Journal of Molecular Biology

Reflectin is a cationic, block copolymeric protein that mediates the dynamic fine-tuning of color and brightness of light reflected from nanostructured Bragg reflectors in iridocyte skin cells of squids. In vivo, neuronally activated phosphorylation of reflectin triggers its assembly, driving osmotic dehydration of the membrane-bounded Bragg lamellae containing the protein to simultaneously shrink the lamellar thickness and spacing while increasing its refractive index contrast, thus tuning the wavelength and increasing the brightness of reflectance. In vitro, we show that reduction in repulsive net charge of the purified, recombinant reflectin - either (for the first time) by generalized anionic screening with salt, or by pH titration - drives a finely tuned, precisely calibrated increase in size of the resulting multimeric assemblies. The calculated effects of phosphorylation in vivo are consistent with these effects observed in vitro. X-ray scattering analyses confirm the sphericity, size and low polydispersity of the assemblies. Precise proportionality between assembly size and charge-neutralization is enabled by the demonstrated rapid dynamic arrest of multimer growth. The resulting stability of reflectin assemblies with time ensures reciprocally precise control of the particle number concentration, thereby encoding a precise calibration between the extent of neuronal signaling, osmotic pressure, and the resulting optical changes. The results presented here strongly suggest that it is charge neutralization, rather than any change in aromatic content, that is the proximate driver of assembly, fine-tuning a colligative property-based nanostructured biological machine. A physical mechanism is proposed.

Sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) and phospholamban (PLB) are essential for intracellular Ca2+ transport in myocytes. Ca2+-dependent activation of SERCA-PLB provides a rheostat function that regulates cytosolic and SR Ca2+ levels. While experimental and computational studies alone have led to a greater insight into the mechanisms for SERCA-PLB regulation, the structural changes induced by Ca2+ binding and how those are communicated to couple enzymatic activity with active transport remain poorly understood. Therefore, we have performed atomistic simulations totaling 32.7 s and cell-based intramolecular fluorescence resonance energy transfer (FRET) experiments to determine structural changes of PLB-bound SERCA in response to Ca2+ binding. Complementary simulations and experiments showed structural disorder underlies PLB inhibition of SERCA, and Ca2+ binding is sufficient to shift the protein population toward a structurally ordered state of the complex. This structural transition results in a redistribution of structural states toward a partially closed conformation of SERCAs cytosolic headpiece. Closure is accompanied by functional interactions between the N-domain {beta}5-{beta}6 loop and the A-domain. Regulation of these key structural elements indicate that Ca2+ is a critical mediator of allosteric signaling that dictates structural changes and motions that pre-activate SERCA-PLB. These findings provide direct support that dynamically driven protein allostery underlies PLB regulation of SERCA. These functional insights at unprecedented spatiotemporal resolution suggest a general modular architecture mechanism for dynamic regulation of the SERCA-PLB complex. Understanding these mechanisms is of paramount importance to guide therapeutic modulation of SERCA and other evolutionarily related ion-motive ATPases.

The nucleocapsid protein (N) of SARS-CoV-2 is essential for virus replication, genome packaging, and maturation. N is comprised of two folded domains that are separated by a highly conserved, disordered, Ser/Arg-rich linker, and flanked by disordered tails. Using NMR spectroscopy and analytical ultracentrifugation we identify an alpha-helical region in the linker that undergoes concentration dependent self-association. NMR and gel shift assays show that the linker binds viral RNA but this binding is dampened by both phosphorylation and a naturally occurring mutation, whereas in contrast, RNA binding to the full-length protein is not affected. Interestingly, phase separation with RNA is significantly reduced upon phosphorylation but enhanced with the mutation. We attribute these differences to changes in the linker helix self-association which dissociates upon phosphorylation but forms more stable higher order oligomers in the variant. These data provide a structural mechanism for how the linker region contributes to protein-protein interactions, RNA-protein interactions, liquid-liquid phase separation and N protein regulation.

1Members of the parvalbumin (PV) family of calcium binding proteins are found in a variety of vertebrates, where can they influence neural functions, muscle contraction and immune responses. It was reported that the -parvalbumin (PV)s AB domain comprising two -helices, dramatically increases the proteins calcium (Ca2+) affinity by {approx}10 kcal/mol. To understand the structural basis of this effect, we conducted all-atom molecular dynamics (MD) simulations of WT PV and truncated -parvalbumin ({Delta}PV) constructs. Additionally, we also examined the binding of magnesium (Mg2+) to these isoforms, which is much weaker than Ca2+ (Mg2+ actually does not bind to the {Delta}PV). Our key finding is that reorganization energies (RE) assessed using molecular mechanics generalized Born approximation (MM/GBSA) correctly rank-order the variants according to their published Ca2+ and Mg2+ affinities. The [Formula] of the {Delta}PV compared to the wild-type (WT) is 415.57{+/-}0.55 kcal/mol, indicating that forming a holo state of {Delta}PV in the presence of Ca2+ incurs a greater reorganization penalty than the WT. This is consistent with the {Delta}PV exhibiting lesser Ca2+ affinity than the WT ({approx}9.5 kcal/mol). Similar trend was observed for Mg2+ bound variants as well. Further, we screened for metrics such as oxygen coordination of EF hand residues with ions and found that the total oxygen coordination number (16 vs. 12 in WT:Ca2+ and {Delta}PV:Ca2+) correlate with the reported ion affinities (-22 vs. -12.6 kcal/mol in WT:Ca2+ and {Delta}PV:Ca2+), which indicates that AB domain is required for the protein to coordinate with maximal efficiency with the binding ions. To our surprise, no significant differences were observed between the Mg2+ bound WT and {Delta}PV isoforms. Additionally, we have screened for factors such as total number of waters, hydrogen bonds, protein helicity and {beta}-content for the entire protein, which enables us to understand the impact of lack of AB domain on the entire structure and not just binding sites. Our data indicate that AB improves the overall helicity ({approx}5%) in apo as well as holo forms. Particularly, AB increases -helicity in the D-helix residues (i.e., 60-65) upon ion binding by {approx}35% (90% vs. 55% in the Ca2+ bound WT and {Delta}PV, 60% vs. 20% in the Mg2+ bound WT and {Delta}PV), which likely contributes to high Ca2+ binding affinity. On the contrary, no significant effect on the overall {beta}-content was observed. Similarly, increased dehydration ({approx}50) and increase in total number of hydrogen bonds ({approx}7) were observed upon ion binding in both the WT and {Delta}PV systems, however, no significant differences were observed between the WT and {Delta}PV variants and also between Ca2+ and Mg2+ isoforms. We speculate that this is due to the partially folded apo state that was captured in our MD simulations, which might not be physiologically relevant as suggested by NMR experiments [1]. Also, we have identified seven different interactions that might play a key role in binding the AB domain with the CDEF helices, particularly the D22(AB)-S78(CDEF) hydrogen bond. Overall, this study indicates that local (i.e., the EF hands) as well as global factors play a role in improved ion binding due to AB domain.

Protein folding has long been regarded as the "holy grail" of biology, typically demanding large models and massive GPU clusters. This study introduces Pro-teusFold, a compact and interpretable model with only 993,408 parameters that achieves state-of-the-art accuracy on modest hardware with an inference time of 0.0011 seconds. By framing folding as an unfolded-to-folded sequence transformation using a novel structural tokenization, ProteusFold reduces regression complexity while preserving bond connectivity through the concept of "Synapses." It achieves near-atomic fidelity (RMSD 0.24,[A], GDT-TS 99.85) and excels in protein-protein docking with a mean DockQ of 0.7675, with 95.5% of complexes above the 0.23 threshold. Compared to AlphaFold2s Predicted Aligned Error (6.28), ProteusFold attains 0.396, representing an order-of-magnitude gain in positional accuracy. Beyond accuracy and efficiency, the model provides residue-level attribution analyses that highlight biologically significant residues, serving as a preliminary guide for experiments. Furthermore, ProteusFold is the first to provide atomic-level attribution of key electronic and thermal properties, offering deeper insight into folding mechanisms and pinpointing the specific atoms responsible for distinct scenarios. Moreover, a meta-analysis suggests the presence of folding hotspots, where critical residues cluster, revealing new avenues for discovery. Thus, ProteusFold delivers accuracy, interpretability, and efficiency, broadening access to protein-folding research.

Norovirus is the most common cause of acute gastroenteritis worldwide. Murine noroviruses (MNV) are often used as model systems for human noroviruses (HuNoV). Therefore, it is important to identify common and divisive properties. Here, we compare the interactions of human and murine norovirus P-domains with bivalent metal ions. Binding of bivalent metal ions and bile acids to MNV P-domains have been shown to stabilize a contracted ("resting") as opposed to an extended ("raised") capsid conformation. This conformational change has been linked to infectivity, diarrheagenic potential, and immune escape. Likewise, the interaction of bivalent metal ions with human norovirus capsids results in contraction, suggesting a similar underlying mechanism. We used methyl TROSY NMR experiments to study the thermodynamics and kinetics of metal ion binding to P-domains, revealing a highly synergistic interaction with the bile acid glycochenodeoxycholic acid (GCDCA) for MNV. Neutralization assays support this synergistic behavior. It turns out that bivalent metal ion binding to MNV and HuNoV P-domains differs significantly. Therefore, although the transition between "raised" and "resting" capsid conformations and consequential modulation of infectivity appears to be triggered by bivalent metal ions in murine and human noroviruses, the underlying mechanisms must be different.

Understanding the mechanisms by which single-stranded RNA viruses regulate capsid assembly around their RNA genomes has become increasingly important for the development of both antiviral treatments and drug delivery systems. Here, we investigate the effects of RNA-induced allostery in a single-stranded RNA virus -- Levivirus bacteriophage MS2 -- using the computational methods of the Dynamic Flexibility Index (DFI) and the Dynamic Coupling Index (DCI). We show that asymmetric binding of RNA to a symmetric MS2 coat protein dimer increases the flexibility of the distant FG-loop and induces a conformational change to an asymmetric dimer that is essential for proper capsid formation. We also show that a point mutation W82R in the FG-loop creates an assembly-deficient dimer in which RNA-binding has no significant effect on FG-loop flexibility. Lastly, we show that the highly flexible disordered FG-loop of the RNA bound asymmetric dimer not only becomes the controller of the rigid FG-loop but enhances its dynamic coupling with all the distal positions in the dimer. This strong dynamic coupling allows highly regulated communication and unidirectional signal transduction that drives the formation of the experimentally observed capsid intermediates. Author summaryThe final stage of an RNA virus life cycle is the assembly of a protein shell encapsulating the viral genome prior to release from the host organism. Despite rapid advancements in both experimental and theoretical biology since the mid-20th century, little is still known about the underlying mechanisms of viral capsid assembly. However, understanding the biophysical principles of viral capsid assembly would bring us one step closer to developing new biotechnologies such as antivirals that inhibit this critical stage of the life cycle or artificial capsids for targeted drug/vaccine delivery. Although we limit the present study to one simple RNA virus that infects bacteria, we propose that the physical implications can extend to other RNA viruses including the human coronavirus SARS-CoV-2. We also propose that the allosteric regulation by specific protein-RNA interactions might be a general mechanism exploited by many other ribonucleoprotein complexes, such as CRISPR-Cas9, spliceosome or ribosome.

The central dogma treats the ribosome as a molecular machine that reads one mRNA codon at a time as it adds each amino acid to its growing peptide chain. However, this and previous studies suggest that ribosomes actually perceive pairs of adjacent codons as they take three-nucleotide steps along the mRNA. We examined GNN codons which we find are surprisingly overrepresented in eukaryote protein-coding open reading frames (ORFs), especially immediately after NNU codons. Ribosome profiling experiments in yeast revealed that ribosomes with NNU at their aminoacyl (A) site have particularly elevated densities when NNU is immediately followed (3) by a GNN codon, indicating slower mRNA threading of the NNU codon from the ribosomes A to peptidyl (P) sites. Moreover, if the assessment was limited to ribosomes that have only recently arrived at the next codon, by examining 21-nucleotide ribosome footprints (21-nt RFPs), elevated densities were observed for multiple codon classes when followed by GNN. This striking translation slowdown at adjacent 5-NNN GNN codon pairs is likely mediated in part by the ribosomes CAR surface which acts as an extension of the A-site tRNA anticodon during ribosome translocation and interacts through hydrogen bonding and pi stacking with the GNN codon. The functional consequences of 5-NNN GNN codon adjacency are expected to influence the evolution of protein coding sequences. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/583757v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@d29087org.highwire.dtl.DTLVardef@e8f7dorg.highwire.dtl.DTLVardef@1ee5792org.highwire.dtl.DTLVardef@b750fb_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

The functional properties of RNA-binding proteins (RBPs) require allosteric regulation through inter-domain communication. Despite the foundational importance of allostery to biological regulation, almost no studies have been conducted to describe the biophysical nature by which inter-domain communication manifests in RBPs. Here, we show through high-pressure studies with hnRNP A1 that inter-domain communication is vital for the unique stability of its N- terminal domain containing a tandem of RNA Recognition Motifs (RRMs). Despite high sequence similarity and nearly identical tertiary structures, the two RRMs exhibit drastically different stability under pressure. RRM2 unfolds completely under high-pressure as an individual domain, but when appended to RRM1, it remains stable. Variants in which inter-domain communication is disrupted between the tandem RRMs show a large decrease in stability under pressure. Carrying these mutations over to the full-length protein for in vivo experiments revealed that the mutations affected the ability of the disordered C-terminus to engage in protein-protein interactions and more importantly, they also influenced the RNA binding capacity. Collectively, this work reveals that thermodynamic coupling between the tandem RRMs of hnRNP A1 accounts for its allosteric regulatory functions.

Protein misfolding and aggregation are a hallmark of various neurodegenerative disorders. However, the underlying mechanisms driving protein misfolding in the cellular context are incompletely understood. Here we show that the restriction of conformational degrees of freedom by a membrane anchor stabilizes the native protein conformation and suppresses liquid-liquid phase separation and protein aggregation. Inherited prion diseases in humans and neurodegeneration in transgenic mice are linked to the expression of anchorless prion protein (PrP), suggesting that the C-terminal glycosylphosphatidylinositol (GPI) anchor of native PrP impedes spontaneous formation of neurotoxic and infectious PrP species. Combining novel in vitro and in vivo approaches, we show that anchoring to membranes prevents spontaneous aggregation of PrP. Upon release from the membrane, PrP undergoes a rapid conformational transition to detergent-insoluble aggregates. Our study supports an essential role of the GPI anchor in preventing spontaneous misfolding of PrPC.

Multidrug ABC transporters harness the energy of ATP binding and hydrolysis to change conformation and thereby translocate substrates out of the cell to detoxify them. While this general access mechanism scheme is well accepted, molecular details of this interplay is still elusive. Rhodamine6G binding on a catalytic mutant of the homodimeric multidrug ABC transporter BmrA triggers a cooperative binding of ATP on the two identical nucleotide-binding-sites, otherwise Michaelian. We investigated this asymmetric behavior via a structural-enzymology approach, solving cryoEM structure of BmrA at defined ATP ratio along the enzymatic transition, highlighting the plasticity of BmrA as it undergoes the transition from inward to outward facing conformations. Analysis of continuous heterogeneity within cryoEM data and structural dynamics, revealed that Rhodamine6G narrows the conformational spectrum explored by the nucleotide-binding-domains, describing the allosteric effect of drug binding that optimizes the ATP-dependent conversion of the transporter to the outward-facing state. Following on these findings, the effect of drug-binding showed an ATPase stimulation and a maximal transport activity of the wild-type protein at the concentration-range where the allosteric transition occurs. Drug diffusion rate is the likely rate-limiting step of the reaction, while drug transport and ATPase activities are in effect uncoupled.

Static structural models often fail to capture the dynamic mechanisms of protein interactions. To address this, we introduce DynaPIN, an open-source pipeline for extracting dynamic interface fingerprints from molecular simulations. DynaPIN unifies quality control metrics, interface prediction accuracy assessment, and atomistic interaction analysis into a single automated workflow, accessible at https://github.com/CSB-KaracaLab/DynaPIN. A key feature is our interface-specific analysis centered on a Dynamic Interface definition, which classifies residues based on the persistence of their interaction status over the simulation. We applied DynaPIN to representative rigid, medium, and difficult targets from the DynaBench dataset, an MD simulation resource for Docking Benchmark 5.5. Our results show that interface flexibility diverges from static accuracy classifications established in Docking Benchmark 5.5, as explored before. All in all, by providing standardized, frame-resolved outputs, DynaPINs aim is to facilitate mechanistic studies and generate standardized unbiased data for future dynamics-aware artificial intelligence models.

Mutational analysis of the cellular prion protein (PrPC) has revealed various regions of the protein that modulate prion propagation. However, most approaches involve deletions, insertions, or replacements in the presence of the wild-type cellular protein, which may mask the true phenotype. Here, site-directed alanine mutagenesis of PrPC was conducted to identify sites particularly a surface patch of the protein pertinent to prion propagation in the absence of the wild-type prion protein. Mutations were targeted to the helical, sheet and loop regions of PrPC, or a combination thereof and the mutated proteins expressed in PK1 cells in which endogenous PrPC had been silenced. PK1 cells are a clone of mouse neuroblastoma cells that are highly susceptible to Rocky Mountain Laboratory mouse prions. Using the scrapie cell assay, a highly sensitive cell culture-based bioassay for quantifying infectious titres of mouse prions, we found that all mutations within the structured 121-230 domain, irrespective of secondary structure, severely reduced prion propagation. The reduction was most pronounced for mutations within conformationally variable regions of the protein (G123A.L124A.G125A and V188A.T191A.T192A) and those neighbouring or within helix 1 (S134A.R135A.M153A and H139A.G141A.D146A). While mutations G123A and G125A would likely disrupt the structure of the prion fibril, the other mutations are unlikely to cause disruption. Our data therefore suggests that conformationally variable regions within the structured domain of PrPC are the major determinants of prion propagation efficacy.

The ATP binding cassette (ABC) family of transporters move diverse small molecules across membranes in nearly all organisms. Transport activity requires conformational switching between inward-facing and outward-facing states driven by ATP-dependent dimerization of two nucleotide binding domains (NBDs). The allosteric mechanism that connects ATP binding and hydrolysis in the NBDs to conformational changes in a substrate binding site in the transmembrane domains (TMDs) presents an unresolved question. Here we use sequence coevolution analyses together with biochemical characterization to investigate the role of a highly conserved motif called the peptide sensor in coordinating domain rearrangements in the heterodimeric peptide exporter from Thermus thermophilus, TmrAB. Mutations in the peptide sensor motif alter ATP hydrolysis rates as well as substrate release. Disulfide crosslinking, evolutionary trace, and evolutionary coupling analysis reveal that these effects likely destabilize a network between the peptide sensor motif and the Q-loop and X-loop, two known allosteric elements in the NBDs. We further find that disruption of this network in TmrA versus TmrB has different functional consequences, hinting at an intrinsic asymmetry in heterodimeric ABC transporters extending beyond that of the NBDs. These results support a mechanism in which the peptide sensor motifs help coordinate the transition of TmrAB to an outward open conformation, and each half of the transporter likely plays a different role in the conformational cycle of TmrAB.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has triggered myriad efforts to dissect and understand the structure and dynamics of this complex pathogen. The Spike glycoprotein of SARS-CoV-2 has received special attention as it is the means by which the virus enters the human host cells. The N-terminal domain (NTD) is one of the targeted regions of the Spike protein for therapeutics and neutralizing antibodies against COVID-19. Though its function is not well-understood, the NTD is reported to acquire mutations and deletions that can accelerate the evolutionary adaptation of the virus driving antibody escape. Cellular processes are known to be regulated by complex interactions at the molecular level, which can be characterized by means of a graph representation facilitating the identification of key residues and critical communication pathways within the molecular complex. From extensive all-atom molecular dynamics simulations of the entire Spike for the wild-type and the dominant variant, we derive a weighted graph representation of the protein in two dominant conformations of the receptor-binding-domain; all-down and one-up. We implement graph theory techniques to characterize the relevance of specific residues at facilitating roles of communication and control, while uncovering key implications for fitness and adaptation. We find that many of the reported high-frequency mutations tend to occur away from the critical residues highlighted by our graph theory analysis, implying that these mutations tend to avoid targeting residues that are most critical for protein allosteric communication. We propose that these critical residues could be candidate targets for novel antibody therapeutics. In addition, our analysis provides quantitative insights of the critical role of the NTD and furin cleavage site and their wide-reaching influence over the protein at large. Many of our conclusions are supported by empirical evidence while others point the way towards crucial simulation-guided experiments.

Interaction of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) with specific single-stranded RNA and its relation to liquid-liquid phase separation were investigated in vitro by magnetic resonance based on site-directed spin labelling. An ensemble model of free hnRNP A1 in the absence of RNA was derived from distance distributions between spin labelled sites and small angle X-ray scattering. This model revealed a compact state of the low-complexity domain and interaction of this domain with the RNA recognition motifs. Paramagnetic relaxation enhancement NMR spectroscopy confirmed this interaction. The addition of RNA to dispersed solutions of hnRNP A1 induced phase separation, observed by formation of liquid droplets. The phase separation depended on the RNA concentration and sequence, with continuous wave EPR spectroscopy showing that local protein dynamics is affected by point mutations in the RNA sequence. We propose that an interplay of sequence-specific RNA binding and phase transition serves as a regulatory mechanism for RNA segregation in the stress response of cells.

Bacteria rely on two-component systems to sense environmental cues and regulate gene expression for adaptation. The PhoQ/PhoP system exemplifies this crucial role, playing a key part in sensing magnesium (Mg2+) levels, antimicrobial peptides, mild acidic pH, osmotic upshift, and long-chain unsaturated fatty acids, promoting virulence in certain bacterial species. However, the precise details of PhoQ activation remain elusive. To elucidate PhoQs signaling mechanism at atomic resolution, we combined AlphaFold2 predictions with molecular modeling and carried out extensive Molecular Dynamics (MD) simulations. Our MD simulations revealed three distinct PhoQ conformations that were validated by experimental data. Notably, one conformation was characterized by Mg2+ bridging the acidic patch in the sensor domain to the membrane, potentially representing a repressed state. Furthermore, the high hydration observed in a putative intermediate state lends support to the hypothesis of water-mediated conformational changes during PhoQ signaling. Our findings not only revealed specific conformations within the PhoQ signaling pathway, but also hold significant promise for understanding the broader histidine kinase family due to their shared structural features. Our approach paves the way for a more comprehensive understanding of histidine kinase signaling mechanisms across various bacterial species and opens the door for developing novel therapeutics that target PhoQ modulation.

Polycomb repressive complex 1 (PRC1) catalyzes monoubiquitination of histone H2A on Lys119, promoting gene silencing. Cells at different developmental stages and in different tissues express different PRC1 isoforms. All isoforms share the same catalytic core (subunits RING1B and PCGF) and vary in the composition of regulatory subunits, clustering in two major classes. Canonical isoforms (cPRC1) are regulated by CBX-like subunits, while variant isoforms (vPRC1) are regulated by RYBP-like subunits. The molecular bases for how regulatory subunits affect the structural assembly of the complex and its catalytic activity are still largely unknown. To fill this knowledge gap, here we have specifically studied how RYBP regulates vPRC1 structure and function. Integrating the machine-learning algorithm AlphaFold2 and NMR, we have identified novel vPRC1 structural motifs in RING1B and RYBP. While the new RING1B motif is dispensable for vPRC1 assembly, the RYBP motif is essential for mediating inter-subunit interactions between RYBP and the catalytic RING1B-PCGF4 heterodimer. Importantly, the RYBP motif harbors cancer-related mutations systematically positioned on the same face of a putative transiently-forming -helix. Biochemical, biophysical and enzymatic characterization of purified cancer-related mutants confirm that this region is crucial for the structural stability of the complex. Overall, our data offer novel insights into the molecular architecture of vPRC1 and the effects of its regulatory subunit on the biochemical, structural, enzymatic, and physio-pathological properties of the complex.

Human immunodeficiency virus type 2 (HIV-2) is a lentivirus closely related to HIV-1 but exhibits distinct molecular and clinical features that influence viral infectivity and efficacy of antiretroviral therapy. The HIV capsid is a critical structural component with multifaceted roles during infection and mediates some of the observed divergence between HIV-1 and HIV-2. Unlike HIV-1, study of the HIV-2 capsid is limited and standard protocols for the in vitro assembly of HIV-1 capsid protein (CA) lattice structures have not been successfully translated to the HIV-2 context. This work identifies effective approaches for the assembly of the HIV-2 CA lattice and leverages this to biochemically characterize HIV-2 CA assemblies and mutant phenotypes. Our findings elaborate on the sensitivity of HIV-2 CA to chemical conditions and reveal that it assembles into a more varied spectrum of particle morphologies compared to HIV-1. Utilizing these assemblies, we tested the hypothesis that HIV-1 and HIV-2 employ divergent mechanisms to stabilize CA oligomer forms and investigate the effects of non-conserved substitutions at the CA inter-protomer interfaces. This work advances our understanding of the key biochemical determinants of HIV-2 CA assembly that are distinct from HIV-1 and may contribute to their divergent virological properties.

Glucocorticoid receptor (GR) activity and maturation are closely regulated by Hsp90 co-chaperones such as FKBP51, FKBP52 and p23. These co-chaperones bind to and regulate the GR, but their mutual interplay, the details of the interactions between them and their temporal dynamics are still unclear. Here we utilized UV-inducible crosslinking in living cells to map the interaction site of p23 and the GR, as well as p23 and FKBP51 at a single residue resolution. Surprisingly, we detected a novel multi-co-chaperone complex consisting of the GR, p23, FKBP51 and Hsp90, where both FKBP51 and p23 bind to the GR simultaneously. In this complex, FKBP51, but not the close homolog FKBP52, stabilizes the GR-p23 interaction. This is mediated in part by direct contacts between the FK1 domain of FKBP51 and the C-terminus of p23. Our findings refine the state of GR prior to activation, add a new layer of GR regulation by chaperones, provide evidence for the functional differences between FKBP51 and FKBP52, and underscore the power of photocrosslinking to functionally probe protein-protein contacts inside living cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/658717v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1431173org.highwire.dtl.DTLVardef@2ebfb9org.highwire.dtl.DTLVardef@12d37e9org.highwire.dtl.DTLVardef@1b1d5e3_HPS_FORMAT_FIGEXP M_FIG C_FIG