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Neuropilin-1 acts as a coreceptor with vascular endothelial growth factor receptors to facilitate binding of its ligand, vascular endothelial growth factor. Neuropilin-1 also binds to heparan sulfate, but the functional significance of this interaction has not been established. A combinatorial library screening using heparin oligosaccharides followed by molecular dynamics simulations of a heparin tetradecasaccharide suggested a highly conserved binding site composed of amino acid residues extending across the b1 and b2 domains of murine neuropilin-1. Mutagenesis studies established the importance of arginine513 and lysine514 for binding of heparin to a recombinant form of Nrp1 composed of the a1, a2, b1, and b2 domains. Recombinant Nrp1 protein bearing R513A,K514A mutations showed a significant loss of heparin-binding, heparin-induced dimerization, and heparin-dependent thermal stabilization. Isothermal calorimetry experiments suggested a 1:2 complex of heparin tetradecasaccharide:Nrp1. To study the impact of altered heparin binding in vivo, a mutant allele of Nrp1 bearing the R513A,K514A mutations was created in mice (Nrp1D) and crossbred to Nrp1+/- mice to examine the impact of altered heparan sulfate binding. Analysis of tumor formation showed variable effects on tumor growth in Nrp1D/D mice, resulting in a frank reduction in tumor growth in Nrp1D/- mice. Expression of mutant Nrp1D protein was normal in tissues, suggesting that the reduction in tumor growth was due to the altered binding of heparin/heparan sulfate to neuropilin-1. These findings suggest that the interaction of neuropilin-1 with heparan sulfate modulates its stability and its role in tumor formation and growth.

Functional crosstalk between DNA methylation, histone H3 lysine-9 trimethylation (H3K9me3) and heterochromatin protein 1 (HP1) is essential for proper heterochromatin assembly and genome stability. However, how repressive chromatin cues guide DNA methyltransferases for region-specific DNA methylation remains largely unknown. Here, we report structure-function characterizations of DNA methyltransferase Defective-In-Methylation-2 (DIM2) in Neurospora. The DNA methylation activity of DIM2 requires the presence of both H3K9me3 and HP1. Our structural study reveals a bipartite DIM2-HP1 interaction, leading to a disorder-to-order transition of the DIM2 target-recognition domain that is essential for substrate binding. Furthermore, the structure of DIM2-HP1-H3K9me3-DNA complex reveals a substrate-binding mechanism distinct from that for its mammalian orthologue DNMT1. In addition, the dual recognition of H3K9me3 peptide by the DIM2 RFTS and BAH1 domains allosterically impacts the DIM2-substrate binding, thereby controlling DIM2-mediated DNA methylation. Together, this study uncovers how multiple heterochromatin factors coordinately orchestrate an activity-switching mechanism for region-specific DNA methylation.

Understanding the normal function of the Huntingtin (HTT) protein is of significance in the design and implementation of therapeutic strategies for Huntington's disease (HD). Expansion of the CAG repeat in the HTT gene, encoding an expanded polyglutamine (polyQ) repeat within the HTT protein, causes HD and may compromise HTT's normal activity contributing to HD pathology. Here, we investigated the previously defined role of HTT in autophagy specifically through studying HTT's association with ubiquitin. We find that HTT interacts directly with ubiquitin in vitro. Tandem affinity purification was used to identify ubiquitinated and ubiquitin-associated proteins that copurify with a HTT N-terminal fragment under basal conditions. Copurification is enhanced by HTT polyQ expansion and reduced by mimicking HTT serine 421 phosphorylation. The identified HTT-interacting proteins include RNA-binding proteins (RBPs) involved in mRNA translation, proteins enriched in stress granules, the nuclear proteome, the defective ribosomal products (DRiPs) proteome and the brain-derived autophagosomal proteome. To determine whether the proteins interacting with HTT are autophagic targets, HTT knockout (KO) cells and immunoprecipitation of lysosomes were used to investigate autophagy in the absence of HTT. HTT KO was associated with reduced abundance of mitochondrial proteins in the lysosome, indicating a potential compromise in basal mitophagy, and increased lysosomal abundance of RBPs which may result from compensatory up-regulation of starvation-induced macroautophagy. We suggest HTT is critical for appropriate basal clearance of mitochondrial proteins and RBPs, hence reduced HTT proteostatic function with mutation may contribute to the neuropathology of HD.

DDX3X regulates the translation of a subset of human transcripts containing complex 5' untranslated regions (5' UTRs). In this study, we developed the helicase activity reporter for translation (HART), which uses DDX3X-sensitive 5' UTRs to measure DDX3X-mediated translational activity in cells. To directly measure RNA structure in DDX3X-dependent mRNAs, we used SHAPE-MaP to determine the secondary structures present in DDX3X-sensitive 5' UTRs and then used HART to investigate how sequence alterations influence DDX3X sensitivity. Additionally, we identified residues 38-44 as potential mediators of DDX3X's interaction with the translational machinery. HART revealed that both DDX3X's association with the translational machinery and its helicase activity are required for its function in promoting the translation of DDX3X-sensitive 5' UTRs. These findings suggest DDX3X plays a crucial role in regulating translation through its interaction with the translational machinery during ribosome scanning and establish the HART reporter as a robust, lentivirally encoded, colorimetric measurement of DDX3X-dependent translation in cells.

Patterns of transcriptional activity are encoded in our genome through regulatory elements such as promoters or enhancers that, paradoxically, contain similar assortments of sequence-specific transcription factor (TF) binding sites1-3. Knowledge of how these sequence motifs encode multiple, often overlapping, gene expression programs is central to understanding gene regulation and how mutations in non-coding DNA manifest in disease4,5. Here, by studying gene regulation from the perspective of individual transcription start sites (TSSs), using natural genetic variation, perturbation of endogenous TF protein levels and massively parallel analysis of natural and synthetic regulatory elements, we show that the effect of TF binding on transcription initiation is position dependent. Analysing TF-binding-site occurrences relative to the TSS, we identified several motifs with highly preferential positioning. We show that these patterns are a combination of a TF's distinct functional profiles-many TFs, including canonical activators such as NRF1, NFY and Sp1, activate or repress transcription initiation depending on their precise position relative to the TSS. As such, TFs and their spacing collectively guide the site and frequency of transcription initiation. More broadly, these findings reveal how similar assortments of TF binding sites can generate distinct gene regulatory outcomes depending on their spatial configuration and how DNA sequence polymorphisms may contribute to transcription variation and disease and underscore a critical role for TSS data in decoding the regulatory information of our genome.

Transcription coupled-nucleotide excision repair (TC-NER) removes DNA lesions that block RNA polymerase II (Pol II) transcription. A key step in TC-NER is the recruitment of the TFIIH complex, which initiates DNA unwinding and damage verification; however, the mechanism by which TFIIH is recruited during TC-NER, particularly in yeast, remains unclear. Here, we show that the C-terminal domain (CTD) of elongation factor-1 (Elf1) plays a critical role in TC-NER in yeast by binding TFIIH. Analysis of genome-wide repair of UV-induced cyclobutane pyrimidine dimers (CPDs) using CPD-seq indicates that the Elf1 CTD in yeast is required for efficient TC-NER. We show that the Elf1 CTD binds to the pleckstrin homology (PH) domain of the p62 subunit of TFIIH in vitro, and identify a putative TFIIH-interaction region (TIR) in the Elf1 CTD that is important for PH binding and TC-NER. The Elf1 TIR shows functional, structural, and sequence similarities to a conserved TIR in the mammalian UV sensitivity syndrome A (UVSSA) protein, which recruits TFIIH during TC-NER in mammalian cells. These findings suggest that the Elf1 CTD acts as a functional counterpart to mammalian UVSSA in TC-NER by recruiting TFIIH in response to Pol II stalling at DNA lesions.

BACKGROUND: Identifying the DNA-binding specificities of transcription factors (TF) is central to understanding gene networks that regulate growth and development. Such knowledge is lacking in oomycetes, a microbial eukaryotic lineage within the stramenopile group. Oomycetes include many important plant and animal pathogens such as the potato and tomato blight agent Phytophthora infestans, which is a tractable model for studying life-stage differentiation within the group. RESULTS: Mining of the P. infestans genome identified 197 genes encoding proteins belonging to 22 TF families. Their chromosomal distribution was consistent with family expansions through unequal crossing-over, which were likely ancient since each family had similar sizes in most oomycetes. Most TFs exhibited dynamic changes in RNA levels through the P. infestans life cycle. The DNA-binding preferences of 123 proteins were assayed using protein-binding oligonucleotide microarrays, which succeeded with 73 proteins from 14 families. Binding sites predicted for representatives of the families were validated by electrophoretic mobility shift or chromatin immunoprecipitation assays. Consistent with the substantial evolutionary distance of oomycetes from traditional model organisms, only a subset of the DNA-binding preferences resembled those of human or plant orthologs. Phylogenetic analyses of the TF families within P. infestans often discriminated clades with canonical and novel DNA targets. Paralogs with similar binding preferences frequently had distinct patterns of expression suggestive of functional divergence. TFs were predicted to either drive life stage-specific expression or serve as general activators based on the representation of their binding sites within total or developmentally-regulated promoters. This projection was confirmed for one TF using synthetic and mutated promoters fused to reporter genes in vivo. CONCLUSIONS: We established a large dataset of binding specificities for P. infestans TFs, representing the first in the stramenopile group. This resource provides a basis for understanding transcriptional regulation by linking TFs with their targets, which should help delineate the molecular components of processes such as sporulation and host infection. Our work also yielded insight into TF evolution during the eukaryotic radiation, revealing both functional conservation as well as diversification across kingdoms.

Isoform 1 of DNA methyltransferase DNMT3A (DNMT3A1) specifically recognizes nucleosome monoubiquitylated at histone H2A lysine-119 (H2AK119ub1) for establishment of DNA methylation. Mis-regulation of this process may cause aberrant DNA methylation and pathogenesis. However, the molecular basis underlying DNMT3A1-nucleosome interaction remains elusive. Here we report the cryo-EM structure of DNMT3A1s ubiquitin-dependent recruitment (UDR) fragment complexed with H2AK119ub1-modified nucleosome. DNMT3A1 UDR occupies an extensive nucleosome surface, involving the H2A-H2B acidic patch, a surface groove formed by H2A and H3, nucleosomal DNA, and H2AK119ub1. The DNMT3A1 UDRs interaction with H2AK119ub1 affects the functionality of DNMT3A1 in cells in a context-dependent manner. Our structural and biochemical analysis also reveals competition between DNMT3A1 and JARID2, a cofactor of polycomb repression complex 2 (PRC2), for nucleosome binding, suggesting the interplay between different epigenetic pathways. Together, this study reports a molecular basis for H2AK119ub1-dependent DNMT3A1-nucleosome association, with important implications in DNMT3A1-mediated DNA methylation in development.

Message passing neural networks (MPNNs) on molecular graphs generate continuous and differentiable encodings of small molecules with state-of-the-art performance on protein-ligand complex scoring tasks. Here, we describe the proximity graph network (PGN) package, an open-source toolkit that constructs ligand-receptor graphs based on atom proximity and allows users to rapidly apply and evaluate MPNN architectures for a broad range of tasks. We demonstrate the utility of PGN by introducing benchmarks for affinity and docking score prediction tasks. Graph networks generalize better than fingerprint-based models and perform strongly for the docking score prediction task. Overall, MPNNs with proximity graph data structures augment the prediction of ligand-receptor complex properties when ligand-receptor data are available.

The five NF-κB family members and three nuclear IκB proteins play important biological roles, but the mechanisms by which distinct members of these protein families contribute to selective gene transcription remain poorly understood, especially at a genome-wide scale. Using nascent transcript RNA-seq, we observed considerable overlap between p50-dependent and IκBζ-dependent genes in Toll-like receptor 4 (TLR4)-activated macrophages. Key immunoregulatory genes, including Il6, Il1b, Nos2, Lcn2, and Batf, are among the p50-IκBζ-codependent genes. IκBζ-bound genomic sites are occupied at earlier time points by NF-κB dimers. However, p50-IκBζ codependence does not coincide with preferential binding of either p50 or IκBζ, as RelA co-occupies hundreds of genomic sites with the two proteins. A common feature of p50-IκBζ-codependent genes is a nearby p50/RelA/IκBζ-cobound site exhibiting p50-dependent binding of both RelA and IκBζ. This and other results suggest that IκBζ acts in concert with RelA:p50 heterodimers. Notably, p50-IκBζ-codependent genes comprise a high percentage of genes exhibiting the greatest differential expression between TLR4-stimulated and tumor necrosis factor receptor (TNFR)-stimulated macrophages. Thus, our genome-centric analysis reveals a defined p50-IκBζ pathway that selectively activates a set of key immunoregulatory genes and serves as an important contributor to differential TNFR and TLR4 responses.

In drug discovery, the in silico prediction of binding affinity is one of the major means to prioritize compounds for synthesis. Alchemical relative binding free energy (RBFE) calculations based on molecular dynamics (MD) simulations are nowadays a popular approach for the accurate affinity ranking of compounds. MD simulations rely on empirical force field parameters, which strongly influence the accuracy of the predicted affinities. Here, we evaluate the ability of six different small-molecule force fields to predict experimental protein-ligand binding affinities in RBFE calculations on a set of 598 ligands and 22 protein targets. The public force fields OpenFF Parsley and Sage, GAFF, and CGenFF show comparable accuracy, while OPLS3e is significantly more accurate. However, a consensus approach using Sage, GAFF, and CGenFF leads to accuracy comparable to OPLS3e. While Parsley and Sage are performing comparably based on aggregated statistics across the whole dataset, there are differences in terms of outliers. Analysis of the force field reveals that improved parameters lead to significant improvement in the accuracy of affinity predictions on subsets of the dataset involving those parameters. Lower accuracy can not only be attributed to the force field parameters but is also dependent on input preparation and sampling convergence of the calculations. Especially large perturbations and nonconverged simulations lead to less accurate predictions. The input structures, Gromacs force field files, as well as the analysis Python notebooks are available on GitHub.

Cells employ many large macromolecular machines for the execution and regulation of processes that are vital for cell and organismal viability. Interestingly, cells cannot synthesize these machines as functioning units. Instead, cells synthesize the molecular parts that must then assemble into the functional complex. Many important machines, including chaperones such as GroEL and proteases such as the proteasome, comprise protein rings that are stacked on top of one another. While there is some experimental data regarding how stacked-ring complexes such as the proteasome self-assemble, a comprehensive understanding of the dynamics of stacked-ring assembly is currently lacking. Here, we developed a mathematical model of stacked-trimer assembly and performed an analysis of the assembly of the stacked homomeric trimer, which is the simplest stacked-ring architecture. We found that stacked rings are particularly susceptible to a form of kinetic trapping that we term deadlock, in which the system gets stuck in a state where there are many large intermediates that are not the fully assembled structure but that cannot productively react. When interaction affinities are uniformly strong, deadlock severely limits assembly yield. We thus predicted that stacked rings would avoid situations where all interfaces in the structure have high affinity. Analysis of available crystal structures indicated that indeed the majority-if not all-of stacked trimers do not contain uniformly strong interactions. Finally, to better understand the origins of deadlock, we developed a formal pathway analysis and showed that, when all the binding affinities are strong, many of the possible pathways are utilized. In contrast, optimal assembly strategies utilize only a small number of pathways. Our work suggests that deadlock is a critical factor influencing the evolution of macromolecular machines and provides general principles for understanding the self-assembly efficiency of existing machines.

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections in the USA and of preventable blindness worldwide. This obligate intracellular pathogen replicates within a membrane-bound inclusion, but how it acquires nutrients from the host while avoiding detection by the innate immune system is incompletely understood. C. trachomatis accomplishes this in part through the translocation of a unique set of effectors into the inclusion membrane, the inclusion membrane proteins (Incs). Incs are ideally positioned at the host-pathogen interface to reprogram host signaling by redirecting proteins or organelles to the inclusion. Using a combination of co-affinity purification, immunofluorescence confocal imaging, and proteomics, we characterize the interaction between an early-expressed Inc of unknown function, Tri1, and tumor necrosis factor receptor-associated factor 7 (TRAF7). TRAF7 is a multi-domain protein with a RING finger ubiquitin ligase domain and a C-terminal WD40 domain. TRAF7 regulates several innate immune signaling pathways associated with C. trachomatis infection and is mutated in a subset of tumors. We demonstrate that Tri1 and TRAF7 specifically interact during infection and that TRAF7 is recruited to the inclusion. We further show that the predicted coiled-coil domain of Tri1 is necessary to interact with the TRAF7 WD40 domain. Finally, we demonstrate that Tri1 displaces the native TRAF7 binding partners, mitogen-activated protein kinase kinase kinase 2 (MEKK2), and MEKK3. Together, our results suggest that by displacing TRAF7 native binding partners, Tri1 has the capacity to alter TRAF7 signaling during C. trachomatis infection.IMPORTANCEChlamydia trachomatis is the leading cause of bacterial sexually transmitted infections in the USA and preventable blindness worldwide. Although easily treated with antibiotics, the vast majority of infections are asymptomatic and therefore go untreated, leading to infertility and blindness. This obligate intracellular pathogen evades the immune response, which contributes to these outcomes. Here, we characterize the interaction between a C. trachomatis-secreted effector, Tri1, and a host protein involved in innate immune signaling, TRAF7. We identified host proteins that bind to TRAF7 and demonstrated that Tri1 can displace these proteins upon binding to TRAF7. Remarkably, the region of TRAF7 to which these host proteins bind is often mutated in a subset of human tumors. Our work suggests a mechanism by which Tri1 may alter TRAF7 signaling and has implications not only in the pathogenesis of C. trachomatis infections but also in understanding the role of TRAF7 in cancer.

Several inflammatory cytokines bind to the allosteric site (site 2) and allosterically activate integrins. Site 2 is also a binding site for 25-hydroxycholesterol, an inflammatory lipid mediator, and is involved in inflammatory signaling (e.g., TNF and IL-6 secretion) in addition to integrin activation. FGF2 is pro-inflammatory and pro-thrombotic, and FGF1, homologous to FGF2, has anti-inflammatory and anti-thrombotic actions, but the mechanism of these actions is unknown. We hypothesized that FGF2 and FGF1 bind to site 2 of integrins and regulate inflammatory signaling. Here, we describe that FGF2 is bound to site 2 and allosterically activated β3 integrins, suggesting that the pro-inflammatory action of FGF2 is mediated by binding to site 2. In contrast, FGF1 bound to site 2 but did not activate these integrins and instead suppressed integrin activation induced by FGF2, indicating that FGF1 acts as an antagonist of site 2 and that the anti-inflammatory action of FGF1 is mediated by blocking site 2. A non-mitogenic FGF1 mutant (R50E), which is defective in binding to site 1 of αvβ3, suppressed β3 integrin activation by FGF2 as effectively as WT FGF1.

Leucine rich repeat kinase 2 (LRRK2) is a large multidomain protein containing two catalytic domains, a kinase and a GTPase, as well as protein interactions domains, including a WD40 domain. The association of increased LRRK2 kinase activity with both the familial and sporadic forms of Parkinsons disease has led to an intense interest in determining its cellular function. However, small molecule probes that can bind to LRRK2 and report on or affect its cellular activity are needed. Here, we report the identification and characterization of the first high-affinity LRRK2-binding designed ankyrin-repeat protein (DARPin), named E11. Using cryo-EM, we show that DARPin E11 binds to the LRRK2 WD40 domain. LRRK2 bound to DARPin E11 showed improved behavior on cryo-EM grids, resulting in higher resolution LRRK2 structures. DARPin E11 did not affect the catalytic activity of a truncated form of LRRK2 in vitro but decreased the phosphorylation of Rab8A, a LRRK2 substrate, in cells. We also found that DARPin E11 disrupts the formation of microtubule-associated LRRK2 filaments in cells, which are known to require WD40-based dimerization. Thus, DARPin E11 is a new tool to explore the function and dysfunction of LRRK2 and guide the development of LRRK2 kinase inhibitors that target the WD40 domain instead of the kinase.

The protein phosphatase 5 (PP5) is normally recruited to its substrates by the molecular chaperones, heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90). This interaction requires the tetratricopeptide repeat (TPR) domain of PP5, which binds to an EEVD motif at the extreme C termini of cytosolic Hsp70 and Hsp90 isoforms. In addition to bringing PP5 into proximity with chaperone-bound substrates, this interaction also relieves autoinhibition in PP5s catalytic domain, promoting its phosphatase activity. To better understand the molecular determinants of this process, we screened a large, pentapeptide library for binding to PP5. This screen identified the amino acid preferences at each position, which we validated by showing that the optimal sequences bind 4- to 7-fold tighter than the natural EEVD motifs and stimulate PP5s enzymatic activity. The enhanced affinity for PP5s TPR domain was confirmed using a protein-adaptive differential scanning fluorimetry assay. Using this increased knowledge of structure-activity relationships, we re-examined affinity proteomics results to look for potential EEVD-like motifs in the C termini of known PP5-binding partners. This search identified elongator acetyltransferase complex subunit 1 (IKBKAP) as a putative partner, and indeed, we found that its C-terminal sequence, LSLLD, binds directly to PP5s TPR domain in vitro. Consistent with this idea, mutation of elongator acetyltransferase complex subunit 1s terminal aspartate was sufficient to interrupt the interaction with PP5 in vitro and in cells. Together, these findings reveal the sequence preferences of PP5s TPR domain and expand the scope of PP5s functions to include chaperone-independent complexes.

Antigenically sequence variable M proteins of the major bacterial pathogen Streptococcus pyogenes (Strep A) are responsible for recruiting human C4b-binding protein (C4BP) to the bacterial surface, which enables Strep A to evade destruction by the immune system. The most sequence divergent portion of M proteins, the hypervariable region (HVR), is responsible for binding C4BP. Structural evidence points to the conservation of two C4BP-binding sequence patterns (M2 and M22) in the HVR of numerous M proteins, with this conservation applicable to vaccine immunogen design. These two patterns, however, only partially explain C4BP binding by Strep A. Here, we identified several M proteins that lack these patterns but still bind C4BP and determined the structures of two, M68 and M87 HVRs, in complex with a C4BP fragment. Mutagenesis of these M proteins led to the identification of amino acids that are crucial for C4BP binding, enabling formulation of new C4BP-binding patterns. Mutagenesis was also carried out on M2 and M22 proteins to refine or generate experimentally grounded C4BP-binding patterns. The M22 pattern was the most prevalent among M proteins, followed by the M87 and M2 patterns, while the M68 pattern was rare. These patterns, except for M68, were also evident in numerous M-like Enn proteins. Binding of C4BP via these patterns to Enn proteins was verified. We conclude that C4BP-binding patterns occur frequently in Strep A strains of differing M types, being present in their M or Enn proteins, or frequently both, providing further impetus for their use as vaccine immunogens.

Using a molecular modeling approach for Tau-binding sites, we modified our previously reported imaging agent, [125I]INFT, for the potential improvement of binding properties to Tau in an Alzheimer's disease (AD) brain. Two new derivatives, namely [125I]ISAS and [125I]NIPZ, were designed, where binding energies at site 1 of Tau were -7.4 and -6.0 kcal/mole, respectively, compared to [125I]INFT (-7.6 kcal/mole). The radiosynthesis of [125I]ISAS and [125I]NIPZ was carried out by using iodine-125 and purified chromatographically to achieve >90% purity. In vitro binding affinities (IC50) for Tau were as follows: INFT = 7.3 × 10-8 M; ISAS = 4.7 × 10-8 M; NIPZ > 10-6 M. The binding of [125I]ISAS to gray matter (GM) correlated with the presence of Tau in the AD brain, confirmed by anti-Tau immunohistochemistry. [125I]NIPZ did not bind to Tau, with similar levels of binding observed in GM and white matter (WM). Four radiotracers were compared and the rank order of binding to Tau was found to be [125I]IPPI > [125I]INFT > [125I]ISAS >>> [125I]NIPZ with GM/WM ratios of [125I]IPPI = 7.74 > [125I]INFT = 4.86 > [125I]ISAS = 3.62 >> [125I]NIPZ = 1.24. The predictive value of Chimera-AutoDock for structurally related compounds binding to the Tau binding sites (measured as binding energy) was good. A binding energy of less than -7 kcal/mole is necessary and less than -8 kcal/mole will be more suitable for developing imaging agents.

Activation of PINK1 and Parkin in response to mitochondrial damage initiates a response that includes phosphorylation of RAB7A at Ser72. Rubicon is a RAB7A binding negative regulator of autophagy. The structure of the Rubicon:RAB7A complex suggests that phosphorylation of RAB7A at Ser72 would block Rubicon binding. Indeed, in vitro phosphorylation of RAB7A by TBK1 abrogates Rubicon:RAB7A binding. Pacer, a positive regulator of autophagy, has an RH domain with a basic triad predicted to bind an introduced phosphate. Consistent with this, Pacer-RH binds to phosho-RAB7A but not to unphosphorylated RAB7A. In cells, mitochondrial depolarization reduces Rubicon:RAB7A colocalization whilst recruiting Pacer to phospho-RAB7A-positive puncta. Pacer knockout reduces Parkin mitophagy with little effect on bulk autophagy or Parkin-independent mitophagy. Rescue of Parkin-dependent mitophagy requires the intact pRAB7A phosphate-binding basic triad of Pacer. Together these structural and functional data support a model in which the TBK1-dependent phosphorylation of RAB7A serves as a switch, promoting mitophagy by relieving Rubicon inhibition and favoring Pacer activation.

Schistosomiasis, caused by a parasitic blood fluke of the genus Schistosoma, is a global health problem for which new chemotherapeutic options are needed. We explored the scaffold of gallinamide A, a natural peptidic metabolite of marine cyanobacteria that has previously been shown to inhibit cathepsin L-type proteases. We screened a library of 19 synthetic gallinamide A analogs and identified nanomolar inhibitors of the cathepsin B-type protease SmCB1, which is a drug target for the treatment of schistosomiasis mansoni. Against cultured S. mansoni schistosomula and adult worms, many of the gallinamides generated a range of deleterious phenotypic responses. Imaging with a fluorescent-activity-based probe derived from gallinamide A demonstrated that SmCB1 is the primary target for gallinamides in the parasite. Furthermore, we solved the high-resolution crystal structures of SmCB1 in complex with gallinamide A and its two analogs and describe the acrylamide covalent warhead and binding mode in the active site. Quantum chemical calculations evaluated the contribution of individual positions in the peptidomimetic scaffold to the inhibition of the target and demonstrated the importance of the P1 and P2 positions. Our study introduces gallinamides as a powerful chemotype that can be exploited for the development of novel antischistosomal chemotherapeutics.

SHP2, a pivotal component downstream of both receptor and non-receptor tyrosine kinases, has been underscored in the progression of various human cancers and neurodevelopmental disorders. Allosteric inhibitors have been proposed to regulate its autoinhibition. However, oncogenic mutations, such as E76K, convert SHP2 into its open state, wherein the catalytic cleft becomes fully exposed to its ligands. This study elucidates the dynamic properties of SHP2 structures across different states, with a focus on the effects of oncogenic mutation on two known binding sites of allosteric inhibitors. Through extensive modeling and simulations, we further identified an alternative allosteric binding pocket in solution structures. Additional analysis provides insights into the dynamics and stability of the potential site. In addition, multi-tier screening was deployed to identify potential binders targeting the potential site. Our efforts to identify a new allosteric site contribute to community-wide initiatives developing therapies using multiple allosteric inhibitors to target distinct pockets on SHP2, in the hope of potentially inhibiting or slowing tumor growth associated with SHP2.

Transmembrane (TM) domains as simple as a single span can perform complex biological functions using entirely lipid-embedded chemical features. Computational design has the potential to generate custom tool molecules directly targeting membrane proteins at their functional TM regions. Thus far, designed TM domain-targeting agents have been limited to mimicking the binding modes and motifs of natural TM interaction partners. Here, we demonstrate the design of de novo TM proteins targeting the erythropoietin receptor (EpoR) TM domain in a custom binding topology competitive with receptor homodimerization. The TM proteins expressed in mammalian cells complex with EpoR and inhibit erythropoietin-induced cell proliferation. In vitro, the synthetic TM domain complex outcompetes EpoR homodimerization. Structural characterization reveals that the complex involves the intended amino acids and agrees with our designed molecular model of antiparallel TM helices at 1:1 stoichiometry. Thus, membrane protein TM regions can now be targeted in custom-designed topologies.

Detailed binding experiments reveal new insights into the Norrin/Wnt signaling pathway that helps to control vascularization in the retina.

COVID-19 infection is associated with a variety of vascular occlusive morbidities. However, a comprehensive understanding of how this virus can induce vascular complications remains lacking. Here, we show that a peptide fragment of SARS-CoV-2 spike protein, S192 (sequence 192-211), is capable of forming amyloid-like aggregates that can induce agglutination of red blood cells, which was not observed with low- and non-aggregated S192 peptide. We subsequently screened eight amyloid-binding molecules and identified BAM1-EG6, a benzothiazole amphiphile, as a promising candidate capable of binding to aggregated S192 and partially inhibiting its agglutination activity. These results provide new insight into a potential molecular mechanism for the capability of spike protein metabolites to contribute to COVID-19-related blood complications and suggest a new therapeutic approach for combating microvascular morbidities in COVID-19 patients.

R2 non-long terminal repeat (non-LTR) retrotransposons are among the most extensively distributed mobile genetic elements in multicellular eukaryotes and show promise for applications in transgene supplementation of the human genome. They insert new gene copies into a conserved site in 28S ribosomal DNA with exquisite specificity. R2 clades are defined by the number of zinc fingers (ZFs) at the N terminus of the retrotransposon-encoded protein, postulated to additively confer DNA site specificity. Here, we illuminate general principles of DNA recognition by R2 N-terminal domains across and between clades, with extensive, specific recognition requiring only one or two compact domains. DNA-binding and protection assays demonstrate broadly shared as well as clade-specific DNA interactions. Gene insertion assays in cells identify the N-terminal domains sufficient for target-site insertion and reveal roles in second-strand cleavage or synthesis for clade-specific ZFs. Our results have implications for understanding evolutionary diversification of non-LTR retrotransposon insertion mechanisms and the design of retrotransposon-based gene therapies.

The F-box domain is a highly conserved structural motif that defines the largest class of ubiquitin ligases, Skp1/Cullin1/F-box protein (SCF) complexes. The only known function of the F-box motif is to form the protein interaction surface with Skp1. Here we show that the F-box domain can function as an environmental sensor. We demonstrate that the F-box domain of Met30 is a cadmium sensor that blocks the activity of the SCFMet30 ubiquitin ligase during cadmium stress. Several highly conserved cysteine residues within the Met30 F-box contribute to binding of cadmium with a KD of 8 µM. Binding induces a conformational change that allows for Met30 autoubiquitylation, which in turn leads to recruitment of the segregase Cdc48/p97/VCP followed by active SCFMet30 disassembly. The resulting inactivation of SCFMet30 protects cells from cadmium stress. Our results show that F-box domains participate in regulation of SCF ligases beyond formation of the Skp1 binding interface.

UNLABELLED: AXIN1 is a major component of the β-catenin destruction complex and is frequently mutated in various cancer types, particularly liver cancers. Truncating AXIN1 mutations are recognized to encode a defective protein that leads to β-catenin stabilization, but the functional consequences of missense mutations are not well characterized. Here, we first identified the GSK3β, β-catenin, and RGS/APC interaction domains of AXIN1 that are the most critical for proper β-catenin regulation. Analysis of 80 tumor-associated variants in these domains identified 18 that significantly affected β-catenin signaling. Coimmunoprecipitation experiments revealed that most of them lost binding to the binding partner corresponding to the mutated domain. A comprehensive protein structure analysis predicted the consequences of these mutations, which largely overlapped with the observed effects on β-catenin signaling in functional experiments. The structure analysis also predicted that loss-of-function mutations within the RGS/APC interaction domain either directly affected the interface for APC binding or were located within the hydrophobic core and destabilized the entire structure. In addition, truncated AXIN1 length inversely correlated with the β-catenin regulatory function, with longer proteins retaining more functionality. These analyses suggest that all AXIN1-truncating mutations at least partially affect β-catenin regulation, whereas this is only the case for a subset of missense mutations. Consistently, most colorectal and liver cancers carrying missense variants acquire mutations in other β-catenin regulatory genes such as APC and CTNNB1. These results will aid the functional annotation of AXIN1 mutations identified in large-scale sequencing efforts or in individual patients. SIGNIFICANCE: Characterization of 80 tumor-associated missense variants of AXIN1 reveals a subset of 18 mutations that disrupt its β-catenin regulatory function, whereas the majority are passenger mutations.

Here we present, utilizing universally accepted relationships for hepatic clearance at steady state, that for all models of hepatic elimination the ratio of unbound liver drug concentration to unbound systemic blood concentration, Kpuu, is a function of or related to the hepatic bioavailability for that drug, FH. According to the derivation for the well-stirred model, Kpuu can never exceed unity, can frequently be a function of hepatic blood flow, and is equivalent to the value of FH as determined following oral dosing. For the parallel tube model, Kpuu will not equal FH but will be a function of FH and will also never be a value greater than 1. When hepatic clearance is rate limited by basolateral transporters, Kpuu will be less than 1, and less than FH. We believe that such outcomes are highly unlikely, and that the error arises from a basic assumption concerning hepatic clearance that leads to the mechanistic models of hepatic elimination, the well-stirred, parallel tube and dispersion models. That basic assumption is that the steady-state systemic concentration multiplied by the hepatic systemic clearance is equal to the product of the average unbound liver steady-state concentration and the intrinsic hepatic clearance (Css · CL = CH,u · CLint). Calculations of Kpuu and FH based on present methods of analysis provide a strong argument as to why this universally accepted relationship is not correct. Alternatively, we have shown in recent publications that hepatic clearance may be adequately determined based on Kirchhoff's Laws where no assumption of the above equality concerning hepatic intrinsic clearance is required, and where Kpuu is independent of hepatic extraction ratio and FH.

Imaging scaffolds composed of designed protein cages fused to designed ankyrin repeat proteins (DARPins) have enabled the structure determination of small proteins by cryogenic electron microscopy (cryo-EM). One particularly well characterized scaffold type is a symmetric tetrahedral assembly composed of 24 subunits, 12 A and 12 B, which has three cargo-binding DARPins positioned on each vertex. Here, the X-ray crystal structure of a representative tetrahedral scaffold in the apo state is reported at 3.8 Å resolution. The X-ray crystal structure complements recent cryo-EM findings on a closely related scaffold, while also suggesting potential utility for crystallographic investigations. As observed in this crystal structure, one of the three DARPins, which serve as modular adaptors for binding diverse `cargo proteins, present on each of the vertices is oriented towards a large solvent channel. The crystal lattice is unusually porous, suggesting that it may be possible to soak crystals of the scaffold with small (≤30 kDa) protein cargo ligands and subsequently determine cage-cargo structures via X-ray crystallography. The results suggest the possibility that cryo-EM scaffolds may be repurposed for structure determination by X-ray crystallography, thus extending the utility of electron-microscopy scaffold designs for alternative structural biology applications.

The self-organization of cells relies on the profound complexity of protein-protein interactions. Challenges in directly observing these events have hindered progress toward understanding their diverse behaviors. One notable example is the interaction between molecular motors and cytoskeletal systems that combine to perform a variety of cellular functions. In this work, we leverage theory and experiments to identify and quantify the rate-limiting mechanism of the initial association between a cargo-bound kinesin motor and a microtubule track. Recent advances in optical tweezers provide binding times for several lengths of kinesin motors trapped at varying distances from a microtubule, empowering the investigation of competing models. We first explore a diffusion-limited model of binding. Through Brownian dynamics simulations and simulation-based inference, we find this simple diffusion model fails to explain the experimental binding times, but an extended model that accounts for the ADP state of the molecular motor agrees closely with the data, even under the scrutiny of penalizing for additional model complexity. We provide quantification of both kinetic rates and biophysical parameters underlying the proposed binding process. Our model suggests that a typical binding event is limited by ADP state rather than physical search. Lastly, we predict how these association rates can be modulated in distinct ways through variation of environmental concentrations and physical properties.

Messenger RNA (mRNA) recruitment to the 40S ribosomal subunit is mediated by eukaryotic initiation factor 4F (eIF4F). This complex includes three subunits: eIF4E (m7G cap-binding protein), eIF4A (DEAD-box helicase), and eIF4G. Mammalian eIF4G is a scaffold that coordinates the activities of eIF4E and eIF4A and provides a bridge to connect the mRNA and 40S ribosomal subunit through its interaction with eIF3. While the roles of many eIF4G binding domains are relatively clear, the precise function of RNA binding by eIF4G remains to be elucidated. In this work, we used an eIF4G-dependent translation assay to reveal that the RNA binding domain (eIF4G-RBD; amino acids 682-720) stimulates translation. This stimulating activity is observed when eIF4G is independently tethered to an internal region of the mRNA, suggesting that the eIF4G-RBD promotes translation by a mechanism that is independent of the m7G cap and mRNA tethering. Using a kinetic helicase assay, we show that the eIF4G-RBD has a minimal effect on eIF4A helicase activity, demonstrating that the eIF4G-RBD is not required to coordinate eIF4F-dependent duplex unwinding. Unexpectedly, native gel electrophoresis and fluorescence polarization assays reveal a previously unidentified direct interaction between eIF4G and the 40S subunit. Using binding assays, our data show that this 40S subunit interaction is separate from the previously characterized interaction between eIF4G and eIF3. Thus, our work reveals how eIF4F can bind to the 40S subunit using eIF3-dependent and eIF3-independent binding domains to promote translation initiation.

Since the onset of the coronavirus disease 2019 (COVID-19) pandemic, SARS-CoV-2 variants capable of breakthrough infections have attracted global attention. These variants have significant mutations in the receptor-binding domain (RBD) of the spike protein and the membrane (M) protein, which may imply an enhanced ability to evade immune responses. In this study, an examination of co-mutations within the spike RBD and their potential correlation with mutations in the M protein was conducted. The EVmutation method was utilized to analyze the distribution of the mutations to elucidate the relationship between the mutations in the spike RBD and the alterations in the M protein. Additionally, the Sequence-to-Sequence Transformer Model (S2STM) was employed to establish mapping between the amino acid sequences of the spike RBD and M proteins, offering a novel and efficient approach for streamlined sequence analysis and the exploration of their interrelationship. Certain mutations in the spike RBD, G339D-S373P-S375F and Q493R-Q498R-Y505, are associated with a heightened propensity for inducing mutations at specific sites within the M protein, especially sites 3 and 19/63. These results shed light on the concept of mutational synergy between the spike RBD and M proteins, illuminating a potential mechanism that could be driving the evolution of SARS-CoV-2.

BACKGROUND: High levels of neutrophil extracellular trap (NET) formation or NETosis and autoantibodies are related to poor prognosis and disease severity of COVID-19 patients. Human angiotensin-converting enzyme 2 (ACE2) cross-reactive anti-severe acute respiratory syndrome coronavirus 2 spike protein receptor-binding domain (SARS-CoV-2 RBD) antibodies (CR Abs) have been reported as one of the sources of anti-ACE2 autoantibodies. However, the pathological implications of CR Abs in NET formation remain unknown. METHODS: In this study, we first assessed the presence of CR Abs in the sera of COVID-19 patients with different severity by serological analysis. Sera and purified IgG from CR Abs positive COVID-19 patients as well as a mouse monoclonal Ab (mAb 127) that can recognize both ACE2 and the RBD were tested for their influence on NETosis and the possible mechanisms involved were studied. RESULTS: An association between CR Abs levels and the severity of COVID-19 in 120 patients was found. The CR Abs-positive sera and IgG from severe COVID-19 patients and mAb 127 significantly activated human leukocytes and triggered NETosis, in the presence of RBD. This NETosis, triggered by the coexistence of CR Abs and RBD, activated thrombus-related cells but was abolished when the interaction between CR Abs and ACE2 or Fc receptors was disrupted. We also revealed that CR Abs-induced NETosis was suppressed in the presence of recombinant ACE2 or the Src family kinase inhibitor, dasatinib. Furthermore, we found that COVID-19 vaccination not only reduced COVID-19 severity but also prevented the production of CR Abs after SARS-CoV-2 infection. CONCLUSIONS: Our findings provide possible pathogenic effects of CR Abs in exacerbating COVID-19 by enhancing NETosis, highlighting ACE2 and dasatinib as potential treatments, and supporting the benefit of vaccination in reducing disease severity and CR Abs production in COVID-19 patients.

The de novo design of small molecule-binding proteins has seen exciting recent progress; however, high-affinity binding and tunable specificity typically require laborious screening and optimization after computational design. We developed a computational procedure to design a protein that recognizes a common pharmacophore in a series of poly(ADP-ribose) polymerase-1 inhibitors. One of three designed proteins bound different inhibitors with affinities ranging from

When proteins evolve new activity, a concomitant decrease in stability is often observed because the mutations that confer new activity can destabilize the native fold. In the conventional model of protein evolution, reduced stability is considered a purely deleterious cost of molecular innovation because unstable proteins are prone to aggregation and are sensitive to environmental stressors. However, recent work has revealed that nonnative, often unstable protein conformations play an important role in mediating evolutionary transitions, raising the question of whether instability can itself potentiate the evolution of new activity. We explored this question in a bacteriophage receptor-binding protein during host-range evolution. We studied the properties of the receptor-binding protein of bacteriophage λ before and after host-range evolution and demonstrated that the evolved protein is relatively unstable and may exist in multiple conformations with unique receptor preferences. Through a combination of structural modeling and in vitro oligomeric state analysis, we found that the instability arises from mutations that interfere with trimer formation. This study raises the intriguing possibility that protein instability might play a previously unrecognized role in mediating host-range expansions in viruses.

RAF kinases are integral to the RAS-MAPK signaling pathway, and proper RAF1 folding relies on its interaction with the chaperone HSP90 and the cochaperone CDC37. Understanding the intricate molecular interactions governing RAF1 folding is crucial for comprehending this process. Here, we present a cryo-EM structure of the closed-state RAF1-HSP90-CDC37 complex, where the C-lobe of the RAF1 kinase domain binds to one side of the HSP90 dimer, and an unfolded N-lobe segment of the RAF1 kinase domain threads through the center of the HSP90 dimer. CDC37 binds to the kinase C-lobe, mimicking the N-lobe with its HxNI motif. We also describe structures of HSP90 dimers without RAF1 and CDC37, displaying only N-terminal and middle domains, which we term the semi-open state. Employing 1 μs atomistic simulations, energetic decomposition, and comparative structural analysis, we elucidate the dynamics and interactions within these complexes. Our quantitative analysis reveals that CDC37 bridges the HSP90-RAF1 interaction, RAF1 binds HSP90 asymmetrically, and that HSP90 structural elements engage RAF1s unfolded region. Additionally, N- and C-terminal interactions stabilize HSP90 dimers, and molecular interactions in HSP90 dimers rearrange between the closed and semi-open states. Our findings provide valuable insight into the contributions of HSP90 and CDC37 in mediating client folding.

Obtaining accurate binding free energies from in silico screens has been a long-standing goal for the computational chemistry community. However, accuracy and computational cost are at odds with one another, limiting the utility of methods that perform this type of calculation. Many methods achieve massive scale by explicitly or implicitly assuming that the target protein adopts a single structure, or undergoes limited fluctuations around that structure, to minimize computational cost. Others simulate each protein-ligand complex of interest, accepting lower throughput in exchange for better predictions of binding affinities. Here, we present the PopShift framework for accounting for the ensemble of structures a protein adopts and their relative probabilities. Protein degrees of freedom are enumerated once, and then arbitrarily many molecules can be screened against this ensemble. Specifically, we use Markov state models (MSMs) as a compressed representation of a proteins thermodynamic ensemble. We start with a ligand-free MSM and then calculate how addition of a ligand shifts the populations of each protein conformational state based on the strength of the interaction between that protein conformation and the ligand. In this work we use docking to estimate the affinity between a given protein structure and ligand, but any estimator of binding affinities could be used in the PopShift framework. We test PopShift on the classic benchmark pocket T4 Lysozyme L99A. We find that PopShift is more accurate than common strategies, such as docking to a single structure and traditional ensemble docking─producing results that compare favorably with alchemical binding free energy calculations in terms of RMSE but not correlation─and may have a more favorable computational cost profile in some applications. In addition to predicting binding free energies and ligand poses, PopShift also provides insight into how the probability of different protein structures is shifted upon addition of various concentrations of ligand, providing a platform for predicting affinities and allosteric effects of ligand binding. Therefore, we expect PopShift will be valuable for hit finding and for providing insight into phenomena like allostery.

Molecular docking is a widely used technique for leveraging protein structure for ligand discovery, but it remains difficult to utilize due to limitations that have not been adequately addressed. Despite some progress toward automation, docking still requires expert guidance, hindering its adoption by a broader range of investigators. To make docking more accessible, we developed a new utility called DockOpt, which automates the creation, evaluation, and optimization of docking models prior to their deployment in large-scale prospective screens. DockOpt outperforms our previous automated pipeline across all 43 targets in the DUDE-Z benchmark data set, and the generated models for 84% of targets demonstrate sufficient enrichment to warrant their use in prospective screens, with normalized LogAUC values of at least 15%. DockOpt is available as part of the Python package Pydock3 included in the UCSF DOCK 3.8 distribution, which is available for free to academic researchers at https://dock.compbio.ucsf.edu and free for everyone upon registration at https://tldr.docking.org.

Eukaryotic transcription factors activate gene expression with their DNA-binding domains and activation domains. DNA-binding domains bind the genome by recognizing structurally related DNA sequences; they are structured, conserved, and predictable from protein sequences. Activation domains recruit chromatin modifiers, coactivator complexes, or basal transcriptional machinery via structurally diverse protein-protein interactions. Activation domains and DNA-binding domains have been called independent, modular units, but there are many departures from modularity, including interactions between these regions and overlap in function. Compared to DNA-binding domains, activation domains are poorly understood because they are poorly conserved, intrinsically disordered, and difficult to predict from protein sequences. This review, organized around commonly asked questions, describes recent progress that the field has made in understanding the sequence features that control activation domains and predicting them from sequence.

The transcription factor and cell cycle regulator p53 is marked for degradation by the ubiquitin ligase MDM2. The interaction between these 2 proteins is mediated by a conserved binding motif in the disordered p53 transactivation domain (p53TAD) and the folded SWIB domain in MDM2. The conserved motif in p53TAD from zebrafish displays a 20-fold weaker interaction with MDM2, compared to the interaction in human and chicken. To investigate this apparent difference, we tracked the molecular evolution of the p53TAD/MDM2 interaction among ray-finned fishes (Actinopterygii), the largest vertebrate clade. Intriguingly, phylogenetic analyses, ancestral sequence reconstructions, and binding experiments showed that different loss-of-affinity changes in the canonical binding motif within p53TAD have occurred repeatedly and convergently in different fish lineages, resulting in relatively low extant affinities (KD = 0.5 to 5 μM). However, for 11 different fish p53TAD/MDM2 interactions, nonconserved regions flanking the canonical motif increased the affinity 4- to 73-fold to be on par with the human interaction. Our findings suggest that compensating changes at conserved and nonconserved positions within the motif, as well as in flanking regions of low conservation, underlie a stabilizing selection of functional affinity in the p53TAD/MDM2 interaction. Such interplay complicates bioinformatic prediction of binding and calls for experimental validation. Motif-mediated protein-protein interactions involving short binding motifs and folded interaction domains are very common across multicellular life. It is likely that the evolution of affinity in motif-mediated interactions often involves an interplay between specific interactions made by conserved motif residues and nonspecific interactions by nonconserved disordered regions.

Enhancers control the location and timing of gene expression and contain the majority of variants associated with disease1-3. The ZRS is arguably the most well-studied vertebrate enhancer and mediates the expression of Shh in the developing limb4. Thirty-one human single-nucleotide variants (SNVs) within the ZRS are associated with polydactyly4-6. However, how this enhancer encodes tissue-specific activity, and the mechanisms by which SNVs alter the number of digits, are poorly understood. Here we show that the ETS sites within the ZRS are low affinity, and identify a functional ETS site, ETS-A, with extremely low affinity. Two human SNVs and a synthetic variant optimize the binding affinity of ETS-A subtly from 15% to around 25% relative to the strongest ETS binding sequence, and cause polydactyly with the same penetrance and severity. A greater increase in affinity results in phenotypes that are more penetrant and more severe. Affinity-optimizing SNVs in other ETS sites in the ZRS, as well as in ETS, interferon regulatory factor (IRF), HOX and activator protein 1 (AP-1) sites within a wide variety of enhancers, cause gain-of-function gene expression. The prevalence of binding sites with suboptimal affinity in enhancers creates a vulnerability in genomes whereby SNVs that optimize affinity, even slightly, can be pathogenic. Searching for affinity-optimizing SNVs in genomes could provide a mechanistic approach to identify causal variants that underlie enhanceropathies.

Discovering ligands for amyloid fibrils, such as those formed by the tau protein, is an area of great current interest. In recent structures, ligands bind in stacks in the tau fibrils to reflect the rotational and translational symmetry of the fibril itself; in these structures, the ligands make few interactions with the protein but interact extensively with each other. To exploit this symmetry and stacking, we developed SymDOCK, a method to dock molecules that follow the proteins symmetry. For each prospective ligand pose, we apply the symmetry operation of the fibril to generate a self-interacting and fibril-interacting stack, checking that doing so will not cause a clash between the original molecule and its image. Absent a clash, we retain that pose and add the ligand-ligand van der Waals energy to the ligands docking score (here using DOCK3.8). We can check these geometries and energies using an implementation of ANI, a neural-network-based quantum-mechanical evaluation of the ligand stacking energies. In retrospective calculations, symmetry docking can reproduce the poses of three tau PET tracers whose structures have been determined. More convincingly, in a prospective study, SymDOCK predicted the structure of the PET tracer MK-6240 bound in a symmetrical stack to AD PHF tau before that structure was determined; the docked pose was used to determine how MK-6240 fit the cryo-EM density. In proof-of-concept studies, SymDOCK enriched known ligands over property-matched decoys in retrospective screens without sacrificing docking speed and can address large library screens that seek new symmetrical stackers. Future applications of this approach will be considered.

In this study, we utilize Protein Residue Networks (PRNs), constructed using Local Spatial Pattern (LSP) alignment, to explore the dynamic behavior of Catabolite Activator Protein (CAP) upon the sequential binding of cAMP. We employed the Degree Centrality of these PRNs to investigate protein dynamics on a sub-nanosecond time scale, hypothesizing that it would reflect changes in CAPs entropy related to its thermal motions. We show that the binding of the first cAMP led to an increase in stability in the Cyclic-Nucleotide Binding Domain A (CNBD-A) and destabilization in CNBD-B, agreeing with previous reports explaining the negative cooperativity of cAMP binding in terms of an entropy-driven allostery. LSP-based PRNs also allow for the study of Betweenness Centrality, another graph-theoretical characteristic of PRNs, providing insights into global residue connectivity within CAP. Using this approach, we were able to correctly identify amino acids that were shown to be critical in mediating allosteric interactions in CAP. The agreement between our studies and previous experimental reports validates our method, particularly with respect to the reliability of Degree Centrality as a proxy for entropy related to protein thermal dynamics. Because LSP-based PRNs can be easily extended to include dynamics of small organic molecules, polynucleotides, or other allosteric proteins, the methods presented here mark a significant advancement in the field, positioning them as vital tools for a fast, cost-effective, and accurate analysis of entropy-driven allostery and identification of allosteric hotspots.

The kinetochore scaffold 1 (KNL1) protein recruits spindle assembly checkpoint (SAC) proteins to ensure accurate chromosome segregation during mitosis. Despite such a conserved function among eukaryotic organisms, its molecular architectures have rapidly evolved so that the functional mode of plant KNL1 is largely unknown. To understand how SAC signaling is regulated at kinetochores, we characterized the function of the KNL1 gene in Arabidopsis thaliana. The KNL1 protein was detected at kinetochores throughout the mitotic cell cycle, and null knl1 mutants were viable and fertile but exhibited severe vegetative and reproductive defects. The mutant cells showed serious impairments of chromosome congression and segregation, that resulted in the formation of micronuclei. In the absence of KNL1, core SAC proteins were no longer detected at the kinetochores, and the SAC was not activated by unattached or misaligned chromosomes. Arabidopsis KNL1 interacted with SAC essential proteins BUB3.3 and BMF3 through specific regions that were not found in known KNL1 proteins of other species, and recruited them independently to kinetochores. Furthermore, we demonstrated that upon ectopic expression, the KNL1 homolog from the dicot tomato was able to functionally substitute KNL1 in A. thaliana, while others from the monocot rice or moss associated with kinetochores but were not functional, as reflected by sequence variations of the kinetochore proteins in different plant lineages. Our results brought insights into understanding the rapid evolution and lineage-specific connection between KNL1 and the SAC signaling molecules.

Membrane protein biogenesis poses acute challenges to protein homeostasis, and how they are selectively escorted to the target membrane is not well understood. Here we address this question in the guided-entry-of-tail-anchored protein (GET) pathway, in which tail-anchored membrane proteins (TAs) are relayed through an Hsp70-Sgt2-Get3 chaperone triad for targeting to the endoplasmic reticulum. We show that the Hsp70 ATPase cycle and TA substrate drive dimeric Sgt2 from a wide-open conformation to a closed state, in which TAs are protected by both substrate binding domains of Sgt2. Get3 is privileged to receive TA from closed Sgt2, whereas off-pathway chaperones remove TAs from open Sgt2. Sgt2 closing is less favorable with suboptimal GET substrates, which are rejected during or after the Hsp70-to-Sgt2 handover. Our results demonstrate how fine-tuned conformational dynamics in Sgt2 enable hydrophobic TAs to be effectively funneled onto their dedicated targeting factor while also providing a mechanism for substrate selection.

Protein homeostasis is a tightly conserved process that is regulated through the ubiquitin proteasome system (UPS) in a ubiquitin-independent or ubiquitin-dependent manner. Over the past two decades, the proteasome has become an excellent therapeutic target through inhibition of the catalytic core particle, inhibition of subunits responsible for recognizing and binding ubiquitinated proteins, and more recently, through targeted protein degradation using proteolysis targeting chimeras (PROTACs). The majority of the developed inhibitors of the proteasome's core particle rely on gaining selectivity through binding interactions within the unprimed substrate channel. Although this has allowed for selective inhibitors and chemical probes to be generated for the different proteasome isoforms, much remains unknown about the interactions that could be harnessed within the primed substrate channel to increase potency or selectivity. Herein, we discuss small molecules that interact with the primed substrate pocket and how their differences may give rise to altered activity. Taking advantage of additional interactions with the primed substrate pocket of the proteasome could allow for the generation of improved chemical tools for perturbing or monitoring proteasome activity.

Translation is regulated mainly in the initiation step, and its dysregulation is implicated in many human diseases. Several proteins have been found to regulate translational initiation, including Pdcd4 (programmed cell death gene 4). Pdcd4 is a tumor suppressor protein that prevents cell growth, invasion, and metastasis. It is downregulated in most tumor cells, while global translation in the cell is upregulated. To understand the mechanisms underlying translational control by Pdcd4, we used single-particle cryo-electron microscopy to determine the structure of human Pdcd4 bound to 40S small ribosomal subunit, including Pdcd4-40S and Pdcd4-40S-eIF4A-eIF3-eIF1 complexes. The structures reveal the binding site of Pdcd4 at the mRNA entry site in the 40S, where the C-terminal domain (CTD) interacts with eIF4A at the mRNA entry site, while the N-terminal domain (NTD) is inserted into the mRNA channel and decoding site. The structures, together with quantitative binding and in vitro translation assays, shed light on the critical role of the NTD for the recruitment of Pdcd4 to the ribosomal complex and suggest a model whereby Pdcd4 blocks the eIF4F-independent role of eIF4A during recruitment and scanning of the 5' UTR of mRNA.

PIP3-dependent Rac exchanger 1 (P-Rex1) is abundantly expressed in neutrophils and plays central roles in chemotaxis and cancer metastasis by serving as a guanine-nucleotide exchange factor (GEF) for Rac. The enzyme is synergistically activated by PIP3 and heterotrimeric Gβγ subunits, but mechanistic details remain poorly understood. While investigating the regulation of P-Rex1 by PIP3, we discovered that Ins(1,3,4,5)P4 (IP4) inhibits P-Rex1 activity and induces large decreases in backbone dynamics in diverse regions of the protein. Cryo-electron microscopy analysis of the P-Rex1·IP4 complex revealed a conformation wherein the pleckstrin homology (PH) domain occludes the active site of the Dbl homology (DH) domain. This configuration is stabilized by interactions between the first DEP domain (DEP1) and the DH domain and between the PH domain and a 4-helix bundle (4HB) subdomain that extends from the C-terminal domain of P-Rex1. Disruption of the DH-DEP1 interface in a DH/PH-DEP1 fragment enhanced activity and led to a more extended conformation in solution, whereas mutations that constrain the occluded conformation led to decreased GEF activity. Variants of full-length P-Rex1 in which the DH-DEP1 and PH-4HB interfaces were disturbed exhibited enhanced activity during chemokine-induced cell migration, confirming that the observed structure represents the autoinhibited state in living cells. Interactions with PIP3-containing liposomes led to disruption of these interfaces and increased dynamics protein-wide. Our results further suggest that inositol phosphates such as IP4 help to inhibit basal P-Rex1 activity in neutrophils, similar to their inhibitory effects on phosphatidylinositol-3-kinase.

RNA-binding proteins (RBPs) have pivotal functions in RNA metabolism, but current methods are limited in retrieving RBP-RNA interactions within endogenous biological contexts. Here, we develop INSCRIBE (IN situ Sensitive Capture of RNA-protein Interactions in Biological Environments), circumventing the challenges through in situ RNA labeling by precisely directing a purified APOBEC1-nanobody fusion to the RBP of interest. This method enables highly specific RNA-binding site identification across a diverse range of fixed biological samples such as HEK293T cells and mouse brain tissue and accurately identifies the canonical binding motifs of RBFOX2 (UGCAUG) and TDP-43 (UGUGUG) in native cellular environments. Applicable to any RBP with available primary antibodies, INSCRIBE enables sensitive capture of RBP-RNA interactions from ultra-low input equivalent to ~5 cells. The robust, versatile, and sensitive INSCRIBE workflow is particularly beneficial for precious tissues such as clinical samples, empowering the exploration of genuine RBP-RNA interactions in RNA-related disease contexts.

Chitin is an abundant biopolymer and pathogen-associated molecular pattern that stimulates a host innate immune response. Mammals express chitin-binding and chitin-degrading proteins to remove chitin from the body. One of these proteins, Acidic Mammalian Chitinase (AMCase), is an enzyme known for its ability to function under acidic conditions in the stomach but is also active in tissues with more neutral pHs, such as the lung. Here, we used a combination of biochemical, structural, and computational modeling approaches to examine how the mouse homolog (mAMCase) can act in both acidic and neutral environments. We measured kinetic properties of mAMCase activity across a broad pH range, quantifying its unusual dual activity optima at pH 2 and 7. We also solved high-resolution crystal structures of mAMCase in complex with oligomeric GlcNAcn, the building block of chitin, where we identified extensive conformational ligand heterogeneity. Leveraging these data, we conducted molecular dynamics simulations that suggest how a key catalytic residue could be protonated via distinct mechanisms in each of the two environmental pH ranges. These results integrate structural, biochemical, and computational approaches to deliver a more complete understanding of the catalytic mechanism governing mAMCase activity at different pH. Engineering proteins with tunable pH optima may provide new opportunities to develop improved enzyme variants, including AMCase, for therapeutic purposes in chitin degradation.