilizing common high-energy folding intermediates. A bioinformatics analysis of IGPS sequences from the three superkingdoms revealed an exceedingly high hydrophobicity and surprising α-helix propensity for β4, preceded by a highly conserved βα-hairpin clamp that links β3 and β4. The conservation of the folding mechanisms for archaeal and bacterial IGPS proteins reflects the conservation of key elements of sequence and structure that first appeared in the last universal common ancestor of these ancient proteins.

Proteins are indispensable workhorses of cellular machinery whose functional diversity is defined by their final folded conformations. The folding pathway of a protein is determined by its energy landscape, whose map is encoded in the amino acid sequence. Partially folded states on the landscape often contain elements of the native topology and connect the nascent unfolded polypeptide chain to the functional folded conformation (1, 2). Proteins and their folding pathways have evolved over billions of years, responding to evolutionary forces such as mutation and natural selection (3–5). Orthologs, proteins that have diverged from a common ancestor but share a common structure and function, provide vehicles for exploring the impact of evolution on folding pathways and the intermediates that guide the folding to the native conformation.The functionally diverse (βα)_1–8 TIM barrel motif is an ideal candidate to decipher evolutionary constraints on protein folding pathways. The motif supports a wide variety of essential enzymatic transformations in all three superkingdoms of life (6–8) and is one of the 10 ancestral protein folds that were instrumental in the transition from RNA–protein world to the last universal common ancestor of life (LUCA) to the present complex DNA–RNA–protein world (9, 10). The βα-repeat architecture produces a cylindrical β-barrel core and an amphipathic α-helical shell whose loops between the β-strands and subsequent α-helices form the canonical active site of this very large family of enzymes. Although the pairwise sequence conservation across the family of TIM barrels is typically ∼30%, their folding mechanisms are complex and highly conserved (11). Folding intermediates, both on the productive folding pathway and as misfolded, kinetic traps have been observed for candidate TIM barrels from several bacterial and archaeal organisms (11–16). The divergence of these two superkingdoms, which occurred ∼4 billion y ago, right after life arose, speaks to the robustness of the TIM barrel folding mechanism across the span of evolutionary time.We have previously examined the relationships between sequence, structure, and fitness in a yeast-based competition assay for three thermophilic indole-3-glycerolphosphate synthase (IGPS) orthologs from the TIM barrel family (17). Significant correlations between the archaeal Sulfolobus solfataricus (SsIGPS) and the bacterial Thermotoga maritima (TmIGPS) and Thermus thermophilus (TtIGPS) proteins revealed that both sequence and structure are critical in defining their fitness landscapes. This observation and the conservation of TIM barrel folding mechanisms motivated the hypothesis that the sequences of TIM barrel orthologs from archaeal and bacterial organisms also conserve the structures of their folding intermediates. If valid, we would obtain detailed insights into the constraints that TIM barrel structure and function impose on the enormous sequence space available in ∼4 billion y of evolution (18, 19). We have previously mapped the structures of the on- and off-pathway intermediates for SsIGPS by hydrogen–deuterium exchange mass spectrometry (HDX-MS) (15, 16), providing an archaeal reference for the present study of a bacterial ortholog (SI Appendix, Fig. S1).Comparison of the structures of the folding intermediates and folding mechanisms for S. solfataricus and T. maritima IGPS confirmed our hypothesis. A bioinformatics analysis of thousands of nonredundant IGPS sequences from the bacterial, archaeal, and eukaryota superkingdoms revealed the conservation of three adjacent structural elements that form a nucleus responsible for defining the folding free energy surface of the IGPS family of TIM barrel proteins. We conclude that the folding mechanism of the IGPS TIM barrel, including the structures of key partially folded states, arose in the LUCA and has persisted for over ∼4 billion y.     

Ion mobility–mass spectrometry reveals the role of peripheral myelin protein dimers in peripheral neuropathy

Peripheral myelin protein (PMP22) is an integral membrane protein that traffics inefficiently even in wild-type (WT) form, with only 20% of the WT protein reaching its final plasma membrane destination in myelinating Schwann cells. Misfolding of PMP22 has been identified as a key factor in multiple peripheral neuropathies, including Charcot-Marie-Tooth disease and Dejerine–Sottas syndrome. While biophysical analyses of disease-associated PMP22 mutants show altered protein stabilities, leading to reduced surface trafficking and loss of PMP22 function, it remains unclear how destabilization of PMP22 mutations causes mistrafficking. Here, native ion mobility–mass spectrometry (IM-MS) is used to compare the gas phase stabilities and abundances for an array of mutant PM22 complexes. We find key differences in the PMP22 mutant stabilities and propensities to form homodimeric complexes. Of particular note, we observe that severely destabilized forms of PMP22 exhibit a higher propensity to dimerize than WT PMP22. Furthermore, we employ lipid raft–mimicking SCOR bicelles to study PMP22 mutants, and find that the differences in dimer abundances are amplified in this medium when compared to micelle-based data, with disease mutants exhibiting up to 4 times more dimer than WT when liberated from SCOR bicelles. We combine our findings with previous cellular data to propose that the formation of PMP22 dimers from destabilized monomers is a key element of PMP22 mistrafficking.

The misfolding of membrane proteins is implicated in the mechanisms of multiple debilitating diseases such as cystic fibrosis and retinitis pigmentosa (1–4). Specific membrane protein mutations are often associated with disease states, with variant forms exhibiting altered stability and cellular trafficking (5). Unfortunately, due to the challenges associated with preparing and handling pure, highly concentrated membrane protein samples, detailed structural information on such targets is often lacking, especially for disease mutant forms. Furthermore, as some membrane proteins associated with misfolding-based diseases have hundreds of mutations of interest (3), there is a clear need for high-throughput methods to assess disease mutation-induced changes in membrane protein stability and structure.Peripheral myelin protein 22 (PMP22) is such a membrane protein, for which misfolding and trafficking of mutant variants have been implicated in disease (6). PMP22 is a tetra-span integral membrane glycoprotein predominately expressed in Schwann cells, which are the principal glial cells of the peripheral nervous system (PNS), where they produce myelin (7–9). In addition to accounting for ∼5% of the protein found in the myelin sheath surrounding PNS nerve axons, PMP22 is thought to regulate intracellular Ca²⁺ levels (10), apoptosis (11), linkage of the actin cytoskeleton with lipid rafts (12), formation of epithelial intercellular junctions (13), myelin formation (14), lipid metabolism, and cholesterol trafficking (15). Dysregulation and misfolding of PMP22 has been identified as a key factor in multiple neurodegenerative disorders, such as Charcot-Marie-Tooth disease types 1A and E, as well as Dejerine–Sottas syndrome (6, 16–18). Like a number of other disease-linked membrane proteins (19), the trafficking of PMP22 is known to be inefficient, with only 20% of the wild-type (WT) protein reaching its final plasma membrane destination in Schwann cells (16, 20). Previously, it has been shown through a range of biophysical analyses that disease-associated PMP22 mutations lower thermodynamic protein stability as the root cause of reduced trafficking and loss of protein function; however, the mechanism by which destabilization of PMP22 causes mistrafficking is still not well understood (6). Additionally, a high-resolution structure of PMP22 has not yet been published.Native mass spectrometry (MS) has recently been demonstrated to overcome sample purity and concentration barriers to reveal critical details of membrane protein structure and function (21–23). Through the use of nano-electrospray (nESI), intact membrane proteins are ionized within detergent micelles or other membrane mimetics (24–27), which can then be removed from the membrane protein ions within the instrument. This method has been used to elucidate oligomeric state (28–30), complex organization (31, 32), and lipid interactions (33–35) of diverse membrane proteins. The addition of ion-mobility separation–mass spectrometry (IM-MS) provides data on the orientationally averaged size of analytes (36) and enables collision induced unfolding (CIU) experiments (37). In CIU, the energies experienced by gas-phase protein ions are increased in a stepwise fashion causing gas-phase protein unfolding to occur. These dynamic measurements have been shown to be sensitive to ligand binding (38, 39), glycosylation (40, 41), and disulfide bonding (40) in soluble proteins, as well as selective lipid and small molecule binding in membrane proteins (42–45). While CIU can clearly capture subtle structural changes in membrane proteins (43, 45, 46) and soluble mutant protein variants (47, 48) its ability to characterize membrane protein variants is only beginning to be explored.Here, we demonstrate the ability of native MS and CIU to detect key differences in the gas-phase stability and homodimer complex formation of PMP22 variants, together leading to insights into the mechanism of PMP22 dysregulation in disease. We quantify the propensity of PMP22 to dimerize across WT and seven disease-associated point mutations. We find that mutations associated with severe disease states form significantly more dimer than WT. Through CIU, we quantify the stability of gas-phase monomeric and dimeric PMP22 and find that variants bearing mutations associated with severe neuropathy exhibit the lowest relative monomer conformational stability. Interestingly, we also observe that dimers formed by various disease mutant forms of PMP22 are all more stable than WT PMP22 dimeric complexes. We continue by comparing our results to previously published biophysical datasets and find that our monomeric PMP22 gas-phase stability values correlate well with cellular trafficking data (6). Finally, we probe the effects of solubilization agents on PMP22 by characterizing its dimerization within sphingomyelin and cholesterol rich (SCOR) bicelles (49). We find that dimeric PMP22 complexes persist within SCOR bicelles and that the mutants resulting in the most severe disease phenotypes form higher population of dimer than WT. We conclude by describing a possible mechanism of PMP22 dysregulation in severe neurodegenerative diseases by which PMP22 monomers are destabilized, leading to dimers that traffic much less efficiently to the plasma membrane than WT PMP22.     

Regulatory mechanisms of tau protein fibrillation under the conditions of liquid–liquid phase separation

One of the hallmarks of Alzheimer’s disease and several other neurodegenerative disorders is the aggregation of tau protein into fibrillar structures. Building on recent reports that tau readily undergoes liquid–liquid phase separation (LLPS), here we explored the relationship between disease-related mutations, LLPS, and tau fibrillation. Our data demonstrate that, in contrast to previous suggestions, pathogenic mutations within the pseudorepeat region do not affect tau441’s propensity to form liquid droplets. LLPS does, however, greatly accelerate formation of fibrillar aggregates, and this effect is especially dramatic for tau441 variants with disease-related mutations. Most important, this study also reveals a previously unrecognized mechanism by which LLPS can regulate the rate of fibrillation in mixtures containing tau isoforms with different aggregation propensities. This regulation results from unique properties of proteins under LLPS conditions, where total concentration of all tau variants in the condensed phase is constant. Therefore, the presence of increasing proportions of the slowly aggregating tau isoform gradually lowers the concentration of the isoform with high aggregation propensity, reducing the rate of its fibrillation. This regulatory mechanism may be of direct relevance to phenotypic variability of tauopathies, as the ratios of fast and slowly aggregating tau isoforms in brain varies substantially in different diseases.

Tau is a major neuronal protein that plays a key role in Alzheimer’s disease (AD) and a number of other neurodegenerative disorders that are collectively classified as tauopathies. The latter include frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy, Pick’s disease, corticobasal degeneration, and chronic traumatic encephalopathy (1–5). Under normal physiological conditions, tau is localized to axons where it is involved in the assembly of microtubules (1–6). In tauopathies, the protein self-associates into different forms of filaments that contain largely hyperphosphorylated tau and have properties of amyloid fibrils (1–5).Alternative splicing of the MAPT gene that encodes tau results in six major isoforms in the human central nervous system. These isoforms differ with respect to the number of N-terminal inserts as well as the number of 31 to 32 residue pseudorepeat sequences in the C-terminal part of the protein (1–5). Structurally, tau is largely an intrinsically disordered protein, with local secondary structures existing only within the pseudorepeat region (1, 7). A large number of mutations have been identified in the latter region that correlate with inherited cases of FTDP-17 (8, 9). These mutations not only diminish the ability of tau to promote microtubule assembly, but many also promote self-association of tau into amyloid fibrils (10–12). This strongly suggests that tau misfolding and aggregation is one of the key events in disease pathogenesis.A number of recent reports indicate that purified full-length tau (tau441) has a high propensity to undergo liquid–liquid phase separation (LLPS) in vitro in the presence of crowding agents that emulate the high concentration of macromolecules in the cell. This was observed both for the phosphorylated (13) and nonphosphorylated protein (14–16), and it was determined that tau LLPS is driven largely by attractive electrostatic intermolecular interactions between the negatively charged N-terminal and positively charged middle/C-terminal regions of the protein (15). Tau condensation into droplets (complex coacervation) was also observed in the presence of polyanions such as RNA or heparin (17, 18). These observations in vitro are partially supported by studies in cells (13, 19–24), especially within the context of tau interaction with microtubules (21). However, it remains unclear whether tau could undergo LLPS in cells on its own or, rather, its recruitment to membraneless organelles such as stress granules is largely driven by interactions with other proteins and/or RNA. These limitations notwithstanding, the observations that tau has a propensity for LLPS have potentially important implications for the pathogenic process in tauopathies, as studies with other proteins involved in neurodegenerative diseases (e.g., TDP-43, FUS) indicate that the environment of liquid droplets is conducive to the pathological aggregation of these proteins (25–32). In line with these findings, it was recently suggested that LLPS can initiate tau aggregation. However, the evidence for this was very limited and largely based on optical microscopy observations (13).In the present study, we explored the relationship between pathogenic mutations of tau, protein LLPS, and aggregation into amyloid fibrils. Our data show that, in contrast to previous suggestions (13), pathogenic mutations within the pseudorepeat region do not affect the propensity of tau to undergo LLPS. These mutations, however, do dramatically accelerate the liquid-to-solid phase transition within the droplets, leading to rapid formation of fibrillar aggregates. Most important, this study also reveals a previously unrecognized mechanism by which LLPS can regulate the rate of amyloid formation in mixtures containing tau isoforms with different aggregation propensities. These findings strongly suggest that LLPS may play a major regulatory role in the formation of pathological tau aggregates in neurodegenerative diseases.     

Integration drives rapid phenotypic evolution in flatfishes

Evolutionary innovations are scattered throughout the tree of life, and have allowed the organisms that possess them to occupy novel adaptive zones. While the impacts of these innovations are well documented, much less is known about how these innovations arise in the first place. Patterns of covariation among traits across macroevolutionary time can offer insights into the generation of innovation. However, to date, there is no consensus on the role that trait covariation plays in this process. The evolution of cranial asymmetry in flatfishes (Pleuronectiformes) from within Carangaria was a rapid evolutionary innovation that preceded the colonization of benthic aquatic habitats by this clade, and resulted in one of the most bizarre body plans observed among extant vertebrates. Here, we use three-dimensional geometric morphometrics and a phylogenetic comparative toolkit to reconstruct the evolution of skull shape in carangarians, and quantify patterns of integration and modularity across the skull. We find that the evolution of asymmetry in flatfishes was a rapid process, resulting in the colonization of novel trait space, that was aided by strong integration that coordinated shape changes across the skull. Our findings suggest that integration plays a major role in the evolution of innovation by synchronizing responses to selective pressures across the organism.

Evolutionary innovations are adaptations or exaptations that result in the colonization of novel regions of trait space, and shift the dynamic of ecosystem interactions in their respective environments. These innovations may involve a dramatic restructuring of an ancestral body plan, allow organisms novel access to ecological resources, and in (the case of key innovations) spur increases in lineage diversification (1–6).Traditionally, much of the study of evolutionary innovation has focused on extrinsic drivers, such as ecological change or environmental stimuli, which have frequently been viewed as the principal triggers for evolutionary novelty (6–9). Recently, focus has shifted toward the effects of intrinsic (e.g., developmental) processes that may structure patterns of trait diversification (10–12). Among these processes, integration and modularity have emerged as important sources of insight as a growing consensus has found that patterns of trait covariation can constrain and facilitate responses to selection and strongly influence patterns of trait diversification at both contemporary and macroevolutionary timescales (13–20).Modularity refers to the pattern whereby traits form complexes (i.e., modules) that exhibit a high degree of covariation within themselves, whereas the strength of covariation is far lower between these trait complexes (21, 22). This can result in the compartmentalization of trait complexes across an organism and has been hypothesized to allow modules to respond semiautonomously to different selective pressures (23–27). On macroevolutionary timescales, this modularization of different traits has been shown to result in mosaic patterns of evolution across organisms and is thought to facilitate morphological diversification as different traits are able to fine-tune responses to different selective pressures (27–29). Conversely, integration refers to a pattern whereby different traits exhibit a high degree of covariation (21, 30). Patterns of integration may be the result of pleiotropy or functional coupling (28, 30–33). There is less of a consensus on the macroevolutionary implications of phenotypic integration. Strong integration between traits has traditionally been hypothesized to constrain patterns of trait diversification along specific directions such that traits may exhibit more variation in some directions than others (30, 31). Furthermore, theoretical studies have found that evolutionary flexibility is negatively correlated with the magnitude of integration (27, 28, 34). However, more recent studies have found that the relationship between evolutionary integration and trait diversification is more complex than originally thought. A recent simulation study (28) found that trait integration can actually promote sizeable responses to selection, along lines that are parallel with the direction of trait covariation. This work also indicates that while a modular system has the freedom to explore a wider range of morphospace, this system is less likely to evolve maximally disparate phenotypes as compared to an integrated one that funnels variation along a more restricted trajectory. Indeed, several empirical studies have found rapid rates of shape evolution and high degrees of morphological disparity in clades that also exhibit tight evolutionary integration across the traits of interest (29, 35, 36). These studies suggest that evolutionary integration may play an important role in the evolution of innovation as it allows for clades to rapidly explore novel regions of morphospace.The majority of our understanding of patterns of vertebrate trait diversification come from tetrapod systems, and primarily studies involving birds and mammals (14, 37–40). While these studies have been deeply informative, they help to explain less than half of the vertebrate story of diversification as these studies frequently exclude ray-finned fishes (Actinopterygii) (24). Ray-finned fishes comprise over half of the species diversity in vertebrates. They also exhibit a diverse array of evolutionary innovations that have allowed them to become arguably the most successful vertebrate radiation on the planet.Flatfishes in particular represent a diverse clade of bottom-dwelling, teleost fishes that possess a striking evolutionary innovation: a degree of cranial asymmetry exceeding that of any other vertebrate lineage (41). Most flatfishes are completely blind on one side of their body, and instead feature both eyes on the same side of the head. This perplexing flatfish phenotype is achieved during the early developmental stages where one eye of a symmetrical larva gradually begins to migrate to the other side of its body, rendering one side “eyed” and the other “blind” (42). Developmental studies indicate that this orbital migration is driven by thyroid hormone expression and is paired with changes in swimming behavior (43), as well as asymmetrical visceral organ rearrangement (44). These developmental patterns recapitulate paleontological trends, with fossils indicating that flatfishes gradually became more asymmetrical from symmetrical ancestors (45). In addition to their cranial asymmetry, flatfishes possess a suite of adaptations that further allow them to exploit their benthic habitats; most nobly among these is their derived form of locomotion that combines strong, whole-body undulations (for fast escapes or burial) with finer-scale, dexterous undulations of their dorsal and anal fins (for slower cruising behaviors) (46–48). The evolution of this derived form of locomotion is coupled with additional changes in the roofing bones of the skull as they evolved projections to support the extended dorsal fin along the supraoccipital crest and the frontal bones (49, 50).The flatfish body plan is thought to have rapidly arisen shortly after the Cretaceous–Paleogene boundary (66 Mya) (49). Comparative analyses show rapid bursts of body-shape diversification in flatfishes and their close relatives within the broader clade Carangaria, a diverse radiation including disparate lineages like billfishes and remoras in addition to flatfishes (49, 51, 52) (Fig. 1). This episode of phenotypic innovation is hypothesized to reflect the filling of newly available ecological roles in the early Cenozoic, matching patterns reported for other groups.Open in a separate windowFig. 1.Phylogeny of 102 carangarian species included in the analyses of skull shape evolution. Insets depict representative skull shapes for each clade. Phylogeny based on Ribeiro et al. (52).The striking and rapid evolutionary dynamics within Carangaria make this clade a tantalizing target for investigating the roles that integration and modularity play in trait diversification and, particularly, evolutionary innovation. Here we use three-dimensional geometric morphometrics and a cutting-edge phylogenetic comparative toolkit to study the evolution of the neurocranium and the evolution of cranial asymmetry across 102 carangarian species. We quantify shifts in the rate of skull shape evolution between species and across the neurocranium as a whole, while also quantifying patterns of integration and modularity between flatfishes and their relatives to test for the effect of integration and modularity on the evolution of innovation. We hypothesize that flatfishes underwent a rapid shift in their rates of skull shape evolution as a result of their orbital migration. We additionally hypothesize that flatfishes will exhibit higher levels of integration compared to their carangarian relatives as a result of their asymmetrical larval metamorphosis that involves coordinated changes across the body, and adaptations associated with their derived locomotory mode.     

Encapsulation of ribozymes inside model protocells leads to faster evolutionary adaptation

Functional biomolecules, such as RNA, encapsulated inside a protocellular membrane are believed to have comprised a very early, critical stage in the evolution of life, since membrane vesicles allow selective permeability and create a unit of selection enabling cooperative phenotypes. The biophysical environment inside a protocell would differ fundamentally from bulk solution due to the microscopic confinement. However, the effect of the encapsulated environment on ribozyme evolution has not been previously studied experimentally. Here, we examine the effect of encapsulation inside model protocells on the self-aminoacylation activity of tens of thousands of RNA sequences using a high-throughput sequencing assay. We find that encapsulation of these ribozymes generally increases their activity, giving encapsulated sequences an advantage over nonencapsulated sequences in an amphiphile-rich environment. In addition, highly active ribozymes benefit disproportionately more from encapsulation. The asymmetry in fitness gain broadens the distribution of fitness in the system. Consistent with Fisher’s fundamental theorem of natural selection, encapsulation therefore leads to faster adaptation when the RNAs are encapsulated inside a protocell during in vitro selection. Thus, protocells would not only provide a compartmentalization function but also promote activity and evolutionary adaptation during the origin of life.

RNA is believed to have been a central constituent of early life (1–3). In the “RNA world” theory, functional RNAs (e.g., ribozymes) would both perform catalytic functions and store and transfer genetic information in a simple living system (4–6). Encapsulation of ribozymes in cell-like compartments, such as protocells, is thought to be an essential feature for the emergence of early life (7–11). In particular, compartmentalization would retain useful metabolites in the vicinity (12) and prevent a cooperative, self-replicating ribozyme system from collapsing under parasitization by selfish RNAs (13, 14). A major model of protocells is lipid vesicles, which consist of an aqueous interior surrounded by a semipermeable membrane (15, 16). However, while the ultimate advantages of compartmentalization may be clear, how encapsulation and confinement inside protocell vesicles would affect the activity and early evolution of ribozymes is not understood well.Confinement by lipid membranes presents a biophysical environment similar to macromolecular crowding (17). The effect of macromolecular crowding on the activity, function, and specificity of biomolecules (i.e., proteins and nucleic acids) has been examined extensively (18–23) using crowding agents such as dextran, polyethylene glycol, and Ficoll in vitro (24–29). In general, macromolecular crowding agents decrease the accessible volume for biomolecules, leading to the excluded-volume effect, in which the relative stability of compacted and folded structures is increased (30, 31). At the same time, chemical interactions between the crowding agents and the biomolecule can also stabilize or destabilize the folded structure, influencing catalytic activity (24, 32). While chemical interactions depend on the properties of the specific molecules under study, the excluded-volume effect resulting from spatial confinement inside vesicles is expected to be general. The effect of confinement can be studied while controlling for chemical interactions by comparing the encapsulated condition to the nonencapsulated but membrane-exposed condition. This comparison represents the prebiotic scenario in which RNAs would be present in the same milieu as lipids (33) and may become encapsulated or not. In this way, confinement inside vesicles was shown to increase the binding affinity of the malachite green RNA aptamer (34). Interestingly, spatial confinement inside a tetrahedral DNA framework has also been shown to increase thermodynamic stability and binding affinity of aptamers by facilitating folding (35).While these and other case studies (17, 25, 36–43) illustrate mechanisms by which RNA activity might be perturbed inside vesicles, understanding how encapsulation would affect evolution requires a broader scale of information. In particular, detailed knowledge of how encapsulation affects the sequence-activity relationship is required. This information is captured in the “fitness landscape,” or the function of fitness over sequence space, which embodies many important evolutionary features [e.g., fitness maxima, epistasis, and the viability of evolutionary trajectories (44–47)]. In practice, the fitness of a ribozyme can be considered to be its chemical activity for a particular function in the given environment (48–53).In the present work, we investigated how encapsulation inside model protocells would affect the catalytic activity and evolution of self-aminoacylating ribozymes. We studied tens of thousands of RNA sequences derived from five previously selected self-aminoacylating ribozyme families (53). These sequences were encapsulated in a mixed fatty acid/phospholipid vesicle system. Fatty acids mixed with phospholipids (1:1 molar ratio) have been used as model protocell membranes, as the vesicles tolerate Mg²⁺ concentrations needed for ribozyme activity and the membrane allows small, charged molecules to permeate while preserving large polynucleotides in the vesicle interior (54, 55). To study the biophysical effect of confinement rather than chemical interactions with the membrane, RNA activity inside vesicles was compared with RNA activity when exposed to the same vesicles without encapsulation. We show that ribozymes generally exhibit higher catalytic activity inside the vesicles and that more active sequences experience greater benefit. Using in vitro selection, we demonstrate that one of the evolutionary consequences of this trend is that encapsulation inside vesicles causes a greater rate of genotypic change due to natural selection.     

Cargo competition for a dimerization interface restricts and stabilizes a bacterial protease adaptor

Bacterial protein degradation is a regulated process aided by protease adaptors that alter specificity of energy-dependent proteases. In Caulobacter crescentus, cell cycle–dependent protein degradation depends on a hierarchy of adaptors, such as the dimeric RcdA adaptor, which binds multiple cargo and delivers substrates to the ClpXP protease. RcdA itself is degraded in the absence of cargo, and how RcdA recognizes its targets is unknown. Here, we show that RcdA dimerization and cargo binding compete for a common interface. Cargo binding separates RcdA dimers, and a monomeric variant of RcdA fails to be degraded, suggesting that RcdA degradation is a result of self-delivery. Based on HDX-MS studies showing that different cargo rely on different regions of the dimerization interface, we generate RcdA variants that are selective for specific cargo and show cellular defects consistent with changes in selectivity. Finally, we show that masking of cargo binding by dimerization also limits substrate delivery to restrain overly prolific degradation. Using the same interface for dimerization and cargo binding offers an ability to limit excess protease adaptors by self-degradation while providing a capacity for binding a range of substrates.

Controlled protein degradation regulates key physiological processes in all domains of life. In bacteria, AAA+ (ATPases with Associated Activities) proteases control the regulated destruction of misfolded and native substrates to manage cellular stress responses, cell cycle progression, physiological development, and general protein quality control maintenance (1). The Hsp100/Clp family of proteases, which includes the AAA+ protease ClpXP, share structural features and are critical for degrading factors to promote normal cell physiology in bacteria and organelles (2, 3). These energy-dependent machines recognize substrates using an oligomeric unfoldase, which translocates these targets into peptidase chambers that nonspecifically cleave proteins into smaller fragments (4).To ensure that only specific proteins are degraded by the protease complex, bacteria make use of additional accessory factors, called adaptors, that tune the substrate specificity of the protease (5). Many adaptors act as scaffolds, tethering specific targets to the protease and increasing local concentration to drive degradation. One such example is the SspB adaptor, which binds and scaffolds ssrA-tagged substrates and the extracytoplasmic stress response factor N-RseA to ClpXP (6–8). In Caulobacter crescentus, adaptors can work additively to provide increasing levels of substrate specificity to the ClpXP protease (9). In this system, the CpdR adaptor activates ClpX and promotes binding of the RcdA adaptor (10–12). RcdA can bind a third adaptor, PopA, which requires cyclic-di-guanosine monophosphate to promote degradation of CtrA, a major regulator of the Caulobacter cell cycle (10, 13–18). In the absence of PopA, RcdA can also deliver substrates, such as the polar cue dependent chromosome segregation protein SpbR (originally annotated as CC2323) (9, 19) and the stalk synthesis protein TacA (9, 20, 21).RcdA was crystallized as a homodimer, where two three-helix bundle subunits dimerize via conserved hydrophobic residues in the second helix (22). The disordered C terminus is necessary for interactions with CpdR/ClpX, for delivery of all RcdA-dependent cargo, and for self-degradation (9, 22, 23). Upon cargo binding, the CpdR-mediated degradation of RcdA is suppressed, but how this self-degradation is regulated is unclear (23). Direct binding between RcdA and its cargo has been shown using purified components (9, 23), but the details of how RcdA binds and regulates the turnover of a diverse range of cargo remains uncertain.Here, we show that cargo binding competes with RcdA dimerization by competition for overlapping interfaces. Based on biophysical measurements, we determine that while RcdA is a dimer in solution on its own, the adaptor binds cargo as a monomer. We generate a constitutively monomeric variant by mutating the predicted dimer interface and show that RcdA dimerization is required for self-degradation. Interestingly, this variant is deficient in delivering the substrates SpbR and TacA for degradation but facilitates PopA-dependent CtrA degradation as normal. We use hydrogen–deuterium exchange mass spectrometry (HDX-MS) to map regions of RcdA important for cargo binding and find that different substrates rely on different sites of the dimer interface. Mutations at these regions result in adaptor variants that are defective for degradation of specific substrates, and expression of these variants alters cell physiology consistent with this change in specificity. Taken together, our data show how RcdA can deliver either cargo or itself for degradation and how a large interface, normally masked by dimerization, can be used to capture a range of substrates.     

An engineered 4-1BBL fusion protein with “activity on demand”

Engineered cytokines are gaining importance in cancer therapy, but these products are often limited by toxicity, especially at early time points after intravenous administration. 4-1BB is a member of the tumor necrosis factor receptor superfamily, which has been considered as a target for therapeutic strategies with agonistic antibodies or using its cognate cytokine ligand, 4-1BBL. Here we describe the engineering of an antibody fusion protein, termed F8-4-1BBL, that does not exhibit cytokine activity in solution but regains biological activity on antigen binding. F8-4-1BBL bound specifically to its cognate antigen, the alternatively spliced EDA domain of fibronectin, and selectively localized to tumors in vivo, as evidenced by quantitative biodistribution experiments. The product promoted a potent antitumor activity in various mouse models of cancer without apparent toxicity at the doses used. F8-4-1BBL represents a prototype for antibody-cytokine fusion proteins, which conditionally display “activity on demand” properties at the site of disease on antigen binding and reduce toxicity to normal tissues.

Cytokines are immunomodulatory proteins that have been considered for pharmaceutical applications in the treatment of cancer patients (1–3) and other types of disease (2). There is a growing interest in the use of engineered cytokine products as anticancer drugs, capable of boosting the action of T cells and natural killer (NK) cells against tumors (3, 4), alone or in combination with immune checkpoint inhibitors (3, 5–7).Recombinant cytokine products on the market include interleukin-2 (IL-2) (Proleukin) (8, 9), IL-11 (Neumega) (10, 11), tumor necrosis factor (TNF; Beromun) (12), interferon (IFN)-α (Roferon A, Intron A) (13, 14), IFN-β (Avonex, Rebif, Betaseron) (15, 16), IFN-γ (Actimmune) (17), granulocyte colony-stimulating factor (Neupogen) (18), and granulocyte macrophage colony-stimulating factor (Leukine) (19, 20). The recommended dose is typically very low (often <1 mg/d) (21–23), as cytokines may exert biological activity in the subnanomolar concentration range (24). Various strategies have been proposed to develop cytokine products with improved therapeutic index. Protein PEGylation or Fc fusions may lead to prolonged circulation time in the bloodstream, allowing the administration of low doses of active payload (25, 26). In some implementations, cleavable polyethylene glycol polymers may be considered, yielding prodrugs that regain activity at later time points (27). Alternatively, tumor-homing antibody fusions have been developed, since the preferential concentration of cytokine payloads at the tumor site has been shown in preclinical models to potentiate therapeutic activity, helping spare normal tissues (28–34). Various antibody-cytokine fusions are currently being investigated in clinical trials for the treatment of cancer and of chronic inflammatory conditions (reviewed in refs. 2, 33, 35–37).Antibody-cytokine fusions display biological activity immediately after injection into patients, which may lead to unwanted toxicity and prevent escalation to therapeutically active dosage regimens (9, 22, 38). In the case of proinflammatory payloads (e.g., IL-2, IL-12, TNF-α), common side effects include hypotension, nausea, and vomiting, as well as flu-like symptoms (24, 39–42). These side effects typically disappear when the cytokine concentration drops below a critical threshold, thus providing a rationale for slow-infusion administration procedures (43). It would be highly desirable to generate antibody-cytokine fusion proteins with excellent tumor-targeting properties and with “activity on demand”— biological activity that is conditionally gained on antigen binding at the site of disease, helping spare normal tissues.Here we describe a fusion protein consisting of the F8 antibody specific to the alternatively spliced extra domain A (EDA) of fibronectin (44, 45) and of murine 4-1BBL, which did not exhibit cytokine activity in solution but could regain potent biological activity on antigen binding. The antigen (EDA+ fibronectin) is conserved from mouse to man (46), is virtually undetectable in normal adult tissues (with the exception of the placenta, endometrium, and some vessels in the ovaries), but is expressed in the majority of human malignancies (44, 45, 47, 48). 4-1BBL, a member of the TNF superfamily (49), is expressed on antigen-presenting cells (50, 51) and binds to its receptor, 4-1BB, which is up-regulated on activated cytotoxic T cells (52), activated dendritic cells (52), activated NK and NKT cells (53), and regulatory T cells (54). Signaling through 4-1BB on cytotoxic T cells protects them from activation-induced cell death and skews the cells toward a more memory-like phenotype (55, 56).We engineered nine formats of the F8-4-1BBL fusion protein, one of which exhibited superior performance in quantitative biodistribution studies and conditional gain of cytokine activity on antigen binding. The antigen-dependent reconstitution of the biological activity of the immunostimulatory payload represents an example of an antibody fusion protein with “activity on demand.” The fusion protein was potently active against different types of cancer without apparent toxicity at the doses used. The EDA of fibronectin is a particularly attractive antigen for cancer therapy in view of its high selectivity, stability, and abundant expression in most tumor types (44, 45, 47, 48).     

De novo biosynthesis of a nonnatural cobalt porphyrin cofactor in E. coli and incorporation into hemoproteins

Enzymes that bear a nonnative or artificially introduced metal center can engender novel reactivity and enable new spectroscopic and structural studies. In the case of metal-organic cofactors, such as metalloporphyrins, no general methods exist to build and incorporate new-to-nature cofactor analogs in vivo. We report here that a common laboratory strain, Escherichia coli BL21(DE3), biosynthesizes cobalt protoporphyrin IX (CoPPIX) under iron-limited, cobalt-rich growth conditions. In supplemented minimal media containing CoCl₂, the metabolically produced CoPPIX is directly incorporated into multiple hemoproteins in place of native heme b (FePPIX). Five cobalt-substituted proteins were successfully expressed with this new-to-nature cobalt porphyrin cofactor: myoglobin H64V V68A, dye decolorizing peroxidase, aldoxime dehydratase, cytochrome P450 119, and catalase. We show conclusively that these proteins incorporate CoPPIX, with the CoPPIX making up at least 95% of the total porphyrin content. In cases in which the native metal ligand is a sulfur or nitrogen, spectroscopic parameters are consistent with retention of native metal ligands. This method is an improvement on previous approaches with respect to both yield and ease-of-implementation. Significantly, this method overcomes a long-standing challenge to incorporate nonnatural cofactors through de novo biosynthesis. By utilizing a ubiquitous laboratory strain, this process will facilitate spectroscopic studies and the development of enzymes for CoPPIX-mediated biocatalysis.

The identity of a metal center often defines enzymatic activity, and swapping the native metal for an alternative one or introducing a new metal center has profound effects. More generally, the chemical utility of natural cofactors has inspired decades of study into synthetic analogs with distinct properties, and researchers have subsequently sought straightforward ways to put these novel cofactors back into proteins (1). Substituted metalloenzymes constitute one of the simplest cases. Changing the identity of the metal ion in metalloproteins has enabled powerful spectroscopic and functional studies of these proteins (2–10) in addition to new biocatalytic activities (11–20). However, most methods for producing such proteins with new-to-nature cofactors are limited by the inability to produce the novel protein–cofactor complex in vivo.Hemoproteins, in particular, have been studied through metal substitution because of their important biological functions and utility as biocatalysts. Heme is a ubiquitous and versatile cofactor in biology, and heme-dependent proteins serve essential gas sensing functions (21), metabolize an array of xenobiotic molecules (22), and perform synthetically useful oxygen activation and radical-based chemistry (23). Metal-substituted hemoproteins have enabled key spectroscopic studies of hemoprotein function and the development of biocatalysts with novel reactivity. For example, electron paramagnetic resonance (EPR) studies on cobalt-substituted sperm whale myoglobin (CoMb) enabled detailed characterization of the paramagnetic CoMbO₂ complex (3, 4, 24, 25). In analogous oxygen-binding studies in CoMb and cobalt-substituted hemoglobin (5, 6, 26), resonance Raman was used to identify the O–O stretching mode because cobalt-substituted proteins exhibit enhancement of this vibrational mode compared to the native iron proteins.Metal substitution has a profound effect on catalytic activity of hemoproteins, enabling numerous synthetic applications. Substitution of the native iron for cobalt in several hemoproteins, including a thermostable cytochrome c variant, enabled the reduction of water to H₂ under aerobic, aqueous conditions (27–29). Reconstitution of apoprotein with selected metalloporphyrins has been used to generate metal-substituted myoglobin and cytochrome P450s variants. These enzymes were effective as biocatalysts for C–H activation and carbene insertion reactions (11–14). In a tour de force of directed evolution, which required purification and cofactor reconstitution of each individual variant, Hartwig and coworkers generated a cytochrome P450 variant that utilizes a nonnative Ir(Me)mesoporphyrin cofactor to perform desirable C–H activation chemistry (14). These activities may not be unique to the Ir-substituted protein, as synthetic cobalt porphyrin complexes have been shown to mediate a variety of Co(III)-aminyl and -alkyl radical transformations, including C–H activation (30–32). Indeed, a number of cobalt porphyrin carbene complexes display significant carbon-centered radical character (33–35), whereas the corresponding Fe-porphyrin complexes are closed shell species (36, 37), indicating that cobalt porphyrins may possess distinct, complementary modes of reactivity (38–40).Inspired by these applications, researchers have sought strategies for generating metal-substituted hemoproteins. For many metalloproteins, metal substitution is carried out by removal of the native metal with a chelator and replacement with an alternate metal of similar coordination preference. This method is inapplicable to hemoproteins, as porphyrins do not readily exchange metal ions. Consequently, diverse methods have been employed to make metal-substituted hemoproteins (41–46). Early on, copper, cobalt, nickel, and manganese-substituted horseradish peroxidase (HRP) were prepared by a multistep process that subjected protein to strong acid and organic solvents (41, 42). Variations of this method have been used repeatedly (24, 43, 47–49). However, this method is applicable only to a narrow range of hemoproteins that tolerate the harsh treatment. With the advent of overexpression methods, significant improvement of metalloporphyrin-substituted protein yield was achieved by direct expression of the apoprotein and reconstitution with the desired metalloporphyrin in lysate prior to purification (50). Although this approach has many virtues, direct expression of apoprotein is ineffective for many hemoproteins, again limiting the utility of this method.As an alternative to the above in vitro approaches, researchers have pursued systems for direct in vivo expression of metal substituted hemoproteins. Two specialty strains of Escherichia coli (E. coli) were engineered to incorporate metalloporphyrin analogs from the growth medium into hemoproteins during protein expression. The engineered RP523 strain cannot biosynthesize heme and bears an uncharacterized heme permeability phenotype. Together, these two features enable this strain to assimilate and incorporate various metalloporphyrins into overexpressed hemoproteins with no background heme incorporation (44, 51–53). However, heme auxotrophy makes RP523 cells exceedingly sensitive to O₂, and, in many situations, RP523 cultures must be grown anaerobically. An alternative BL21(DE3)-based engineered strain harbors a plasmid bearing the heme transporter ChuA, which facilitates import of exogenous heme analogs (45). Production of metalloporphyrin-substituted protein with this ChuA-containing strain relies on growth in iron-limited minimal media, thereby diminishing heme biosynthesis. This method was used successfully to express metal-substituted versions of the heme domain of cytochrome P450 BM3 (45) and several myoglobin variants (11, 12). Because these cells biosynthesize a small quantity of their own heme, they are far more robust than the RP523 cells. Unfortunately, this advantage comes at the cost of increased heme contamination in the product protein (2 to 5%) (45).A set of intriguing papers reported the production of cobalt-substituted human cystathionine β-synthase (CoCBS) that relies on the de novo biosynthesis of CoPPIX from CoCl₂ and δ-aminolevulinic acid (δALA), a biosynthetic precursor to heme (46, 54). This method yielded significant amounts of CoCBS—albeit with modest heme contamination (7.4%)—sufficient for spectroscopic and functional characterization of the CoPPIX-substituted protein (8, 46). As cobalt is known to be toxic to E. coli, the researchers passaged the CBS expression strain through cobalt-containing minimal media for 12 d, enabling the cells to adapt to high concentrations of cobalt prior to protein expression. It is plausible that this serial passaging alters the E. coli cells, enabling the biosynthesis of CoPPIX and in vivo production of metal-substituted protein. The adaptation process is slow (>10 d), and it is unknown how genomic instability under these mutagenic conditions affects the reproducibility of this passaging approach.The possibility of facile CoPPIX production is particularly attractive for future biocatalysis efforts. As described above, synthetic cobalt porphyrins have been shown to perform a range of radical-mediated reactions. The ability to produce a CoPPIX center in vivo may enable engineering these unusual reactivities via directed evolution in addition to spectroscopic applications. We therefore set out to explore the unusual phenotype of CoPPIX production by E. coli and to ascertain whether it was possible to efficiently biosynthesize cobalt-containing hemoproteins in vivo from a single “generalist” cell line. Our goal was to achieve an efficient and facile method of cobalt-substituted hemoprotein production with minimal contamination of the native cofactor. Herein, we report the surprising discovery that native E. coli BL21(DE3) can biosynthesize a new-to-nature CoPPIX cofactor (Fig. 1). We use this insight to produce cobalt-substituted hemoproteins in vivo without requirement for complex expression methods or specialized strains.Open in a separate windowFig. 1.Chemical structures of iron protoporphyrin IX (FePPIX or heme b), cobalt protoporphyrin IX (CoPPIX), and free base protoporphyrin IX (H₂PPIX).     

Competitive history shapes rapid evolution in a seasonal climate

Eco-evolutionary dynamics will play a critical role in determining species’ fates as climatic conditions change. Unfortunately, we have little understanding of how rapid evolutionary responses to climate play out when species are embedded in the competitive communities that they inhabit in nature. We tested the effects of rapid evolution in response to interspecific competition on subsequent ecological and evolutionary trajectories in a seasonally changing climate using a field-based evolution experiment with Drosophila melanogaster. Populations of D. melanogaster were either exposed, or not exposed, to interspecific competition with an invasive competitor, Zaprionus indianus, over the summer. We then quantified these populations’ ecological trajectories (abundances) and evolutionary trajectories (heritable phenotypic change) when exposed to a cooling fall climate. We found that competition with Z. indianus in the summer affected the subsequent evolutionary trajectory of D. melanogaster populations in the fall, after all interspecific competition had ceased. Specifically, flies with a history of interspecific competition evolved under fall conditions to be larger and have lower cold fecundity and faster development than flies without a history of interspecific competition. Surprisingly, this divergent fall evolutionary trajectory occurred in the absence of any detectible effect of the summer competitive environment on phenotypic evolution over the summer or population dynamics in the fall. This study demonstrates that competitive interactions can leave a legacy that shapes evolutionary responses to climate even after competition has ceased, and more broadly, that evolution in response to one selective pressure can fundamentally alter evolution in response to subsequent agents of selection.

Although ecological and evolutionary dynamics have traditionally been studied as independent processes assumed to proceed on fundamentally different timescales, it is now widely recognized that evolution often occurs rapidly enough to shape ecological outcomes (1–3). There is a growing interest in understanding the eco-evolutionary dynamics that result (4, 5), motivated in part by their potential importance in determining species’ fates under global environmental change (6, 7).Climate is a principal abiotic pressure that species face in the wild that can exert strong selection capable of driving rapid ecological and evolutionary change (8, 9). Understanding species’ evolutionary responses to climatic conditions has become essential, as temperature, its variability, and the frequency of extreme weather events increase under global change (10). Unfortunately, this understanding remains limited by a lack of experimental tests that place species in the complex and competitive environments in which ecology and evolution actually occur (11, 12). This represents a critical knowledge gap, as species confronted with changing climatic regimes not only face native competitors, but may also face novel competitors in the form of invasive species and species migrating in response to climate change (13, 14).We have several reasons to expect that selection imposed by competitors could shape species’ ecological and evolutionary responses to climate. First, most species live embedded in communities of competitors, rendering these interactions a likely source of selection in nature. Second, interspecific competition is widely recognized as a key driver of ecological (15, 16) and macroevolutionary dynamics (17, 18). Finally, a handful of experiments have demonstrated that species can rapidly adapt to interspecific competition (2, 19–21). Nonetheless, given that experimental evaluations of rapid evolution tend to focus on single-species populations (22, 23) or selection imposed by consumers or disease (24, 25), we have little understanding of how evolution in response to interspecific competition affects species’ abilities to persist in or adapt to new thermal regimes.Through changes in the genetic composition and phenotypic traits of populations, rapid evolution in response to competition could alter a species’ ecological trajectory, evolutionary trajectory, or both. We would expect rapid adaptation to competition to influence ecological trajectories under a shifting climate if competition drives the evolution of a phenotype, such as body size, that also influences individual performance and therefore population dynamics as temperatures change (26, 27). Selection from competition could be exerted directly via aggressive interactions with a competitor or indirectly through changes in the availability of shared resources. Studies that have experimentally demonstrated the effects of rapid evolution in response to interspecific competition have identified shifts in phenotypic traits (19, 28) that can affect population dynamics by altering birth and death rates (2, 29). Moreover, adaptive responses to competition have been shown to alter species’ population trajectories when they are also faced with changing environmental conditions, including CO₂ enrichment (23, 30).In addition to these ecological consequences of adapting to competitors, such adaptation could also alter species’ evolutionary trajectories when faced with shifting climatic conditions (31). This could arise through several mechanisms. First, theory indicates that a reduction in population size and strong selection caused by competition can reduce standing genetic variation, which could hinder adaptation to a changing climate (31–33). Second, by altering the genetic composition of populations (2, 34), adaptation to interspecific competition could influence both the magnitude and the direction of evolutionary change when organisms are exposed to novel climatic conditions (31). Traits that link genetic change and competitive performance are likely to be complex and polygenic (35–38), and, as such, the evolution of these traits may be particularly affected by epistasis and pleiotropy (39, 40). As a result, adaptation to interspecific competition could have cryptic but far-reaching consequences for subsequent evolutionary trajectories in response to changing climate if competition drives changes in allele frequencies at loci underlying variation in climate-relevant traits, or if genetic correlations link phenotypes selected under competition with those that affect fitness in a changing climate (41, 42). However, theory examining how evolutionary responses to competition can affect subsequent evolutionary responses to a changing climate remains scarce (27, 43), and the more general links between rapid adaptation in response to the changing selective agents described above have yet to be tested in a natural context.We tested how rapid evolution in response to interspecific competition influences ecological and evolutionary dynamics in a seasonal climate using a large-scale field-based experimental evolution study with the vinegar fly Drosophila melanogaster and its invasive competitor Zaprionus indianus. The interactions between D. melanogaster and Z. indianus in the seasonal climate of the northeastern United States provide an excellent natural context in which to evaluate the eco-evolutionary interactions between competition and climate. D. melanogaster maintains resident populations throughout the year in temperate North American orchards (35, 44). After emerging from diapause each spring, populations expand and rapidly evolve under warm summer conditions while feeding and laying eggs on fallen fruit (36, 37, 45). Then in fall and early winter, populations gradually decline and evolve under cooling conditions (35, 36, 46).In contrast, Z. indianus has invaded tropical regions across the globe and now seasonally invades the northeastern United States from more southern latitudes (47). Compared to D. melanogaster, Z. indianus is larger-bodied, less cold-tolerant, and slower to develop (48). In both its native and invasive range, it competes with D. melanogaster adults for food and oviposition space on rotting fruit and with D. melanogaster larvae for food during development (48). Because of its cold intolerance, Z. indianus suffers high mortality and reproductive arrest as temperatures drop in the fall (49, 50), leaving fall D. melanogaster populations to continue to reproduce and adapt to fall conditions in the absence of their interspecific competitor. It is not known how selection imposed by competition with Z. indianus over the summer affects D. melanogaster and shapes its ecology and evolution in the cooler fall.We conducted an experimental evolution study with replicate fly populations in an experimental orchard that mimics our focal species’ primary northeastern US habitat. The field mesocosms that we used experience natural temperature fluctuations and contain many of the predators and microbes that co-occur with local natural populations of D. melanogaster (37, 45). To examine the consequences of rapid evolution in response to interspecific competition on ecological and evolutionary dynamics in the fall, we first allowed replicate populations of D. melanogaster to grow and evolve in the presence or absence of Z. indianus for approximately six generations over the summer (Fig. 1). At the end of summer, we removed Z. indianus, equalized abundances of D. melanogaster across populations, and allowed the populations to continue their ecological and evolutionary dynamics through the fall (approximately three generations). We quantified the ecological (population dynamic) consequences of our treatments with weekly censuses of relative fly abundances throughout the summer and fall. We quantified the evolutionary consequences of our treatments by measuring 10 key phenotypes of D. melanogaster collected at the end of summer and end of the fall and then reared for two generations in a common garden to remove plastic responses to treatments or field conditions.Open in a separate windowFig. 1.Experimental design to determine the effect of rapid evolution in response to interspecific competition on the ecological and evolutionary trajectory of D. melanogaster in a cool fall climate. Each replicate population consisted of a large outdoor cage containing thousands (up to 100,000) of genetically diverse flies. At each “phenotyping” time point,10 fly phenotypes were measured on each replicate population after two generations in a common garden environment. In evolving populations, eggs laid in the field experiment were allowed to develop into adult flies, whereas in replacement populations, eggs laid in the field experiment were replaced by eggs laid by laboratory populations in order to prevent intergenerational adaptation to fall conditions. Colors and dashing of lines to distinguish treatments are also used in Figs. 2–4.The mechanisms that drive ecological and evolutionary patterns can be difficult to untangle in cases where ecological and evolutionary dynamics occur simultaneously (1, 51), and this is further complicated by the polygenic and multiphenotypic nature of D. melanogaster’s adaptive responses to climatic and biotic conditions (35, 36, 45, 52, 53). We therefore implemented an additional treatment in the fall phase of the experiment to provide insight into the mechanisms underlying the effects of competition on ecological and evolutionary responses to fall climate. In the fall, we effectively stopped intergenerational adaptation to fall conditions in half of our populations by replacing all eggs laid in field mesocosms with eggs laid by populations of flies collected from the experiment at the end of the summer and maintained in a nonseasonal laboratory environment (hereafter called “replacement” populations) (2, 45) (Fig. 1 and Methods). From an ecological perspective, this replacement treatment allowed us to determine the effect of adaptation to competition on fall population dynamics in both the presence and absence of further intergenerational adaptation to fall conditions. From an evolutionary perspective, it allowed us to evaluate the extent to which responses depended on intergenerational genetic change (e.g., recombination reducing negative epistatic or pleiotropic effects of adaptation or cumulative effects of selection across generations) versus recurrent selection of standing genetic variation within individual cohorts.We predicted that if competition with Z. indianus and cold fall temperatures exert opposing selection on D. melanogaster (e.g., opposing effects on body size or development time), evolution to interspecific competition would accelerate fall population decline. This could arise if, for example, the presence of slower-developing Z. indianus exerts selection for faster larval development that allows D. melanogaster to avoid larval competition but is detrimental under cold conditions (54, 55). If, instead, competition and climate were to select in the same direction, evolution to interspecific competition could slow fall population decline. This could occur if, for example, competition with the large-bodied Z. indianus for oviposition space selects for large adult body size in D. melanogaster that is beneficial under cold conditions. These expectations, of course, depend on simple relationships between genetic change, trait change, and success under competition and climate. Because the complex genetic architecture underlying fitness-associated traits is likely to generate complex links between adaptation to different selective pressures, we also predicted more generally that any divergent phenotypic and genetic changes resulting from adaptation to the summer competitive environment would shape the outcome of adaptation to subsequent fall conditions.     

Periscope Proteins are variable-length regulators of bacterial cell surface interactions

Changes at the cell surface enable bacteria to survive in dynamic environments, such as diverse niches of the human host. Here, we reveal “Periscope Proteins” as a widespread mechanism of bacterial surface alteration mediated through protein length variation. Tandem arrays of highly similar folded domains can form an elongated rod-like structure; thus, variation in the number of domains determines how far an N-terminal host ligand binding domain projects from the cell surface. Supported by newly available long-read genome sequencing data, we propose that this class could contain over 50 distinct proteins, including those implicated in host colonization and biofilm formation by human pathogens. In large multidomain proteins, sequence divergence between adjacent domains appears to reduce interdomain misfolding. Periscope Proteins break this “rule,” suggesting that their length variability plays an important role in regulating bacterial interactions with host surfaces, other bacteria, and the immune system.

Bacteria encounter complex and dynamic environments, including within human hosts, and have thus evolved various mechanisms that enable a rapid response for survival within, and exploitation of, new conditions. In addition to classical control by regulation of gene expression, bacteria exploit mechanisms that give rise to random variation to facilitate adaptation [e.g., phase and antigenic variation (1)]. In Gram-positive and Gram-negative human pathogens, DNA inversions (2, 3), homologous recombination (4), DNA methylation (1), and promoter sequence polymorphisms (5) govern changes in bacterial surface components, including capsular polysaccharide and protein adhesins, which can impact bacterial survival and virulence in the host (1, 6). Many of these mechanisms are very well studied and widespread across bacteria.A less well-studied mechanism is length variation in bacterial surface proteins. Variability in the number of sequence repeats in the Rib domain (7)–containing proteins on the surface of Group B streptococci has been linked to pathogenicity and immune evasion (8). The repetitive regions of the Staphylococcus aureus surface protein G (SasG) (9) and Staphylococcus epidermidis SasG homolog, Aap (10), also demonstrate sequence repeat number variability. In SasG, this variability regulates ligand binding by other bacterial proteins in vitro (11) in a process that has been proposed to enable bacterial dissemination in the host. Variations in repeat number have also been noted in the biofilm forming proteins Esp from Enterococcus faecalis (12) and, more recently, CdrA from Pseudomonas aeruiginosa (13). High DNA sequence identity in the genes that encode these proteins is likely to facilitate intragenic recombination events that would lead to repeat number variation (14) and, in turn, to protein sequence repetition. However, such sequence repetition is usually highly disfavored in large multidomain proteins (15), so its existence in these bacterial surface proteins suggests that protein length variation provides an evolutionary benefit. SasG, Aap, and Rib contain N-terminal host ligand binding domains and C-terminal wall attachment motifs; thus our recent demonstration that the repetitive regions of both SasG (16) and Rib (17) form unusual highly elongated rods suggests that host-colonization domains will be projected differing distances from the bacterial surface.Here, we show that repeat number variation in predicted bacterial surface proteins is more widespread and we characterize a third rod-like repetitive region in the Streptococcus gordonii protein (Sgo_0707) formed by tandem array of Streptococcal High Identity Repeats in Tandem (SHIRT) domains. Thus, we propose a growing class of “Periscope Proteins,” in which long, highly similar DNA repeats facilitate expression of surface protein stalks of variable length. This mechanism could enable changes in response to selection pressures and confer key advantages to the organism that include evasion of the host immune system (8) and regulation of surface interactions (11) involved in biofilm formation and host colonization.     

Robust folding of a de novo designed ideal protein even with most of the core mutated to valine

Protein design provides a stringent test for our understanding of protein folding. We previously described principles for designing ideal protein structures stabilized by consistent local and nonlocal interactions, based on a set of rules relating local backbone structures to tertiary packing motifs. The principles have made possible the design of protein structures having various topologies with high thermal stability. Whereas nonlocal interactions such as tight hydrophobic core packing have traditionally been considered to be crucial for protein folding and stability, the rules proposed by our previous studies suggest the importance of local backbone structures to protein folding. In this study, we investigated the robustness of folding of de novo designed proteins to the reduction of the hydrophobic core, by extensive mutation of large hydrophobic residues (Leu, Ile) to smaller ones (Val) for one of the designs. Surprisingly, even after 10 Leu and Ile residues were mutated to Val, this mutant with the core mostly filled with Val was found to not be in a molten globule state and fold into the same backbone structure as the original design, with high stability. These results indicate the importance of local backbone structures to the folding ability and high thermal stability of designed proteins and suggest a method for engineering thermally stabilized natural proteins.

The de novo design of protein structures, starting from pioneering work (1, 2), has been achieved in tandem with our understanding of how amino acid sequences determine folded structures (3–16). A breakthrough in protein design methodology was a finding of principles for encoding funnel-shaped energy landscapes into amino acid sequences (7, 10, 17, 18). Based on studies of protein folding, it had been suggested that naturally occurring proteins have evolved to have funnel-shaped energy landscapes toward their folded structures (19–23). However, complicated structures of naturally occurring proteins with nonideal features for folding—for example, kinked α-helices, bulged β-strands, long or strained loops, and buried polar groups—make it difficult to understand how the funnels are encoded in amino acid sequences. By focusing on protein structures without such nonideal features, we proposed principles for designing ideal protein structures stabilized by completely consistent local and nonlocal interactions (24), based on a set of rules relating local backbone structures to preferred tertiary motifs (7, 10). These design rules describe the relation of the lengths or torsion patterns of two secondary structure elements and the connecting loop to favorable packing geometries (SI Appendix, Fig. S1A). The design principles enable to encode strongly funneled energy landscapes into amino acid sequences, by the stabilization of folded structures (positive design) and by the destabilization of nonnative conformations (negative design) due to the restriction of folding conformational space by the rules (SI Appendix, Fig. S1C). In the design procedure, backbone structures for a target topology are generated based on a blueprint (SI Appendix, Fig. S1B), in which either the lengths or backbone torsion patterns of the secondary structures and loops are determined using the rules so that the tertiary motifs present in the target topology are favored, and then amino acid sequences stabilizing the generated backbone structures are designed. The designed amino acid sequences stabilize their folded structures both with nonlocal interactions such as hydrophobic core packing and with local interactions favoring the secondary structures and loops specified in the blueprint, which destabilize a myriad of nonnative topologies through local backbone strain captured by the rules, thereby resulting in funnel-shaped energy landscapes (SI Appendix, Fig. S1C). The principles have enabled the de novo design of ideal protein structures for various topologies with atomic-level accuracy (Fig. 1) (6, 7, 10, 13).Open in a separate windowFig. 1.In silico energy landscapes and far-UV circular dichroism (CD) spectra for 10 de novo designed ideal proteins. (A–E) Five designs by Koga et al. in 2012 (7). (F–I) Four designs by Lin et al. in 2015 (10). (J) Top7 by Kuhlman et al. in 2003 (6). (Top) Design models. (Middle) Energy landscapes obtained from Rosetta ab initio structure prediction simulations (41). Red points represent the lowest energy structures obtained in independent Monte Carlo structure prediction trajectories starting from an extended chain for each sequence; the y axis is the Rosetta all-atom energy; the x axis is the Cα root-mean-square deviation (RMSD) to the design model. Green points represent the lowest energy structures obtained in trajectories starting from the design model. (Bottom) The far-UV CD spectra during thermal denaturation with the melting temperature T_m, which is obtained by fitting to the denaturation curves shown in SI Appendix, Fig. S2.Interestingly, the de novo designs exhibit prominent characteristics in terms of thermal stability when compared with naturally occurring proteins. The circular dichroism (CD) measurements up to 170 °C conducted in this study revealed the melting temperature (T_m), which was above 100 °C for most of the designs (Fig. 1) (6, 7, 10). Therefore, the designs have great potential for use as scaffolds to engineer proteins with specific functions of interest. Indeed, miniprotein structures (∼40 residues) designed de novo according to the rules were applied as scaffolds for creating protein binders specific for influenza hemagglutinin and botulinum neurotoxin, displaying high thermal stability (>70 °C) despite the small size (25).The rules in the principles described above emphasize the importance of local backbone structures not the details of amino acid side chains to protein folding, which is also supported by studies using simple calculations with the hydrophobic-polar lattice model or the snake-cube model (26, 27). On the other hand, it is known that hydrophobic interactions are the dominant driving force for folding (28, 29) and the cores of naturally occurring proteins are tightly packed with hydrophobic amino acid residues (30, 31) like a jigsaw puzzle. Indeed, in our design principles, protein cores were designed to be tightly packed and as “fat” as possible with larger hydrophobic residues so that energy landscapes were sculpted to be deeply funneled into a target topology by lowering its energy (SI Appendix, Fig. S1C).Which factor, the local backbone structures encoded by the rules or the tight core packing with fat hydrophobic residues, contributes more to the generation of funnels in the designs? Here, we studied the contribution of hydrophobic core packing to folding ability and thermal stability by investigating the robustness of folding against the reduction of packing, using the design with the highest thermal stability among our nine de novo designs (Fig. 1, except Top7), Rsmn2x2_5_6 (10). We started to study single-residue mutants from Leu or Ile to Val that prune one carbon atom from the aliphatic side chain, which lose the tight packing like a jigsaw puzzle and decrease the hydrophobicity, and then, we combined the mutations. Consequently, we found that a mutant with 10 residue substitutions of Leu or Ile with Val still has the folding ability and high thermal stability despite its reduced and loosened hydrophobic core packing. This result suggests the importance of the local backbone structures for the folding ability and stability of the de novo designs.     

The battle between harvest and natural selection creates small and shy fish

Harvest of fish and wildlife, both commercial and recreational, is a selective force that can induce evolutionary changes to life history and behavior. Naturally selective forces may create countering selection pressures. Assessing natural fitness represents a considerable challenge in broadcast spawners. Thus, our understanding about the relative strength of natural and fisheries selection is slim. In the field, we compared the strength and shape of harvest selection to natural selection on body size over four years and behavior over one year in a natural population of a freshwater top predator, the northern pike (Esox lucius). Natural selection was approximated by relative reproductive success via parent–offspring genetic assignments over four years. Harvest selection was measured by comparing individuals susceptible to recreational angling with individuals never captured by this gear type. Individual behavior was measured by high-resolution acoustic telemetry. Harvest and natural size selection operated with equal strength but opposing directions, and harvest size selection was consistently negative in all study years. Harvest selection also had a substantial behavioral component independent of body length, while natural behavioral selection was not documented, suggesting the potential for directional harvest selection favoring inactive, timid fish. Simulations of the outcomes of different fishing regulations showed that traditional minimum size-based harvest limits are unlikely to counteract harvest selection without being completely restrictive. Our study suggests harvest selection may be inevitable and recreational fisheries may thus favor small, inactive, shy, and difficult-to-capture fish. Increasing fractions of shy fish in angling-exploited stocks would have consequences for stock assessment and all fisheries operating with hook and line.

Anticipating and preparing for future evolutionary changes within harvested populations whether by fishing or hunting is critical for sustainable natural resource management and successful conservation of ecosystems (1–6). Harvest-induced evolution is a concern for both commercial and recreational fisheries, and harvest from recreational fisheries now frequently exceeds harvest from commercial fisheries in some marine fish and most inland fish populations (7). Harvesting, firstly, elevates adult mortality which favors the evolution of life history adaptations that maximize current as opposed to future reproduction [i.e., a fast life history characterized by early reproduction at a small size and elevated reproductive effort (1, 2)]. Additionally, harvesting is trait selective. Most individuals in harvested populations are not captured or hunted randomly (8). Instead, a suite of traits elevates the probability of harvest (8–13). In fisheries, vulnerability to harvest and fish body size are positively related across most fishing gears, and the relationship is exacerbated by the widespread use of minimum landing sizes (14, 15). Consequently, the average body size of individuals within fish stocks is commonly observed to decrease (15, 16).Decreasing average body size in fish stocks first results from demographic truncation by direct removal of large individuals within a generation but may also result from evolutionary adaptation to a new fitness landscape (17). Positively size-selective harvesting alters the fitness landscape by favoring early reproduction at smaller sizes, in turn slowing down postmaturation growth due to altered allocation of energy from soma to gonads (2, 18). Additionally, reduced postmaturation growth may arise from evolutionary adaptations in energy acquisition–related behaviors [e.g., evolution of risk-sensitive foraging in response to the selective removal of bold, active, or aggressive behavioral phenotypes (19, 20)]. There is considerable debate whether any observed phenotypic changes, derived from monitoring data from wild fisheries, in life history traits such as maturation timing or growth rate are indeed evolutionary (i.e., genetic) or an effect of phenotypic plasticity (21), and a recent review concluded that no conclusive example for fisheries-induced evolution exists at the scale of wild fisheries (21).Most research on fisheries-induced selection and evolution has been focused on life history traits (2). However, fisheries can also induce adaptive changes in behavior through at least two mechanisms. First, by creating selection pressures that favor fast life histories, fisheries may indirectly alter correlated behavioral traits like aggressive and bold behaviors (22–24). Second, passive gear types such as gill nets, traps, or hooks heavily rely on a behavioral response by individual fish for successful capture (25). Fish that are able to forage more, at the expense of taking more risks, are able to grow faster and may produce more offspring (26–28), but they may also be more vulnerable to capture (10, 27) and mortality by predation (29). Accordingly, models comparing life history outcomes emerging from either purely behavioral to purely size-dependent vulnerability to capture demonstrate that behavioral selection can create the same pressures and ultimately evolutionary outcomes as size-selective capture and, depending on context, either favor bold or shy fish (30, 31). As personality traits are known to have a heritable component (32, 33) and vary consistently among individuals (34, 35), the selective capture of active, aggressive, and bold fish may ultimately promote the emergence of timid populations (10, 19, 27). Independent of life history adaptations, these changes may also disrupt the “pace-of-life” syndrome and the correlation of behavior and life history (24, 36, 37). A widespread increase in timidity implies that fish will become harder to catch (10). If this is the case, challenges in stock assessments will arise as they are built on assumptions of consistent fish availability to sampling gear over time to serve as indices of abundance (19, 38, 39).Our understanding of selective harvest’s impact on phenotypic change has not yet been able to fully explain empirical observations from fisheries in the wild (40, 41). Indeed, the rate and impacts of harvest-induced evolution continues to attract controversy despite more than 20 y of research (2, 21, 41). Models of harvest-induced life history evolution consistently underestimate rates of phenotypic change observed in empirical studies from the wild, while experimental studies in the laboratory tend to overestimate empirical rates of evolution (40–42). The discrepancy between models or laboratory studies and empirical data in the wild may partly result from plastic, rather than evolutionary, impacts on phenotypes collected in the wild (43), from inappropriate assumptions of fitness trade-offs in models (30, 31), from exaggerated fishing mortality induced in selection line experiments (44), or from inadvertent selection on other traits correlated with growth, such as behavioral traits, rather than direct selection on size (30, 31). To understand the potential for harvest-induced evolution, a key first step is to understand the selection pressures induced by exploitation in the wild (42, 45). This is because following the breeder’s equation from quantitative genetics, the selection response in any trait is a product of the selection differentials acting on a trait and the trait’s heritability (46). We focus here on estimating selection acting on adaptive traits in a wild fish population and compare the selection to natural selective forces on the same traits.In particular, the counteracting forces of natural selection must be considered to understand the total selective forces acting on a phenotype (47, 48). However, natural selection has rarely been empirically measured in the context of harvest selection in wild fisheries (45, 47–49). Meta-analyses on selection in the wild indicate that fishing is one of the few anthropogenic selective forces consistently stronger than natural selection (49). Yet, natural selection compared to size-selective fisheries has, so far, only been quantified by fitness proxies such as survival (45), growth rate, or female body size (47, 48), assumed to be positively correlated with lifetime reproductive success (RS) (50). As the RS of fish is challenging to measure in the wild, it is unclear how body size and fitness actually scale (50), and consequently it is largely unclear what natural selection on body size or other traits looks like in exploited stocks. Further, the fitness landscape of behavioral traits has rarely been assessed in the wild, although behavior commonly relates to growth (51), survival (52, 53), and RS (26, 27).Our aim was to quantify the strength and direction of harvest and natural selection in the wild using an experimentally exploited top predatory fish and to improve our understanding of whether a portion of harvest size selection is actually the result of undetected behavioral selection (54, 55). To that end, we investigated the strength and direction of harvest selection on body size and activity in northern pike, Esox lucius, measuring fitness in the context of natural selection as relative reproductive success (RRS) over four years and classification of movement behavior over one year using high-resolution acoustic telemetry (56) covering an entire natural ecosystem. We used hook and line fishing as an example of a widespread fishing gear used by both recreational and commercial fisheries. We predicted that harvest and natural size selection act in opposition in which larger fish would have higher RRS (50) but would also be more likely to be captured by angling (57, 58). Furthermore, we expected that fishing selection on size would be much stronger than natural selection (49). However, we also predicted additional harvest selection on behavior (55) because recreational fishing gear is known to be related to behavioral phenotypes (10, 55, 59–61). Finally, through simulations, we investigated how regulations could alter the relationship between harvest and natural selection and potentially counteract fishing selection considering minimum length limits and harvest slots based on established models (42).