Structural basis for p50RhoGAP BCH domain–mediated regulation of Rho inactivation

Spatiotemporal regulation of signaling cascades is crucial for various biological pathways, under the control of a range of scaffolding proteins. The BNIP-2 and Cdc42GAP Homology (BCH) domain is a highly conserved module that targets small GTPases and their regulators. Proteins bearing BCH domains are key for driving cell elongation, retraction, membrane protrusion, and other aspects of active morphogenesis during cell migration, myoblast differentiation, and neuritogenesis. We previously showed that the BCH domain of p50RhoGAP (ARHGAP1) sequesters RhoA from inactivation by its adjacent GAP domain; however, the underlying molecular mechanism for RhoA inactivation by p50RhoGAP remains unknown. Here, we report the crystal structure of the BCH domain of p50RhoGAP Schizosaccharomyces pombe and model the human p50RhoGAP BCH domain to understand its regulatory function using in vitro and cell line studies. We show that the BCH domain adopts an intertwined dimeric structure with asymmetric monomers and harbors a unique RhoA-binding loop and a lipid-binding pocket that anchors prenylated RhoA. Interestingly, the β5-strand of the BCH domain is involved in an intermolecular β-sheet, which is crucial for inhibition of the adjacent GAP domain. A destabilizing mutation in the β5-strand triggers the release of the GAP domain from autoinhibition. This renders p50RhoGAP active, thereby leading to RhoA inactivation and increased self-association of p50RhoGAP molecules via their BCH domains. Our results offer key insight into the concerted spatiotemporal regulation of Rho activity by BCH domain–containing proteins.

Small GTPases are molecular switches that cycle between an active GTP-bound state and an inactive GDP-bound state and are primarily involved in cytoskeletal reorganization during cell motility, morphogenesis, and cytokinesis (1, 2). These small GTPases are tightly controlled by activators and inactivators, such as guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), respectively (3, 4), which are multidomain proteins that are themselves regulated through their interactions with other proteins, lipids, secondary messengers, and/or by posttranslational modifications (5–7). Despite our understanding of the mechanisms of action of GTPases, GAPs, and GEFs, little is known about how they are further regulated by other cellular proteins in tightly controlled local environments.The BNIP-2 and Cdc42GAP Homology (BCH) domain has emerged as a highly conserved and versatile scaffold protein domain that targets small GTPases, their GEFs, and GAPs to carry out various cellular processes in a spatial, temporal, and kinetic manner (8–15). BCH domain–containing proteins are classified into a distinct functional subclass of the CRAL_TRIO/Sec14 superfamily, with ∼175 BCH domain–containing proteins (in which 14 of them are in human) present across a range of eukaryotic species (16). Some well-studied BCH domain–containing proteins include BNIP-2, BNIP-H (CAYTAXIN), BNIP-XL, BNIP-Sα, p50RhoGAP (ARHGAP1), and BPGAP1 (ARHGAP8), with evidence to show their involvement in cell elongation, retraction, membrane protrusion, and other aspects of active morphogenesis during cell migration, growth activation and suppression, myoblast differentiation, and neuritogenesis (17–21). Aside from interacting with small GTPases and their regulators, some of these proteins can also associate with other signaling proteins, such as fibroblast growth factor receptor tyrosine kinases, myogenic Cdo receptor, p38-MAP kinase, Mek2/MP1, and metabolic enzymes, such as glutaminase and ATP-citrate lyase (17–26). Despite the functional diversity and versatility of BCH domain–containing proteins, the structure of the BCH domain and its various modes of interaction remain unknown. The BCH domain resembles the Sec14 domain (from the CRAL-TRIO family) (16, 27, 28), a domain with lipid-binding characteristics, which may suggest that the BCH domain could have a similar binding strategy. However, to date, the binding and the role of lipids in BCH domain function remain inconclusive.Of the BCH domain–containing proteins, we have focused on the structure and function of p50RhoGAP. p50RhoGAP comprises an N-terminal BCH domain and a C-terminal GAP domain separated by a proline-rich region. We found that p50RhoGAP contains a noncanonical RhoA-binding motif in its BCH domain and is associated with GAP-mediated cell rounding (13). Further, we showed previously that deletion of the BCH domain dramatically enhanced the activity of the adjacent GAP domain (13); however, the full dynamics of this interaction is unclear. Previously, it has been reported that the BCH and other domains regulate GAP activity in an autoinhibited manner (18, 21, 29, 30) involving the interactions of both the BCH and GAP domains, albeit the mechanism remains to be investigated. It has also been shown that a lipid moiety on Rac1 (a Rho GTPase) is necessary for its inactivation by p50RhoGAP (29, 31), which may imply a role in lipid binding. An understanding of how the BCH domain coordinates with the GAP domain to affect the local activity of RhoA and other GTPases would offer a previously unknown insight into the multifaceted regulation of Rho GTPase inactivation.To understand the BCH domain–mediated regulation of p50RhoGAP and RhoA activities, we have determined the crystal structure of a homologous p50RhoGAP BCH domain from S. pombe for functional interrogation. We show that the BCH domain adopts an intertwined dimeric structure with asymmetric monomers and harbors a unique RhoA-interacting loop and a lipid-binding pocket. Our results show that the lipid-binding region of the BCH domain helps to anchor the prenylation tail of RhoA while the loop interacts directly with RhoA. Moreover, we show that a mutation in the β5-strand releases the autoinhibition of the GAP domain by the BCH domain. This renders the GAP domain active, leading to RhoA inactivation and the associated phenotypic effects in yeast and HeLa cells. The released BCH domain also contributes to enhanced p50RhoGAP–p50RhoGAP interaction. Our findings offer crucial insights into the regulation of Rho signaling by BCH domain–containing proteins.     

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.     

Membrane bending by protein phase separation

Membrane bending is a ubiquitous cellular process that is required for membrane traffic, cell motility, organelle biogenesis, and cell division. Proteins that bind to membranes using specific structural features, such as wedge-like amphipathic helices and crescent-shaped scaffolds, are thought to be the primary drivers of membrane bending. However, many membrane-binding proteins have substantial regions of intrinsic disorder which lack a stable three-dimensional structure. Interestingly, many of these disordered domains have recently been found to form networks stabilized by weak, multivalent contacts, leading to assembly of protein liquid phases on membrane surfaces. Here we ask how membrane-associated protein liquids impact membrane curvature. We find that protein phase separation on the surfaces of synthetic and cell-derived membrane vesicles creates a substantial compressive stress in the plane of the membrane. This stress drives the membrane to bend inward, creating protein-lined membrane tubules. A simple mechanical model of this process accurately predicts the experimentally measured relationship between the rigidity of the membrane and the diameter of the membrane tubules. Discovery of this mechanism, which may be relevant to a broad range of cellular protrusions, illustrates that membrane remodeling is not exclusive to structured scaffolds but can also be driven by the rapidly emerging class of liquid-like protein networks that assemble at membranes.

From endocytic buds (1) to needle-like filopodial protrusions (2), curved membrane surfaces play critical roles in many cellular processes (3). The energetic cost of creating these highly curved surfaces is considerable, such that spontaneous membrane fluctuations are insufficient to establish and stabilize the shapes of cellular membranes (4). Instead, work during the past two decades has revealed that interactions between proteins and lipids drive membrane curvature (5). Multiple physical mechanisms underlie the ability of proteins to shape membrane surfaces. These include amphipathic helices that insert like wedges into one leaflet of the membrane, creating an interleaflet area mismatch that drives curvature (6). Alternatively, proteins with inherently curved membrane binding domains such as BAR domains, dynamin, and ESCRTs act as scaffolds that can stabilize curved membrane geometries (7, 8). While each of these mechanisms relies on structured protein domains, we have recently reported that intrinsically disordered proteins, which lack a stable three-dimensional structure, can also be potent drivers of membrane bending (9, 10). Specifically, when noninteracting disordered domains are crowded together in cellular structures, steric repulsion among them drives the membrane to buckle outward, taking on a curved shape.Interestingly, rather than repelling one another, many disordered proteins have recently been found to assemble together via weak, multivalent interactions, forming networks that have the physical properties of liquids (11). Notably, recent studies have suggested that liquid–liquid phase separation of membrane-bound proteins plays an important role in diverse cellular processes including nucleation of actin filaments (12), immunological signaling (13), and assembly of virions (14).How might liquid–liquid phase separation of proteins at membrane surfaces impact membrane curvature? To address this question, we examined phase separation of the N-terminal low-complexity domain of fused in sarcoma, FUS LC, on the surfaces of synthetic and cell-derived membrane vesicles. FUS LC was chosen as a model protein for this study because it is among the most thoroughly characterized examples of a domain that undergoes liquid–liquid protein phase separation in solution (15). Here, we assemble FUS LC on membrane surfaces using an N-terminal histidine tag (16) that binds strongly to lipids with Ni-NTA headgroups. As FUS LC accumulated at the membrane surface, we observed protein phase separation in the two-dimensional plane of the membrane followed by spontaneous inward bending of the membrane, such that protein-lined tubules were created. Similar tubules were observed with two other domains implicated in liquid–liquid phase separation, the low-complexity domain of hnRNPA2 (17) and the RGG domain of LAF-1 (18). Interestingly, the tubules had undulating morphologies, similar to a string of pearls. This phenomenon is associated with an area mismatch between the two membrane leaflets (19, 20), suggesting that protein phase separation pulls lipids toward one another, creating a net compressive stress on one side of the membrane. In line with this hypothesis, a continuum mechanics model, built on the standard Helfrich framework, recreated the tubule morphology when a compressive stress was imposed using spontaneous curvature on the outer membrane surface. Further, the model predicted that tubule diameter should increase with increasing membrane rigidity and increasing rigidity ratio, trends confirmed by our experiments. Collectively, these findings suggest that protein phase separation on membrane surfaces generates considerable stresses that can drive the spontaneous assembly of membrane buds and tubules with physiologically relevant dimensions.     

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.     

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.     

Type III secretion system effector proteins are mechanically labile

Multiple gram-negative bacteria encode type III secretion systems (T3SS) that allow them to inject effector proteins directly into host cells to facilitate colonization. To be secreted, effector proteins must be at least partially unfolded to pass through the narrow needle-like channel (diameter <2 nm) of the T3SS. Fusion of effector proteins to tightly packed proteins—such as GFP, ubiquitin, or dihydrofolate reductase (DHFR)—impairs secretion and results in obstruction of the T3SS. Prior observation that unfolding can become rate-limiting for secretion has led to the model that T3SS effector proteins have low thermodynamic stability, facilitating their secretion. Here, we first show that the unfolding free energy (ΔGunfold0) of two Salmonella effector proteins, SptP and SopE2, are 6.9 and 6.0 kcal/mol, respectively, typical for globular proteins and similar to published ΔGunfold0 for GFP, ubiquitin, and DHFR. Next, we mechanically unfolded individual SptP and SopE2 molecules by atomic force microscopy (AFM)-based force spectroscopy. SptP and SopE2 unfolded at low force (F_unfold ≤ 17 pN at 100 nm/s), making them among the most mechanically labile proteins studied to date by AFM. Moreover, their mechanical compliance is large, as measured by the distance to the transition state (Δx^‡ = 1.6 and 1.5 nm for SptP and SopE2, respectively). In contrast, prior measurements of GFP, ubiquitin, and DHFR show them to be mechanically robust (F_unfold > 80 pN) and brittle (Δx^‡ < 0.4 nm). These results suggest that effector protein unfolding by T3SS is a mechanical process and that mechanical lability facilitates efficient effector protein secretion.

Type III secretion systems (T3SS) are large nanomachines utilized by both pathogenic and symbiotic bacteria to inject effector proteins directly into the cytoplasm of host cells (1–3). Once delivered, effector proteins facilitate host cell colonization through a variety of mechanisms (4–7), including down-regulation of the host immune response (8) and rearrangement of the cytoskeleton (9, 10). The T3SS apparatus, known as the injectisome, is a syringe-like structure with a hollow needle that spans the inner and outer bacterial membranes, the extracellular space, and the host membrane, enabling proteins to pass directly from bacteria to host cells (Fig. 1A) (2). Specialized bacterial chaperones often bind the N-terminal 50 to 100 amino acids (aa) of the effector proteins, known as the chaperone binding domain, and help maintain the effector N-terminal domain in an extended conformation. C-terminal to the chaperone binding domain, effector proteins contain one or more globular domains, which adopt their folded conformations even when in complex with their cognate chaperone (4, 11, 12). The effector proteins, or their chaperone complexes, are recognized by the base of the injectisome prior to secretion (13). At its narrowest point, the injectisome needle’s inner diameter is less than 2 nm (14–16). As a result, effector proteins must be mostly unfolded to be secreted (17–20). Secretion is thus thought to proceed by a “threading-the-needle mechanism,” where the N-terminal extended domain is released from the chaperone and fed to the injectisome, followed by unfolding of the C-terminal effector domain (21).Open in a separate windowFig. 1.Thermodynamic stability of T3SS effector proteins SptP_CD and SopE2_CD. (A) Schematic depiction of protein transport through the T3SS showing effector proteins, which are at least partially folded in the bacterial cytoplasm. Such effector proteins interact with an associated unfoldase to passage through the T3SS, which has an inner channel with a diameter <2 nm. Once inside the host cytoplasm, effector proteins refold to carry out their function. (B) Crystal structures of SptP_CD (Protein Data Bank [PDB] ID code 1G4U) and SopE2_CD (PDB ID code 1R9K). (C) Ellipticity from CD at λ = 222 nm plotted as a function of urea concentrations for SptP_CD (orange) and SopE2_CD (green). A fit of the data with Eq. 1 yielded the free energy of unfolding ΔGunfold0 for SptP_CD (6.9 ± 0.2 kcal/mol [mean ± fit error]) and SopE2_CD (6.0 ± 0.2 kcal/mol [mean ± fit error]). Data points are the result of at least three independent measurements. Error bars represent SD.Before proteins are secreted through the T3SS, they interact with a hexameric ATPase at the base of the T3SS that is capable of mediating chaperone release from effector proteins and effector-protein unfolding (15, 22). Indeed, most in vivo unfolding is catalyzed by unfoldases that work from one end of the substrate protein in stark contrast to the global effects of temperature, pH, or chemical denaturants. The most common examples of targeted protein unfolding are catalyzed by ATPases of the AAA(+) family that mechanically unfold their substrates (23, 24). For example, the AAA(+) ATPase ClpX forms a ring-shaped hexamer that mechanically pulls its substrates through its narrow central pore to unfold them (25). These are powerful unfoldases that can unfold even tightly packed proteins such as GFP, ubiquitin, and dihydrofolate reductase (DHFR) (23, 24, 26, 27). However, the T3SS ATPase does not belong to the AAA(+) family of ATPases. Instead, it is structurally similar to the catalytic β-subunit of the F₁F₀ ATP synthase, a rotary motor that normally couples proton gradient dissipation to ATP synthesis but can also run in reverse and hydrolyze ATP to do work (15, 28–30). The T3SS ATPase is not as powerful an unfoldase as the AAA(+) family, as fusions of effector proteins with GFP, ubiquitin, or DHFR stall in the injectisome and are poorly secreted (20, 22, 31, 32). These observations have led to the current model that T3SS effector proteins have low thermodynamic stability to facilitate their secretion (22, 31–33).While thermodynamic stability is the most common metric of protein stability, mechanical stability is a distinct metric that quantifies how easily a protein unfolds under force (F_unfold). Mechanical stability is typically measured by pulling across the N and C termini of single molecules via force spectroscopy using optical tweezers (34, 35) or an atomic force microscope (AFM) (36). Early force spectroscopy studies showed that thermodynamic stability does not correlate with mechanical stability (37–41). For example, titin’s I28 domain requires ∼20% more force to unfold than titin’s I27 domain [I85 and I91, respectively, in the new nomenclature (42)], despite I27 having approximately twofold higher thermodynamic stability (43). Importantly, AFM studies have shown that GFP (44), ubiquitin (45), and DHFR (46) are mechanically robust, requiring high forces to unfold despite their typical thermodynamic stabilities. These three proteins each stall the T3SS; thus, mechanical stability may be the physical determinant to proteins being secreted by the T3SS, rather than thermodynamic stability.Here, we determine the thermodynamic and mechanical stabilities of SptP and SopE2, two effector proteins from Salmonella enterica. These effectors are ideal candidates for this study as they have known crystal structures (10, 47), have characterized in vivo secretion kinetics (48), and represent effector proteins of different size and structure (Fig. 1B). We show that the catalytic domains of SptP and SopE2 have unremarkable thermodynamic stabilities, similar to many other previously characterized proteins, including GFP, ubiquitin, and DHFR. Conversely, our AFM-based force spectroscopy measurements demonstrate that SptP and SopE2 are among the most mechanically labile proteins studied to date by AFM. These two T3SS effector proteins are therefore mechanically labile while being thermodynamically stable, supporting the hypothesis that it is mechanical stability, not thermodynamic stability, that predicts efficient protein secretion by the T3SS.     

Micellar TIA1 with folded RNA binding domains as a model for reversible stress granule formation

TIA1, a protein critical for eukaryotic stress response and stress granule formation, is structurally characterized in full-length form. TIA1 contains three RNA recognition motifs (RRMs) and a C-terminal low-complexity domain, sometimes referred to as a “prion-related domain” or associated with amyloid formation. Under mild conditions, full-length (fl) mouse TIA1 spontaneously oligomerizes to form a metastable colloid-like suspension. RRM2 and RRM3, known to be critical for function, are folded similarly in excised domains and this oligomeric form of apo fl TIA1, based on NMR chemical shifts. By contrast, the termini were not detected by NMR and are unlikely to be amyloid-like. We were able to assign the NMR shifts with the aid of previously assigned solution-state shifts for the RRM2,3 isolated domains and homology modeling. We present a micellar model of fl TIA1 wherein RRM2 and RRM3 are colocalized, ordered, hydrated, and available for nucleotide binding. At the same time, the termini are disordered and phase separated, reminiscent of stress granule substructure or nanoscale liquid droplets.

T cell intracellular antigen-1 (TIA1) has multiple roles within cells, including a critical role in stress granule (SG) formation during eukaryotic cellular stress response (1–3) and translation regulation (4–6). SGs appear in cells exposed to stressors, such as pH, oxidation, and temperature changes, and contain stalled preinitiation RNA–protein complexes. They have been hypothesized to act as a decision point in mRNA processing by helping to guide homeostasis-restoring protein expression or begin apoptosis. Although sometimes associated with misfolded protein aggregates, SG components dissolve and regain function more quickly than other aggregates after the stress is removed (7). TIA1 has three RNA recognition motifs (RRM1, RRM2, RRM3) known to bind RNA with relatively little sequence specificity. TIA1 has a C-terminal low-complexity domain (LCD) enriched with asparagine and glutamine that has been referred to in the literature as a prion-related domain (PRD) because of its sequence similarity to amyloid- or prion-forming proteins. Proteins associated with SGs have also been linked to several human diseases, some characterized as protein misfolding disorders. A mutation within the LCD of TIA1 is the diagnostic marker for Welander distal myopathy (8, 9), and several other TIA1 mutations are linked to amyotrophic lateral sclerosis (10).Despite its importance, little is known about the full-length (fl) or oligomeric form(s) of TIA1 or about the LCD. The RRM domains have been structurally characterized (11–13), giving insight into structure, dynamics, binding, and function. Several excised TIA1-RRM domain constructs were characterized with small-angle scattering and liquid-state NMR; the LCD was excluded from the constructs used in prior published structural studies (11, 13). NMR was used to solve the structure of TIA1-RRM1 (Protein Data Bank [PDB] ID code 5O2V), TIA1-RRM2 bound to the dinucleotide UU-RNA (PDB ID code 5O3J), and TIA1-RRM2,3 (PDB ID code 2MJN). RRM1 appears to contribute little to RNA binding, which may be explained by the negatively charged residues in the RNP1 motif within RRM1. A model of rigid RRM domains with flexible linkers was used to interpret scattering data and show that RRM2 and RRM3 associate more closely with each other and more so after RNA binding than with RRM1. However, the effects of the LCD on the fl structure have remained elusive due to experimental challenges.It has been reported that the LCD of TIA1 can cause phase separation (14). In vivo, phase separation of groups of functionally related, locally concentrated proteins and nucleic acids (14) is believed to lead to the formation of membraneless organelles (biomolecular condensates), such as stress granules (15). Many cellular condensates contain proteins with RNA-binding domains, including Cajal bodies, P bodies, and SGs (15). Misregulation of phase separation has been implicated in several human disease-related functions (8, 9).Many proteins with LCDs also spontaneously partition into separate phases or form gels at high concentrations in vitro, potentially providing a model for in vivo phase separation. The in vitro systems share important properties with the corresponding in vivo systems. Both can undergo transitions and display a continuum of mechanical properties from liquid-like droplets to glassy (16), solid-like particles. Liquid–liquid droplets are typically micrometer, morphologically spherical domains that exhibit liquid-like dynamics in their rapid recovery from photobleaching and solution-state NMR spectra (17). Many liquid droplets or condensates in vitro are metastable and transform over time (16, 18) in a process referred to as hardening or maturation (7, 10, 16, 19). Analogously, membraneless organelles can undergo transitions in vivo during regulated maturation processes (15, 20, 21). Thus, it has been suggested that misfolded, amyloid-like fibrils and aggregates form during maturation of the membraneless organelles (7), suggesting a role for condensates in templating the formation of disease-related fibrils (22). Furthermore, the multiphase in vitro suspensions can be compared to colloids, in that they are homogeneously distributed, stable multiphase suspensions whose formation is controlled by salt concentration, viscogens, and temperature. However useful these analogies are, it is important to note that the in vitro systems are, of course, highly simplified compared to the in vivo situation. The in vivo systems are subject to important biological control over their formation and dissolution and include many other components such as nucleic acids and other proteins.The LCD/PRD of TIA1 has primary sequence similarity to the better-characterized SUP35 prion protein and is also similar to other amyloid-forming proteins (23–25). Atomic force and electron microscopy (EM) have been used to show that TIA1 forms fibers under some conditions (26, 27). Congo red and thioflavin T binding assays have been used to suggest TIA1 forms a cross-β amyloid (26, 28). Sup35 and FUS are proteins with a domain structure and an LCD analogous to TIA1; both have been reported to form amyloids (29). Alternatively, it has been hypothesized that the LCD in multidomain proteins can be unfolded (intrinsically disordered) even in the functional form and that oligomerization might be driven by nonspecific intermolecular interactions between several LCDs on different monomers (7, 30, 31). A dominant hypothesis in the literature has been that the LCD induces disease-related amyloid formation. Many proposed functions of TIA1, such as RNA sequestration into SGs (16), raise the question of whether the RRM domains are folded in the condensates or high-order oligomeric forms.Here we report structural studies of fl apo TIA1 prepared without the use of harsh solvents or denaturants. High-order oligomeric systems such as amyloid fibrils are often challenging systems for traditional structural biology methods. However, solid-state NMR (SSNMR) and EM have been powerful tools for studying these systems. We characterize fl TIA1 with EM and SSNMR to test for the presence of a solid-like phase (fibril), a liquid-like phase (intrinsically disordered protein), or some other structure. We address which domains are folded or ordered, which are solvent exposed, and whether the oligomeric structure is compatible with binding at the RRMs. The answers to these intensely debated questions have consequences for future studies of biomolecular condensates.     

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.     

A unique C2 domain at the C terminus of Munc13 promotes synaptic vesicle priming

Neurotransmitter release during synaptic transmission comprises a tightly orchestrated sequence of molecular events, and Munc13-1 is a cornerstone of the fusion machinery. A forward genetic screen for defects in neurotransmitter release in Caenorhabditis elegans identified a mutation in the Munc13-1 ortholog UNC-13 that eliminated its unique and deeply conserved C-terminal module (referred to as HC2M) containing a Ca²⁺-insensitive C2 domain flanked by membrane-binding helices. The HC2M module could be functionally replaced in vivo by protein domains that localize to synaptic vesicles but not to the plasma membrane. HC2M is broadly conserved in other Unc13 family members and is required for efficient synaptic vesicle priming. We propose that the HC2M domain evolved as a vesicle/endosome adaptor and acquired synaptic vesicle specificity in the Unc13ABC protein family.

Chemical synaptic transmission is the primary mode of cellular communication within the nervous system. The presynaptic piece of this process encompasses a remarkable set of sequential and highly regulated interactions between a host of proteins, synaptic vesicles (SV), the plasma membrane, and calcium ions (Ca²⁺). Fusion of neurotransmitter-containing vesicles with the presynaptic plasma membrane is driven by the assembly of the neuronal SNAREs SNAP-25 and Syntaxin 1 on the plasma membrane and Synaptobrevin-2/VAMP2 on the SV. The assembly process and its coupling to intracellular Ca²⁺ are choreographed by a deeply conserved group of proteins including Munc13, Munc18, Synaptotagmin 1, and Complexin (1–4). Together with the SNAREs, these proteins form the core of the fusion apparatus across all metazoan nervous systems (5–7).First identified in a landmark genetic screen for nervous system mutants in the nematode Caenorhabditis elegans, UNC-13 is the founding member of the highly conserved metazoan Unc13 secretory protein family that includes Unc13ABC in humans (Munc13-1/2/3 in mice) (8–10). Munc13-1/UNC-13 localizes to the presynaptic active zone and is implicated in numerous presynaptic functions including initiation of release site assembly, SV docking and priming, Ca²⁺- and lipid-dependent forms of short-term synaptic plasticity, opening and positioning Syntaxin 1 for SNARE assembly, and protecting SNARE complexes from disassembly by NSF/alpha-SNAP (3, 11–13). Loss of Munc13-1 orthologs in the nervous system almost entirely eliminates all forms of chemical synaptic transmission, establishing the Unc13 family as essential to this process (14–16). All UNC-13 orthologs contain a large Syntaxin-binding MUN domain flanked by a Ca²⁺- and lipid-binding C1-C2 module and an additional C2 domain on its C terminus referred to as C2C (5, 10, 17).The C-terminal end of UNC-13 is the least understood domain within the Unc13 protein family in terms of both structure and mechanism (18, 19). Recent work on the MUN and C2C domains of Munc13-1 both in vitro and in cultured hippocampal synapses supports the notion that the MUN-C2C region attaches Munc13-1 to SVs as a means of preparing SVs for fusion (20, 21), but several questions remain unresolved. Is the SV interaction mediated by direct membrane binding? Does the C2C domain itself bind to SVs or does the MUN domain serve this role? Does either domain provide cargo specificity as part of the priming process? Interestingly, the C-terminal end of the MUN domain of CAPS, another Unc13 family member, can bind dense-core vesicles (DCVs) although it lacks a C-terminal C2 domain (22). Moreover, the MUN domain without the C2C domain has also been demonstrated to bind liposomes through an interaction with Synaptobrevin 2 (23). These observations bring up several possibilities for interactions with the C terminus of Munc13 including direct MUN–membrane interactions, C2C–membrane interactions, or protein–protein interactions involving either or both domains. Other Unc13 family members possessing a MUN domain with a C-terminal C2 domain such as Unc13D/Munc13-4 and BAIAP3 have been proposed to tether specific cargo such as endosomes, secretory granules, and large DCVs (24, 25). How Unc13 proteins select among different cargos remains largely unanswered (24, 26, 27).Through behavioral, electrophysiological, biochemical, and genetic approaches, we uncover a deeply conserved C-terminal membrane-binding domain within Munc13-1/UNC-13 termed the Munc13 C-terminal (MCT) domain. This region, together with C2C and a neighboring N-terminal helix fold together into a stable membrane-binding protein domain in vitro, and loss of any part of this module in vivo impairs SV priming and nervous system function. Moreover, the C-terminal domain can be replaced by foreign domains that bind SVs but not the plasma membrane, demonstrating a role in SV interactions at the synapse. Phylogenetic protein sequence comparisons suggest that the ancestral Unc13/BAIAP3 homolog possessed a similar C-terminal domain prior to the emergence of metazoa, and subsequently, the UNC-13ABC subfamily domain evolved as an SV adaptor that plays a critical role in neurotransmission in all animals.     

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.     

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).     

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.     

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).     

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.     

Gain-of-function factor H–related 5 protein impairs glomerular complement regulation resulting in kidney damage

Genetic variation within the factor H–related (FHR) genes is associated with the complement-mediated kidney disease, C3 glomerulopathy (C3G). There is no definitive treatment for C3G, and a significant proportion of patients develop end-stage renal disease. The prototypical example is CFHR5 nephropathy, through which an internal duplication within a single CFHR5 gene generates a mutant FHR5 protein (FHR5mut) that leads to accumulation of complement C3 within glomeruli. To elucidate how abnormal FHR proteins cause C3G, we modeled CFHR5 nephropathy in mice. Animals lacking the murine factor H (FH) and FHR proteins, but coexpressing human FH and FHR5mut (hFH-FHR5mut), developed glomerular C3 deposition, whereas mice coexpressing human FH with the normal FHR5 protein (hFH-FHR5) did not. Like in patients, the FHR5mut had a dominant gain-of-function effect, and when administered in hFH-FHR5 mice, it triggered C3 deposition. Importantly, adeno-associated virus vector-delivered homodimeric mini-FH, a molecule with superior surface C3 binding compared to FH, reduced glomerular C3 deposition in the presence of the FHR5mut. Our data demonstrate that FHR5mut causes C3G by disrupting the homeostatic regulation of complement within the kidney and is directly pathogenic in C3G. These results support the use of FH-derived molecules with enhanced C3 binding for treating C3G associated with abnormal FHR proteins. They also suggest that targeting FHR5 represents a way to treat complement-mediated kidney injury.

The complement system is an important component of the immune response to pathogens, particularly meningococcal infection. Complement activation is tightly regulated to prevent its effectors from damaging host tissue. Impaired control of activation, termed complement dysregulation, is associated with tissue injury, including age-related macular degeneration and renal disease. Complement-mediated kidney damage is exemplified by thrombotic microangiopathy in atypical hemolytic uraemic syndrome and glomerular damage in C3 glomerulopathy (C3G) and IgA nephropathy (IgAN).Complement factor H (FH) is a plasma protein that down-regulates C3 activation through the complement alternative pathway. The essential role of FH is illustrated by homozygous FH-deficient patients who have acquired severe C3 deficiency due to uncontrolled C3 consumption (1). The FH protein family includes five factor H–related proteins (FHR1 through 5), and all are composed of subunits called short consensus repeat (SCR) domains. While FH contains both regulatory and binding SCR domains for the activated C3 fragment C3b, the FHR proteins contain only binding domains, suggesting different functions. The importance of FHR proteins in renal pathology is derived from the associations between susceptibility to C3G and abnormal FHR proteins (2–8). C3G is characterized by dominant glomerular C3 deposition and glomerular damage (9). The prototypic example of FHR-associated C3G is CFHR5 nephropathy (3, 10). Affected individuals have a heterozygous internal duplication within the CFHR5 gene. The normal FHR5 protein consists of 9 SCR domains, whereas the abnormal FHR5 protein (FHR5mut), due to duplicated exons encoding the first two SCR domains, consists of 11 SCR domains. There are now several examples of abnormal FHR proteins and C3G (2, 4–8).Both FHR1 (11–13) and FHR5 (14, 15) influence susceptibility to IgAN, a glomerular disorder characterized by galactose-deficient IgA1 immune deposits and C3 deposition. How FHR proteins influence glomerular C3 deposition in both C3G and IgAN remains poorly understood. FHR1 and FHR5 proteins can antagonize the ability of FH to down-regulate C3 activation in vitro (7, 16). However, FHR1, FHR4, and FHR5 can also promote C3 activation in vitro independently of FH (17–19). From these in vitro observations, it can be hypothesized that the degree of C3 deposition in response to a complement-activating trigger within the kidney (e.g., IgA1 immune deposits) depends on the relative interactions between local complement activation and either FH (inhibition of activation) or the FHR proteins (promotion of activation). The degree of complexity in this system is also governed by context-specific interactions between the FHR proteins and surface glycans (20). However, the lack of appropriate in vivo models (21) due to interspecies differences in the FHR proteins has prevented researchers from modeling FHR-associated renal pathology to identify mechanisms of injury and therapeutics, an approach that has been very successful for FH-associated renal pathologies (22–30).To overcome these limitations, we developed murine strains consisting of 1) mice lacking the entire 664 kb FH-FHR locus and therefore deficient in FH and all the FHR proteins (delFH-FHR), 2) mice lacking the 537 kb FHR locus and therefore expressing normal FH but not the FHR proteins (delFHR), and 3) mice expressing human FH (hFH) and FHR5/FHR5mut in the absence of the mouse proteins. Using these unique models, we show that delFH-FHR animals develop a spontaneous C3G, that the absence of the FHR proteins in the delFHR strain did not result in spontaneous renal disease, and that delFH-FHR animals transgenically expressing hFH with the FHR5mut associated with CFHR5 nephropathy, spontaneously develop C3G, recapitulating the key features of the human disease. Finally, we demonstrate that adeno-associated virus (AAV) vector delivery of a homodimeric mini-FH (HDM-FH) molecule, previously shown to be efficacious in reducing glomerular C3 in FH-deficient mice (30), can significantly reduce glomerular C3 in our CFHR5 nephropathy mouse model and, thus, could be a future therapeutic approach for treating C3G associated with abnormal FHR proteins.