The intracellular environment affects protein–protein interactions

Protein–protein interactions are essential for life but rarely thermodynamically quantified in living cells. In vitro efforts show that protein complex stability is modulated by high concentrations of cosolutes, including synthetic polymers, proteins, and cell lysates via a combination of hard-core repulsions and chemical interactions. We quantified the stability of a model protein complex, the A34F GB1 homodimer, in buffer, Escherichia coli cells and Xenopus laevis oocytes. The complex is more stable in cells than in buffer and more stable in oocytes than E. coli. Studies of several variants show that increasing the negative charge on the homodimer surface increases stability in cells. These data, taken together with the fact that oocytes are less crowded than E. coli cells, lead to the conclusion that chemical interactions are more important than hard-core repulsions under physiological conditions, a conclusion also gleaned from studies of protein stability in cells. Our studies have implications for understanding how promiscuous—and specific—interactions coherently evolve for a protein to properly function in the crowded cellular environment.

Proteins rarely work alone. Networks of protein–protein interactions turn a myriad of chemical signals into physiological responses that maintain intracellular homeostasis (1). Therefore, it is unsurprising that nearly two-thirds of disease-causing missense mutations involve protein complexes (2). However, nearly all equilibrium thermodynamic and kinetic studies of protein–protein interactions have been performed in dilute buffer. Acquiring quantitative equilibrium data on protein complexes in cells, despite the fact that living things are not at equilibrium, is important for two reasons. First, the equilibrium condition is the starting point for estimating the driving force under nonequilibrium conditions (3). Second, recent results on protein stability show that conclusions from experiments conducted in dilute solution cannot be extrapolated to the crowded and complex intracellular environment (4).The cellular interior contains a variety of proteins, nucleic acids, and small molecules. In the bacterium Escherichia coli, the concentration of macromolecules can exceed 300 g/L and occupy up to 30% of volume (5). The macromolecule concentration in eukaryotic cells is smaller (6) but still hundreds of times larger than the solute concentrations used in most in vitro studies. Furthermore, the majority of proteins in the cells studied here, E. coli and oocytes, are net polyanions with mean isoelectric points of 6.6 and 6.7, respectively (7). Understanding how the intracellular environment modulates protein—and protein complex—stability is important because the totality of weak interactions in cells form the so-called quinary structure that organizes the crowded cellular interior (8–10). Importantly, these interactions cannot be measured in dilute solutions.The physical consequences of macromolecular crowding arise from two components: hard-core repulsions and so-called “soft” chemical interactions (11). Hard-core repulsion is a steric effect, arising from the impenetrable nature of atoms. Steric repulsions reduce the volume available to reactants and products. Simple theory predicts that sterics favor compact states (12). For small, folded proteins, stability is described by the two-state equilibrium between the biologically active folded state and the inactive, higher-energy, and less-compact unfolded state (13). For protein complex formation involving folded proteins, which comprises an equilibrium between constituent proteins and the complex, the complex exposes less surface than the sum of the two monomers (14). Thus, simple theory predicts stabilization of proteins and protein complexes, although more sophisticated theoretical efforts focused on the size of crowding molecule predict stabilization or destabilization (15, 16).Soft interactions include hydrogen bonds and charge–charge, water–protein, solute–protein, and hydrophobic interactions. Of these, the only strong repulsive interaction is that from the opposition of like charges. These repulsions add to the hard-core effect and are therefore stabilizing. The other soft interactions are attractive and destabilizing because they favor expanded conformations that allow access to the attractive surfaces.Until recently the focus was on hard-core interactions and crowding (17) because studies of protein stability (quantified as the free energy of unfolding, ΔGUo'') in uncharged synthetic polymers tended to show only a stabilizing influence compared to buffer alone (11). However, recent studies of stability under more physiologically relevant crowded conditions and in living cells show, by and large, a decrease in stability (4). Studies of protein–protein interactions in vitro under physiologically relevant crowded conditions also show the importance of chemical interactions (18, 19).Yeast two-hybrid techniques, coimmunoprecipitation, and split systems provide simple yes/no information about protein interactions in cells, however, these methodologies are also prone to false positives. The protein–protein interface can also be characterized by in-cell NMR. However, quantification of the equilibrium thermodynamics of complex stability is challenging in cells. The few reports tend to involve heterodimerization (20), which can be complicated to assess because the concentration of both reactants must be controlled, fluorescence-resonance-energy transfer because labels adding hundreds to thousands of daltons must be included, or because competition with natural, unlabeled proteins in cells must be considered (21–27).     

Funneled energy landscape unifies principles of protein binding and evolution

Most proteins have evolved to spontaneously fold into native structure and specifically bind with their partners for the purpose of fulfilling biological functions. According to Darwin, protein sequences evolve through random mutations, and only the fittest survives. The understanding of how the evolutionary selection sculpts the interaction patterns for both biomolecular folding and binding is still challenging. In this study, we incorporated the constraint of functional binding into the selection fitness based on the principle of minimal frustration for the underlying biomolecular interactions. Thermodynamic stability and kinetic accessibility were derived and quantified from a global funneled energy landscape that satisfies the requirements of both the folding into the stable structure and binding with the specific partner. The evolution proceeds via a bowl-like evolution energy landscape in the sequence space with a closed-ring attractor at the bottom. The sequence space is increasingly reduced until this ring attractor is reached. The molecular-interaction patterns responsible for folding and binding are identified from the evolved sequences, respectively. The residual positions participating in the interactions responsible for folding are highly conserved and maintain the hydrophobic core under additional evolutionary constraints of functional binding. The positions responsible for binding constitute a distributed network via coupling conservations that determine the specificity of binding with the partner. This work unifies the principles of protein binding and evolution under minimal frustration and sheds light on the evolutionary design of proteins for functions.

Proteins in nature have a high degree of thermodynamic and kinetic specificities different from random heteropolymers of amino acids (1–3). Except for intrinsically disordered proteins, naturally occurring proteins are believed to evolve to spontaneously fold into stable native structure and specifically bind with partners for fulfilling the biological functions (4–6). Directed evolution, which mimics natural evolution via rounds of mutagenesis and selections in the laboratory, has also successfully obtained desired protein functions (7–11). According to Darwin, protein sequences evolve through random mutations for the fitness (12). The evolutionary constraint to fold into a particular, stable three-dimensional structure has been considered as the fitness to greatly restrict the sequence space of protein evolution (13–18). However, the biological functions of the proteins are often performed through binding with their partners. The evolutionary selection ultimately operates on the functions other than the structures.A protein’s biological function, such as binding/recognition, conformation dynamics, and activity, can be described by its thermodynamics and kinetics, which are determined by the underlying interactions between the residues. The principle of minimal frustration has been fruitful in illustrating how the global pattern of interactions determines thermodynamic stability and kinetic accessibility of protein folding and binding (3, 19–25). The principle requires that energetic conflicts are minimized in folded native states, so that a sequence can spontaneously fold. Because of the functional necessity, naturally occurring sequences are actually in the tradeoff for coding the capacity to simultaneously satisfy stable folding and functional binding. From the view of localized frustration (23–27), naturally occurring proteins maintain a conserved network of minimally frustrated interactions at the hydrophobic core. In contrast, highly frustrated interactions tend to be clustered on the surface, often near binding sites that become less frustrated upon binding. A natural question is how the evolution sculpts the interaction patterns that conflict with the overall folding of minimal frustration but are specific for protein binding.Extensive statistical analysis of the evolutionary information has shown that native structures of protein folding and binding can be reliably predicted from the global pattern of interactions between amino acids extracted from homologous native sequences (NSs) (28–34). This indicates that thermodynamic and kinetic specificities of protein folding and binding are encoded as the evolutionary footprints on the NSs. In this sense, thermodynamic and kinetic specificities should be not only the evolutionary outcomes but also the selection pressures on protein evolution. The proposed selection fitness quantified by the folding requirement of the thermodynamic stability and the kinetic accessibility has successfully evolved sequences and structures of small domains with strong protein characteristics, including the hydrophobic core, high designability, and fast folding (35). The principle of minimal frustration as a rule to quantify the selection fitness has provided the physical mechanism and mathematical formations for the theoretical and computational studies of protein-folding evolution.Different from our previous study, which concentrated on the evolution of individual domain folding (35), here, we incorporated the constraint of functional binding into the selection fitness under the principle of minimal frustration. Thermodynamic stability and kinetic accessibility were derived and quantified from the global funneled energy landscape, which satisfies the requirements of both folding into the stable structure and binding with the specific partner. The evolution under the selection fitness of optimizing both folding and binding requirements is realized through a bowl-like energy landscape with a closed-ring attractor at the bottom. The sequence space is increasingly reduced until this ring attractor is reached. The interaction patterns respectively responsible for the folding and binding are extracted from the evolved sequences. The residual positions participating in the interactions responsible for folding are highly conserved and maintain the hydrophobic core under additional evolutionary constraints of functional binding. The positions responsible for binding constitute a distributed network via coupling conservations of the residual positions. This distributed network with coupling conservations determines the specificity of the binding with the partner, and the interactions involving the positions of the network can be influenced and adjusted depending on the binding partner. This work unifies the principles of protein binding and evolution and provides an evolution strategy to generate evolved sequences similar to naturally occurring sequences.     

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.     

Human species D adenovirus hexon capsid protein mediates cell entry through a direct interaction with CD46

Human adenovirus species D (HAdV-D) types are currently being explored as vaccine vectors for coronavirus disease 2019 (COVID-19) and other severe infectious diseases. The efficacy of such vector-based vaccines depends on functional interactions with receptors on host cells. Adenoviruses of different species are assumed to enter host cells mainly by interactions between the knob domain of the protruding fiber capsid protein and cellular receptors. Using a cell-based receptor-screening assay, we identified CD46 as a receptor for HAdV-D56. The function of CD46 was validated in infection experiments using cells lacking and overexpressing CD46, and by competition infection experiments using soluble CD46. Remarkably, unlike HAdV-B types that engage CD46 through interactions with the knob domain of the fiber protein, HAdV-D types infect host cells through a direct interaction between CD46 and the hexon protein. Soluble hexon proteins (but not fiber knob) inhibited HAdV-D56 infection, and surface plasmon analyses demonstrated that CD46 binds to HAdV-D hexon (but not fiber knob) proteins. Cryoelectron microscopy analysis of the HAdV-D56 virion–CD46 complex confirmed the interaction and showed that CD46 binds to the central cavity of hexon trimers. Finally, soluble CD46 inhibited infection by 16 out of 17 investigated HAdV-D types, suggesting that CD46 is an important receptor for a large group of adenoviruses. In conclusion, this study identifies a noncanonical entry mechanism used by human adenoviruses, which adds to the knowledge of adenovirus biology and can also be useful for development of adenovirus-based vaccine vectors.

Wild-type human adenoviruses (HAdVs) are associated with a broad range of more or less severe clinical manifestations in respiratory, lymphoid, gastrointestinal, and ocular tissues (1). To date, more than 100 HAdV types have been identified, and classified into seven species, A through G (2). HAdV species D comprises two-thirds of all known types and exhibits features that make these types attractive as vaccine vectors (3–7). Thus, HAdV-D types are currently being explored as vaccine vectors for prevention against infections caused by, for example, severe acute respiratory syndrome coronavirus 2 (8–10), HIV (11), respiratory syncytial virus (12), Ebola virus (13), and Zika virus (14). The presence and engagement of functional receptors on relevant host cells are features that are crucial for the efficacy of adenovirus-based vector vaccines, and knowledge about such interactions is of importance since this allows detargeting of vectors to native receptors present on less relevant cells but also opens up for retargeting of vectors to relevant host cells.HAdV-A and C through G types enter host cells through interactions with the Coxsackievirus and adenovirus receptor (CAR) (15–17), and HAdV-B types interact with CD46 and/or desmoglein-2 (DSG2) (18–21). HAdV-D37 and a few other HAdV-D types interact with sialic acid-containing glycans (22, 23). These interactions are all mediated by the knob domain of the fiber capsid protein. HAdV-D37 and several other distinct HAdV-D types have also been reported to use CD46 as a cellular receptor (3, 5, 24–31), a protein that is expressed on all human nucleated cells including dendritic cells (3). Remarkably, whereas the interactions between HAdV-B types and CD46 are well-characterized and clearly depend on the fiber protein (18–20, 32), little is known about the mechanism whereby HAdV-D types engage CD46. One study suggests that HAdV-D37 interacts with CD46 through the fiber knob domain (27), but this interaction has not been functionally or structurally validated. In this study, we demonstrate that HAdV-D56 and D26 engage CD46 through a nonconventional interaction involving the hexon instead of the fiber, and that 16 out of 17 randomly selected HAdV-D types use CD46 as a cellular receptor.     

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.     

Cholinergic suppression of hippocampal sharp-wave ripples impairs working memory

Learning and memory are assumed to be supported by mechanisms that involve cholinergic transmission and hippocampal theta. Using G protein–coupled receptor-activation–based acetylcholine sensor (GRAB_ACh3.0) with a fiber-photometric fluorescence readout in mice, we found that cholinergic signaling in the hippocampus increased in parallel with theta/gamma power during walking and REM sleep, while ACh3.0 signal reached a minimum during hippocampal sharp-wave ripples (SPW-R). Unexpectedly, memory performance was impaired in a hippocampus-dependent spontaneous alternation task by selective optogenetic stimulation of medial septal cholinergic neurons when the stimulation was applied in the delay area but not in the central (choice) arm of the maze. Parallel with the decreased performance, optogenetic stimulation decreased the incidence of SPW-Rs. These findings suggest that septo–hippocampal interactions play a task-phase–dependent dual role in the maintenance of memory performance, including not only theta mechanisms but also SPW-Rs.

The neurotransmitter acetylcholine is thought to be critical for hippocampus-dependent declarative memories (1, 2). Reduction in cholinergic neurotransmission, either in Alzheimer’s disease or in experiments with cholinergic antagonists, such as scopolamine, impairs memory function (3–8). Acetylcholine may bring about its beneficial effects on memory encoding by enhancing theta rhythm oscillations, decreasing recurrent excitation, and increasing synaptic plasticity (9–11). Conversely, drugs which activate cholinergic receptors enhance learning and, therefore, are a neuropharmacological target for the treatment of memory deficits in Alzheimer’s disease (5, 12, 13).The contribution of cholinergic mechanisms in the acquisition of long-term memories and the role of the hippocampal–entorhinal–cortical interactions are well supported by experimental data (5, 12, 13). In addition, working memory or “short-term” memory is also supported by the hippocampal–entorhinal–prefrontal cortex (14–16). Working memory in humans is postulated to be a conscious process to “keep things in mind” transiently (16). In rodents, matching to sample task, spontaneous alternation between reward locations, and the radial maze task have been suggested to function as a homolog of working memory [“working memory like” (17)].Cholinergic activity is a critical requirement for working memory (18, 19) and for sustaining theta oscillations (10, 20–22). In support of this contention, theta–gamma coupling and gamma power are significantly higher in the choice arm of the maze, compared with those in the side arms where working memory is no longer needed for correct performance (23–26). It has long been hypothesized that working memory is maintained by persistent firing of neurons, which keep the presented items in a transient store in the prefrontal cortex and hippocampal–entorhinal system (27–31), although the exact mechanisms are debated (32–37). An alternative hypothesis holds that items of working memory are stored in theta-nested gamma cycles (38). Common in these models of working memory is the need for an active, cholinergic system–dependent mechanism (39–41). However, in spontaneous alternation tasks, the animals are not moving continuously during the delay, and theta oscillations are not sustained either. During the immobility epochs, theta is replaced by intermittent sharp-wave ripples (SPW-R), yet memory performance does not deteriorate. On the contrary, artificial blockade of SPW-Rs can impair memory performance (42, 43), and prolongation of SPW-Rs improves performance (44). Under the cholinergic hypothesis of working memory, such a result is unexpected.To address the relationship between cholinergic/theta versus SPW-R mechanism in spontaneous alternation, we used a G protein–coupled receptor-activation–based acetylcholine sensor (GRAB_ACh3.0) (45) to monitor acetylcholine (ACh) activity during memory performance in mice. In addition, we optogenetically enhanced cholinergic tone, which suppresses SPW-Rs by a different mechanism than electrically or optogenetically induced silencing of neurons in the hippocampus (43, 44). We show that cholinergic signaling in the hippocampus increases in parallel with theta power/score during walking and rapid eye movement (REM) sleep and reaches a transient minimum during SPW-Rs. Selective optogenetic stimulation of medial septal cholinergic neurons decreased the incidence of SPW-Rs during non-REM sleep (46–48), as well as during the delay epoch of a working memory task and impaired memory performance. These findings demonstrate that memory performance is supported by complementary theta and SPW-R mechanisms.     

Phosphorylation-dependent subfunctionalization of the calcium-dependent protein kinase CPK28

Calcium (Ca²⁺)-dependent protein kinases (CDPKs or CPKs) are a unique family of Ca²⁺ sensor/kinase-effector proteins with diverse functions in plants. In Arabidopsis thaliana, CPK28 contributes to immune homeostasis by promoting degradation of the key immune signaling receptor-like cytoplasmic kinase BOTRYTIS-INDUCED KINASE 1 (BIK1) and additionally functions in vegetative-to-reproductive stage transition. How CPK28 controls these seemingly disparate pathways is unknown. Here, we identify a single phosphorylation site in the kinase domain of CPK28 (Ser318) that is differentially required for its function in immune homeostasis and stem elongation. We show that CPK28 undergoes intermolecular autophosphorylation on Ser318 and can additionally be transphosphorylated on this residue by BIK1. Analysis of several other phosphorylation sites demonstrates that Ser318 phosphorylation is uniquely required to prime CPK28 for Ca²⁺ activation at physiological concentrations of Ca²⁺, possibly through stabilization of the Ca²⁺-bound active state as indicated by intrinsic fluorescence experiments. Together, our data indicate that phosphorylation of Ser318 is required for the activation of CPK28 at low intracellular [Ca²⁺] to prevent initiation of an immune response in the absence of infection. By comparison, phosphorylation of Ser318 is not required for stem elongation, indicating pathway-specific requirements for phosphorylation-based Ca²⁺-sensitivity priming. We additionally provide evidence for a conserved function for Ser318 phosphorylation in related group IV CDPKs, which holds promise for biotechnological applications by generating CDPK alleles that enhance resistance to microbial pathogens without consequences to yield.

Protein kinases represent one of the largest eukaryotic protein superfamilies. While roughly 500 protein kinases have been identified in humans (1), the genomes of Arabidopsis thaliana (hereafter, Arabidopsis) (2) and Oryza sativa (3) encode more than 1,000 and 1,500 protein kinases, respectively, including several families unique to plants. Among these protein kinases are the receptor-like kinases (RLKs), receptor-like cytoplasmic kinases (RLCKs), and calcium-dependent protein kinases (CDPKs or CPKs) that have emerged as key regulators of plant immunity (4–6). Despite encompassing only 2% of most eukaryotic genomes, protein kinases phosphorylate more than 40% of cellular proteins (7, 8), reflecting their diverse roles in coordinating intracellular signaling events. Reversible phosphorylation of serine (Ser), threonine (Thr), and tyrosine (Tyr) residues can serve an array of functions including changes in protein conformation and activation state (9, 10), protein stability and degradation (11, 12), subcellular localization (13–15), and interaction with protein substrates (16–18).Calcium (Ca²⁺) is a ubiquitous secondary messenger that acts cooperatively with protein phosphorylation to propagate intracellular signals. Spatial and temporal changes in intracellular Ca²⁺ levels occur in response to environmental and developmental cues (19–23). In plants, Ca²⁺ transients are decoded by four major groups of calcium sensor proteins, which possess one or more Ca²⁺-binding EF-hand motifs (24, 25): calmodulins (CaM), CaM-like proteins, calcineurin B–like proteins, CDPKs, and Ca²⁺/CaM-dependent protein kinases.At the intersection of phosphorylation cascades and Ca²⁺ signaling are CDPKs, a unique family of Ca²⁺ sensor/kinase-effector proteins. CDPKs have been identified in all land plants and green algae, as well as certain protozoan ciliates and apicomplexan parasites (26, 27). CDPKs have a conserved domain architecture, consisting of a canonical Ser/Thr protein kinase domain and an EF-hand containing Ca²⁺-binding CaM-like domain (CLD), linked together by an autoinhibitory junction (AIJ) and flanked by variable regions on both the amino (N) and carboxyl (C) termini (28, 29). As their name implies, most CDPKs require Ca²⁺ for their activation (30). Upon Ca²⁺ binding to all EF-hands in the CaM-like domain, a dramatic conformational change occurs, freeing the AIJ from the catalytic site of the kinase, rendering the enzyme active (31–33). CDPKs vary in their sensitivity to Ca²⁺ (30), presumably allowing proteins to perceive distinct stimuli through differences in Ca²⁺-binding affinity. For example, Arabidopsis CPK4 displays half maximal kinase activity in the presence of ∼3 μM free Ca²⁺ (30) while CPK5 only requires ∼100 nM (34). Importantly, CDPKs are signaling hubs with documented roles in multiple distinct pathways (4, 24, 35–38) and are therefore likely regulated beyond Ca²⁺ activation.Subfunctionalization is at least partially mediated by protein localization and interaction with pathway-specific binding partners, as is well documented for Arabidopsis CPK3 which functions in response to biotic and abiotic stimuli in distinct cellular compartments (39). Recent attention has been drawn to site-specific phosphorylation as a mechanism to regulate the activity of multifunctional kinases. For example, phosphorylation sites on the RLK BRASSINOSTEROID INSENSITIVE 1–ASSOCIATED KINASE 1 (BAK1) are differentially required for its function as a coreceptor with a subset of leucine-rich repeat –RLKs (40). Phosphoproteomic analyses indicate that CDPKs are differentially phosphorylated following exposure to distinct stimuli (41–48); however, the biochemical mechanisms by which site-specific phosphorylation regulates multifunctional CDPKs is still poorly understood.Arabidopsis CPK28 is a plasma membrane–localized protein kinase with dual roles in plant immune homeostasis (49–51) and phytohormone-mediated reproductive growth (52, 53). In vegetative plants, CPK28 serves as a negative regulator of immune signal amplitude by phosphorylating and activating two PLANT U-BOX–type E3 ubiquitin ligases, PUB25 and PUB26, which target the key immune RLCK BOTRYTIS-INDUCED KINASE 1 (BIK1) for proteasomal degradation (50). Owing to elevated levels of BIK1, CPK28 null plants (cpk28-1) have heightened immune responses and enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pto DC3000) (51). Upon transition to the reproductive stage, cpk28-1 plants additionally present shorter leaf petioles, enhanced anthocyanin production, and a reduction in stem elongation (52, 53). The molecular basis for developmental phenotypes in the cpk28-1 knockout mutant, beyond hormonal imbalance (52, 53), are comparatively unknown.Our recent work demonstrated that autophosphorylation status dictates Ca²⁺-sensitivity of CPK28 peptide kinase activity in vitro (54). While dephosphorylated CPK28 is stimulated by the addition of 100 μM CaCl₂ compared to untreated protein, hyperphosphorylated CPK28 displayed similar levels of activity at basal Ca²⁺ concentrations (54). These results highlight the interesting possibility that phosphorylation status may control the activation of multifunctional kinases in distinct pathways by allowing CDPKs to respond to stimulus-specific Ca²⁺ signatures.In the present study, we identify a single autophosphorylation site, Ser318, that decouples the activity of CPK28 in immune signaling from its role in reproductive growth. We show that expression of a nonphosphorylatable Ser-to-Ala variant (CPK28^S318A) is unable to complement the immune phenotypes of cpk28-1 mutants but is able to complement defects in stem growth. Further, we uncover a functional role for phosphorylation of Ser318 in priming CPK28 for activation at low free [Ca²⁺]. Together, we demonstrate that site-specific phosphorylation can direct the activity of a multifunctional kinase in distinct pathways and provide evidence for a conserved mechanism in orthologous group IV CDPKs.     

ICAM-1 induced rearrangements of capsid and genome prime rhinovirus 14 for activation and uncoating

Most rhinoviruses, which are the leading cause of the common cold, utilize intercellular adhesion molecule-1 (ICAM-1) as a receptor to infect cells. To release their genomes, rhinoviruses convert to activated particles that contain pores in the capsid, lack minor capsid protein VP4, and have an altered genome organization. The binding of rhinoviruses to ICAM-1 promotes virus activation; however, the molecular details of the process remain unknown. Here, we present the structures of virion of rhinovirus 14 and its complex with ICAM-1 determined to resolutions of 2.6 and 2.4 Å, respectively. The cryo-electron microscopy reconstruction of rhinovirus 14 virions contains the resolved density of octanucleotide segments from the RNA genome that interact with VP2 subunits. We show that the binding of ICAM-1 to rhinovirus 14 is required to prime the virus for activation and genome release at acidic pH. Formation of the rhinovirus 14–ICAM-1 complex induces conformational changes to the rhinovirus 14 capsid, including translocation of the C termini of VP4 subunits, which become poised for release through pores that open in the capsids of activated particles. VP4 subunits with altered conformation block the RNA–VP2 interactions and expose patches of positively charged residues. The conformational changes to the capsid induce the redistribution of the virus genome by altering the capsid–RNA interactions. The restructuring of the rhinovirus 14 capsid and genome prepares the virions for conversion to activated particles. The high-resolution structure of rhinovirus 14 in complex with ICAM-1 explains how the binding of uncoating receptors enables enterovirus genome release.

Human rhinoviruses are the cause of more than half of common colds (1). Medical visits and missed days of school and work cost tens of billions of US dollars annually (2, 3). There is currently no cure for rhinovirus infections, and the available treatments are only symptomatic. Rhinoviruses belong to the family Picornaviridae, genus Enterovirus, and are classified into species A, B, and C (4). Rhinoviruses A and B can belong to either “major” or “minor” groups, based on their utilization of intercellular adhesion molecule-1 (ICAM-1) or low-density lipoprotein receptor for cell entry (5–7). Type C rhinoviruses use CDHR3 as a receptor (8). Rhinovirus 14 belongs to the species rhinovirus B and uses ICAM-1 as a receptor. Receptors recognized by rhinoviruses and other enteroviruses can be divided into two groups based on their function in the infection process (9). Attachment receptors such as DAF, PSGL1, KREMEN1, CDHR3, and sialic acid enable the binding and endocytosis of virus particles into cells (10–13). In contrast, uncoating receptors including ICAM-1, CD155, CAR, and SCARB2 enable virus cell entry but also promote genome release from virus particles (5, 14–16).Virions of rhinoviruses are nonenveloped and have icosahedral capsids (17). Genomes of rhinoviruses are 7,000 to 9,000 nucleotide-long single-stranded positive-sense RNA molecules (1, 17). The rhinovirus genome encodes a single polyprotein that is co- and posttranslationally cleaved into functional protein subunits. Capsid proteins VP1, VP3, and VP0, originating from one polyprotein, form a protomer, 60 of which assemble into a pseudo-T = 3 icosahedral capsid. To render the virions mature and infectious, VP0 subunits are cleaved into VP2 and VP4 (18, 19). VP1 subunits form pentamers around fivefold symmetry axes, whereas subunits VP2 and VP3 form heterohexamers centered on threefold symmetry axes. The major capsid proteins VP1 through 3 have a jelly roll β-sandwich fold formed by two β-sheets, each containing four antiparallel β-strands, which are conventionally named B to I (20–22). The two β-sheets contain the strands BIDG and CHEF, respectively. The C termini of the capsid proteins are located at the virion surface, whereas the N termini mediate interactions between the capsid proteins and the RNA genome on the inner surface of the capsid. VP4 subunits are attached to the inner face of the capsid formed by the major capsid proteins. The surfaces of rhinovirus virions are characterized by circular depressions called canyons, which are centered around fivefold symmetry axes of the capsids (21).The VP1 subunits of most rhinoviruses, but not those of rhinovirus 14, contain hydrophobic pockets, which are filled by molecules called pocket factors (17, 21, 23, 24). It has been speculated that pocket factors are fatty acids or lipids (25). The pockets are positioned immediately below the canyons. The exposure of rhinoviruses to acidic pH induces expulsion of the pocket factors, which leads to the formation of activated particles and genome release (17, 26–32). The activated particles are characterized by capsid expansion, a reduction in interpentamer contacts, the release of VP4 subunits, externalization of N termini of VP1 subunits, and changes in the distribution of RNA genomes (17, 26–29, 33, 34). Artificial hydrophobic compounds that bind to VP1 pockets with high affinity inhibit infection by rhinoviruses (35, 36).ICAM-1 is an endothelial- and leukocyte-associated protein that stabilizes cell–cell interactions and facilitates the movement of leukocytes through endothelia (37). ICAM-1 can be divided into an extracellular amino-terminal part composed of five immunoglobulin domains, a single transmembrane helix, and a 29-residue–long carboxyl-terminal cytoplasmic domain. The immunoglobulin domains are characterized by a specific fold that consists of seven to eight β-strands, which form two antiparallel β-sheets in a sandwich arrangement (38–40). The immunoglobulin domains of ICAM-1 are stabilized by disulfide bonds and glycosylation (38–41). The connections between the immunoglobulin domains are formed by flexible linkers that enable bending of the extracellular part of ICAM-1. For example, the angle between domains 1 and 2 differs by 8° between molecules in distinct crystal forms (38, 42). As a virus receptor, ICAM-1 enables the virus particles to sequester at the cell surface and mediates their endocytosis (5). The structures of complexes of rhinoviruses 3, 14, and 16, and CVA21 with ICAM-1 have been determined to resolutions of 9 to 28 Å (42–46). It was shown that ICAM-1 molecules bind into the canyons at the rhinovirus surface, approximately between fivefold and twofold symmetry axes (42–46). ICAM-1 interacts with residues from all three major capsid proteins. It has been speculated that the binding of ICAM-1 triggers the transition of virions of rhinovirus 14 to activated particles and initiates genome release (45, 47). However, the limited resolution of the previous studies prevented characterization of the corresponding molecular mechanism.Here, we present the cryo-electron microscopy (cryo-EM) reconstruction of the rhinovirus 14 virion, which contains resolved density of octanucleotide segments of the RNA genome that interact with VP2 subunits. Furthermore, we show that the binding of ICAM-1 to rhinovirus 14 induces changes in its capsid and genome, which are required for subsequent virus activation and genome release at acidic pH.     

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.     

Determinism and contingencies shaped the evolution of mitochondrial protein import

Mitochondrial protein import requires outer membrane receptors that evolved independently in different lineages. Here we used quantitative proteomics and in vitro binding assays to investigate the substrate preferences of ATOM46 and ATOM69, the two mitochondrial import receptors of Trypanosoma brucei. The results show that ATOM46 prefers presequence-containing, hydrophilic proteins that lack transmembrane domains (TMDs), whereas ATOM69 prefers presequence-lacking, hydrophobic substrates that have TMDs. Thus, the ATOM46/yeast Tom20 and the ATOM69/yeast Tom70 pairs have similar substrate preferences. However, ATOM46 mainly uses electrostatic, and Tom20 hydrophobic, interactions for substrate binding. In vivo replacement of T. brucei ATOM46 by yeast Tom20 did not restore import. However, replacement of ATOM69 by the recently discovered Tom36 receptor of Trichomonas hydrogenosomes, while not allowing for growth, restored import of a large subset of trypanosomal proteins that lack TMDs. Thus, even though ATOM69 and Tom36 share the same domain structure and topology, they have different substrate preferences. The study establishes complementation experiments, combined with quantitative proteomics, as a highly versatile and sensitive method to compare in vivo preferences of protein import receptors. Moreover, it illustrates the role determinism and contingencies played in the evolution of mitochondrial protein import receptors.

Intracellular endosymbionts lack protein import systems, whereas such systems are a defining feature of mitochondria and plastids, both of which evolved from bacterial endosymbionts (1–3). Today, more than 95% of all mitochondrial proteins are imported from the cytosol, which makes mitochondrial protein import a key process required for mitochondrial biogenesis (4–6). The question of how mitochondrial protein import evolved is therefore central to understand how the endosymbiotic bacterial ancestor of mitochondria converted into an organelle that is genetically integrated into the host cell (7–9).Proteins are targeted to mitochondria by internal or external import signals, the most frequent one of which is the N-terminal presequence found in 60 to 70% of all imported proteins (10, 11). Interestingly, the various mitochondrial import signals are conserved even between highly diverged eukaryotes (6). The import signals are decoded by receptors, which are integral mitochondrial outer membrane (OM) proteins that are associated with the heterooligomeric protein translocase of the OM (TOM complex) (6, 12). Contrary to the core components of the TOM complex (Tom40, Tom22, and Tom7), which are highly conserved in essentially all eukaryotes, these receptors evolved independently in different eukaryotic lineages, even though they recognize the same conserved import signals (6).The best studied prototypical import receptors are Tom20 and Tom70 of yeast, orthologs of which are found in all members of the eukaryotic supergroup of the opisthokonts (13). Tom20 is an N-terminally anchored OM membrane protein, and its cytosolic domain contains a single tetratricopeptide repeat (TPR). Tom20 preferentially recognizes precursor proteins that have N-terminal presequences. It binds to the hydrophobic surface of the presequence and transfers the precursors to the highly conserved Tom22 that functions as a secondary receptor (14–17). Tom70 is the primary receptor for proteins that have multiple membrane spanning domains, such as mitochondrial carrier proteins, but also binds to hydrophobic precursor proteins that have presequences (18–20). Moreover, it has been shown that binding of Tom70 to the mitochondrial presequence-like stretches that are present in the mature part of many precursor proteins increases the import efficiency (21). Tom70 is N-terminally anchored in the membrane. Its large cytosolic domain consists of 11 TPR motifs. The three TPR motifs proximal to the membrane interact with cytosolic Hsp70 or Hsp90, from which Tom70 can receive precursor proteins (22, 23). The remaining eight TPR motifs directly recognize substrate proteins (24, 25). In yeast, Tom20 and Tom70 have partially redundant functions. Tom70 is not essential for growth and respiration. Loss of Tom20 causes a stronger phenotype; it abolishes respiration but is not lethal. Finally, even the deletion of Tom70 and Tom20 does not kill the cells, provided that the secondary receptor Tom22 is still present (15, 26–29).A single import receptor, termed Tom20, is associated with the TOM complex of plant mitochondria. Yeast and plant Tom20 (30) are superficially similar: both have a single transmembrane domain (TMD) and a soluble domain containing one (in yeast) and two TPR motifs (in plants). Furthermore, both proteins have the same domain organization provided that they are aligned in an antiparallel way. Thus, whereas yeast Tom20 is N-terminally anchored, plant Tom20 is a C-terminally anchored protein. This strongly suggests that yeast and plant Tom20, while both being import receptors, have different evolutionary origins (31, 32). Moreover, plants have another TPR domain-containing OM protein, termed OM64, that is not associated with the TOM complex, but implicated in protein import (31, 33).ATOM46 and ATOM69 are the two receptor subunits of the atypical translocase of the OM (ATOM) of trypanosomatids (34). ATOM69 is superficially similar to yeast Tom70. Both have the same molecular mass and multiple TPR-like motifs. ATOM69, in addition, has an N-terminal CS/Hsp20-like domain, which potentially can bind to cytosolic chaperones. Analogous to plant Tom20, ATOM69 is C-terminally membrane-anchored, whereas yeast Tom70 has an N-terminal TMD. ATOM46 also has an N-terminal membrane anchor and a cytosolic armadillo (ARM) repeat domain, a protein–protein interaction module specific for eukaryotes. The cytosolic domains of ATOM69 and ATOM46 were shown to bind a number of different precursor proteins and are essential for normal growth (34). ATOM69 and ATOM46 have been found in all kinetoplastids as well as in euglenoids (35). Except for the TPR domain in ATOM69, the two import receptors of trypanosomes do not resemble the TOM subunits of other species, indicating that they evolved independently from both the yeast and the plant receptors.Recently, an analysis of the TOM complex in Trichomonas vaginalis hydrogenosomes, which are mitochondria-derived hydrogen-producing organelles that lack their own genome (36), identified Tom36 and Tom46 (37). The two proteins are paralogues and consist of an N-terminal CS/Hsp20-like domain, three TPR-like sequences, and a C-terminal membrane anchor, which is reminiscent of trypanosomal ATOM69, although the mass of both hydrogenosomal proteins is much lower than that of ATOM69. Moreover, HHpred analysis, using Tom36 as a query, retrieved ATOM69 as the first hit (37). The cytosolic domains of Tom36 and Tom46 were able to bind hydrogenosomal precursor proteins, suggesting they may function as protein import receptors. However, despite the similarities between ATOM69 and Trichomonas Tom36/Tom46, phylogenetic analysis suggests that they evolved independently of each other, and therefore reflect yet another example of convergent evolution, although a diversification of a common ancestor cannot be ruled out (37).Here, we have investigated the substrate specificity of the trypanosomal import receptors ATOM46 and ATOM69 using inducible RNA interference (RNAi) cell lines and biochemical methods. We could correlate the observed receptor preference with specific features of the recognized substrate proteins, such as the presence of a predicted presequence, average hydrophobicity, and presence of TMDs. Moreover, we devised a method that allows for identification of which trypanosomal precursor proteins can be recognized by heterologous import receptors. Using this method, the mitochondrial proteomes are quantitatively compared between Trypanosoma brucei cell lines lacking either ATOM46 or ATOM69 and with T. brucei cell lines in which ATOM46 or ATOM69 were replaced by either Tom20 from yeast or Tom36 from Trichomonas.     

Transferrin receptor targeting by de novo sheet extension

The de novo design of polar protein–protein interactions is challenging because of the thermodynamic cost of stripping water away from the polar groups. Here, we describe a general approach for designing proteins which complement exposed polar backbone groups at the edge of beta sheets with geometrically matched beta strands. We used this approach to computationally design small proteins that bind to an exposed beta sheet on the human transferrin receptor (hTfR), which shuttles interacting proteins across the blood–brain barrier (BBB), opening up avenues for drug delivery into the brain. We describe a design which binds hTfR with a 20 nM K_d, is hyperstable, and crosses an in vitro microfluidic organ-on-a-chip model of the human BBB. Our design approach provides a general strategy for creating binders to protein targets with exposed surface beta edge strands.

While most protein–protein interfaces are composed primarily of sidechain–sidechain interactions, backbone hydrogen bonding can also play a role. For example, beta sheet hydrogen bonds across protein–protein interfaces are present in complexes of PDZ domains with their peptide targets, SUMO–SIM interactions, and Serpin-protease complexes among others (1, 2); in each case, the result is an extended beta sheet that spans both partners. Such backbone interactions can contribute to interaction specificity even though backbone hydrogen bonding groups are present on all residues: Formation of hydrogen bonds with the correct geometry requires precise alignment of often twisted or curved beta strands—the structure of the interacting edge strand in a binder must be matched to the structure of the edge strand it interacts with in the target. Pathological processes such as the formation of amyloid fibrils also involve beta sheet extension (3), and inhibitors that hydrogen bond to the beta sheet have been developed to block such extension (4, 5). Design approaches have been used to create homodimeric structures with extended beta sheets (6, 7) that rely on symmetrical/self-docking of scaffolds to form homodimers. But to date, methods for designing heterodimeric complexes through beta sheet extension in which one component (the target protein) is fixed have not been described, even though targets with an exposed edge strand constitute a substantial class of therapeutically interesting molecules (1, 2).A challenge in designing binding proteins is how to form an extensive binding interface while avoiding the energetically unfavorable burial of nonhydrogen-bonded polar groups on the target. Indeed, previous de novo binder design efforts have focused primarily on sidechain–sidechain interactions with hydrophobic patches on target protein surfaces with few polar groups (8, 9). With this approach, polar regions of a target protein surface are difficult to design binders against as it is very challenging to make sidechain-mediated hydrogen bonds to all the exposed polar groups simultaneously; in particular, the many exposed C=O and N–H groups at the edges of beta sheets are difficult to fully engage with sidechain hydrogen bonds. We reasoned that designed binding proteins with edge beta strands complementary in shape to an exposed beta strand in the target protein could overcome this challenge, as the multiple strand–strand hydrogen bonds could compensate for the loss of interactions with water.     

Myristoylation alone is sufficient for PKA catalytic subunits to associate with the plasma membrane to regulate neuronal functions

Myristoylation is a posttranslational modification that plays diverse functional roles in many protein species. The myristate moiety is considered insufficient for protein–membrane associations unless additional membrane-affinity motifs, such as a stretch of positively charged residues, are present. Here, we report that the electrically neutral N-terminal fragment of the protein kinase A catalytic subunit (PKA-C), in which myristoylation is the only functional motif, is sufficient for membrane association. This myristoylation can associate a fraction of PKA-C molecules or fluorescent proteins (FPs) to the plasma membrane in neuronal dendrites. The net neutral charge of the PKA-C N terminus is evolutionally conserved, even though its membrane affinity can be readily tuned by changing charges near the myristoylation site. The observed membrane association, while moderate, is sufficient to concentrate PKA activity at the membrane by nearly 20-fold and is required for PKA regulation of AMPA receptors at neuronal synapses. Our results indicate that myristoylation may be sufficient to drive functionally significant membrane association in the absence of canonical assisting motifs. This provides a revised conceptual base for the understanding of how myristoylation regulates protein functions.

Myristoylation is a major type of posttranslational modification that occurs at the N terminus of a myriad of proteins (1–4). Depending on the target, myristoylation can contribute to the structure, stability, protein–protein interactions, and subcellular localization of the modified proteins (2–4). In particular, myristoylation often facilitates protein association with the membrane. However, it is thought that, with an acyl chain of only 14 carbons, myristate confers insufficient energy for stable association of a protein with the membrane (5, 6). Subsequent studies have shown that a second membrane-affinity motif, such as a stretch of basic residues or a second lipid modification, is required for the membrane association of several myristoylated proteins (reviewed in refs. 2–4). When the second membrane-affinity motif is removed or neutralized, either physiologically or via mutagenesis, the membrane localization of the protein is disrupted. Thus, the canonical view is that myristoylation alone is not sufficient to provide a functionally significant association of a protein with the plasma membrane, even though myristoylation has been observed to be associated with reconstituted lipid bilayers (7).Myristoylation was first discovered in the catalytic subunit of protein kinase A (PKA) (1, 8), which is a primary mediator of the second messenger cAMP that plays diverse essential roles in nearly all organisms, from bacteria to humans. At rest, PKA is a tetrameric protein that consists of two regulatory subunits (PKA-Rs) and two catalytic subunits (PKA-Cs). PKA holoenzymes are anchored to specific subcellular locations via the binding of PKA-R with A-Kinase anchoring proteins (9–12). In the presence of cAMP, PKA-C is released from PKA-R and becomes an active kinase (8, 13–16).Despite being myristoylated, PKA-C is thought to function as a cytosolic protein because of its high solubility (14, 15). Consistently, a PKA-C mutant with disrupted myristoylation has been shown to support the phosphorylation of certain substrates and to maintain several PKA functions in heterologous cells (17). Structural studies found that the PKA-C myristoylation is folded into a hydrophobic pocket, and it was proposed that this myristoylation serves a structural role (18–20). This view has started to shift based on recent reports showing that activated PKA-C can associate with the membrane in a myristoylation-dependent manner (16, 21, 22), including in neurons. However, the extent to which PKA-C associates with the plasma membrane in living cells and its functional significance are not known. Furthermore, as discussed above, myristolyation-mediated membrane association is thought to require a second-membrane motif. The identity of this second membrane-affinity motif has not been determined. Therefore, we set out to address these questions.     

Cadherin cis and trans interactions are mutually cooperative

Cadherin transmembrane proteins are responsible for intercellular adhesion in all biological tissues and modulate tissue morphogenesis, cell motility, force transduction, and macromolecular transport. The protein-mediated adhesions consist of adhesive trans interactions and lateral cis interactions. Although theory suggests cooperativity between cis and trans bonds, direct experimental evidence of such cooperativity has not been demonstrated. Here, the use of superresolution microscopy, in conjunction with intermolecular single-molecule Förster resonance energy transfer, demonstrated the mutual cooperativity of cis and trans interactions. Results further demonstrate the consequent assembly of large intermembrane junctions, using a biomimetic lipid bilayer cell adhesion model. Notably, the presence of cis interactions resulted in a nearly 30-fold increase in trans-binding lifetimes between epithelial-cadherin extracellular domains. In turn, the presence of trans interactions increased the lifetime of cis bonds. Importantly, comparison of trans-binding lifetimes of small and large cadherin clusters suggests that this cooperativity is primarily due to allostery. The direct quantitative demonstration of strong mutual cooperativity between cis and trans interactions at intermembrane adhesions provides insights into the long-standing controversy of how weak cis and trans interactions act in concert to create strong macroscopic cell adhesions.

Cadherin adhesion proteins are essential for the hierarchical organization of all multicellular organisms, and their dysfunction is associated with several pathologies (1–8). For example, deficiencies in cadherin-mediated adhesion are correlated with the onset and metastasis of multiple cancers and tissue diseases (6–8). Cadherin-mediated adhesion involves the formation of adherens junctions (9, 10), which entail interactions between cadherin extracellular domains in cis and trans configurations, where cis interactions occur between proteins on the same cell membrane, and trans interactions occur between proteins on opposing membranes (11–26). Theory suggests that these cis and trans bonds form cooperatively (11–26). For example, cis interactions are believed to enhance molecular ordering (15) and may increase intercellular adhesion through cluster avidity (16, 27, 28), with potential applications related to angiogenesis and therefore cancer therapies (29–31). Early studies suggested that cis interactions enhanced the cadherin adhesive function (16). However, observations of lateral cis interactions between cadherin extracellular domains have been elusive because of their low affinity and the challenges of studying membrane-bound proteins (32–34). Observations of cis interactions in crystal structures and the disruption of cadherin organization within junctions by putative cis mutants suggested that they operate in tandem with trans interactions (15, 35). One hypothesis was that initial trans binding enhances the cis-binding affinity, leading to lateral clustering, junction nucleation, and growth (36). Such trans → cis cooperativity was predicted theoretically but not verified experimentally (37).Recent single-molecule (SM) studies successfully demonstrated that cis interactions induced clustering between cadherin extracellular domains on a supported lipid bilayer (SLB) (38, 39). The latter result suggested that conformational constraints associated with membrane immobilization increased the cis-binding affinity sufficiently to induce clustering, even in the absence of trans interactions. This observation raised the possibility of the reciprocal cooperativity (i.e., cis → trans), in which initial cis binding may enhance adhesion. Although the probability (but not the strength) of trans binding was found to increase for cadherin dimer constructs, relative to the monomer (34), the connection to cis interactions, if any, remains unclear. Demonstrating cis/trans cooperativity would require demonstrating that the presence of cis interactions alters the strength of trans bonds quantitatively and vice versa.In this study, we systematically identified and quantified cis/trans cooperativity, using dynamic SM Förster resonance energy transfer (FRET). These measurements determine whether cis interactions increase trans-binding lifetimes and, conversely, whether trans interactions increase cis-binding lifetimes. They further elucidated the putative role of cis/trans cooperativity in the formation and growth of cadherin junctions between opposing membranes. We find that cis and trans interactions are strongly and mutually cooperative. Most importantly, results show that cis interactions dramatically increase trans-binding lifetimes by more than an order of magnitude, and these cooperative interactions are shown to facilitate the assembly of large junctions. A detailed analysis of trans-binding kinetics as a function of cluster size provide insight into the molecular mechanism of the elevated trans lifetimes. The results presented suggest that specific cis interactions allosterically activate trans-binding interactions.     

Replisome bypass of a protein-based R-loop block by Pif1

Efficient and faithful replication of the genome is essential to maintain genome stability. Replication is carried out by a multiprotein complex called the replisome, which encounters numerous obstacles to its progression. Failure to bypass these obstacles results in genome instability and may facilitate errors leading to disease. Cells use accessory helicases that help the replisome bypass difficult barriers. All eukaryotes contain the accessory helicase Pif1, which tracks in a 5′–3′ direction on single-stranded DNA and plays a role in genome maintenance processes. Here, we reveal a previously unknown role for Pif1 in replication barrier bypass. We use an in vitro reconstituted Saccharomyces cerevisiae replisome to demonstrate that Pif1 enables the replisome to bypass an inactive (i.e., dead) Cas9 (dCas9) R-loop barrier. Interestingly, dCas9 R-loops targeted to either strand are bypassed with similar efficiency. Furthermore, we employed a single-molecule fluorescence visualization technique to show that Pif1 facilitates this bypass by enabling the simultaneous removal of the dCas9 protein and the R-loop. We propose that Pif1 is a general displacement helicase for replication bypass of both R-loops and protein blocks.

Efficient and faithful replication of the genome is essential to maintain genome stability and is carried out by a multiprotein complex called the replisome (1–4). There are numerous obstacles to progression of the replisome during the process of chromosome duplication. These obstacles include RNA-DNA hybrids (R-loops), DNA secondary structures, transcribing RNA polymerases, and other tightly bound proteins (5–9). Failure to bypass these barriers may result in genome instability, which can lead to cellular abnormalities and genetic disease. Cells contain various accessory helicases that help the replisome bypass these difficult barriers (10–20). A subset of these helicases act on the opposite strand of the replicative helicase (1, 2, 14, 19).All eukaryotes contain an accessory helicase, Pif1, which tracks in a 5′–3′ direction on single-stranded DNA (ssDNA) (11–16). Pif1 is important in pathways such as Okazaki-fragment processing and break-induced repair that require the removal of DNA-binding proteins as well as potential displacement of R-loops (11–13, 21, 15–18, 22–25). Genetic studies and immunoprecipitation pull-down assays indicate that Pif1 interacts with PCNA (the DNA sliding clamp), Pol ε (the leading-strand polymerase), the MCMs (the motor subunits of the replicative helicase CMG), and RPA (the single-stranded DNA-binding protein) (15, 26, 27). Pif1 activity in break-induced repair strongly depends on its interaction with PCNA (26). These interactions with replisomal components suggest that Pif1 could interact with the replisome during replication. In Escherichia coli, the replicative helicase is the DnaB homohexamer that encircles the lagging strand and moves in a 5′–3′ direction (20). E. coli accessory helicases include the monomeric UvrD (helicase II) and Rep, which move in the 3′–5′ direction and operate on the opposite strand from the DnaB hexamer. It is known that these monomeric helicases promote the bypass of barriers during replication such as stalled RNA polymerases (5). The eukaryotic replicative helicase is the 11-subunit CMG (Cdc45, Mcm2–7, GINS) and tracks in the 3′–5′ direction, opposite to the direction of Pif1 (25, 28). Once activated by Mcm10, the MCM motor domains of CMG encircle the leading strand (29–32). We hypothesized that, similar to UvrD and Rep in E. coli, Pif1 interacts with the replisome tracking in the opposite direction to enable bypass of replication obstacles.In this report, we use an in vitro reconstituted Saccharomyces cerevisiae replisome to study the role of Pif1 in bypass of a “dead” Cas9 (dCas9), which is a Cas9 protein that is deactivated in DNA cleavage but otherwise fully functional in DNA binding. As with Cas9, dCas9 is a single-turnover enzyme that can be programmed with a guide RNA (gRNA) to target either strand. The dCas9–gRNA complex forms a roadblock consisting of an R-loop and a tightly bound protein (dCas9), a construct that is similar to a stalled RNA polymerase. This roadblock (hereafter dCas9 R-loop) arrests replisomes independent of whether the dCas9 R-loop is targeted to the leading or lagging strand (30). Besides its utility due to its programmable nature (33), the use of the dCas9 R-loop allows us to answer several mechanistic questions. For example, the ability to program the dCas9 R-loop block to any specific sequence enables us to observe whether block removal is different depending on whether the block is on the leading or lagging strand. Furthermore, the inner diameter of CMG can accommodate double-stranded DNA (dsDNA) and possibly an R-loop, but not a dCas9 protein. Using the dCas9 R-loop block allows us to determine the fate of each of its components.Here, we report that Pif1 enables the bypass of the dCas9 R-loop by the replisome. Interestingly, dCas9 R-loops targeted to either the leading or lagging strand are bypassed with similar efficiency. In addition, the PCNA clamp is not required for bypass of the block, indicating that Pif1 does not need to interact with PCNA during bypass of the block. We used a single-molecule fluorescence imaging to show that both the dCas9 and the R-loop are displaced as an intact nucleoprotein complex. We propose that Pif1 is a general displacement helicase for replication bypass of both R-loops and protein blocks.