Structural Homology Search Research Articles

Unanticipated broad phylogeny of BEN DNA-binding domains revealed by structural homology searches

Organization of protein sequences into domain families is a foundation for cataloging and investigating protein functions. However, long-standing strategies based on primary amino acid sequences are blind to the possibility that proteins with dissimilar sequences could have comparable tertiary structures. Building on our recent findings that in silico structural predictions of BEN family DNA-binding domains closely resemble their experimentally determined crystal structures, we exploited the AlphaFold2 database for comprehensive identification of BEN domains. Indeed, we identified numerous novel BEN domains, including members of new subfamilies. For example, while no BEN domain factors had previously been annotated in C.elegans, this species actually encodes multiple BEN proteins. These include key developmental timing genes of orphan domain status, sel-7 and lin-14, the latter being the central target of the founding miRNA lin-4. We also reveal that the domain of unknown function 4806 (DUF4806), which is widely distributed across metazoans, is structurally similar to BEN and comprises a new subtype. Surprisingly, we find that BEN domains resemble both metazoan and non-metazoan homeodomains in 3D conformation and preserve characteristic residues, indicating that despite their inability to be aligned by conventional methods, these DNA-binding modules are probably evolutionarily related. Finally, we broaden the application of structural homology searches by revealing novel human members of DUF3504, which exists on diverse proteins with presumed or known nuclear functions. Overall, our work strongly expands this recently identified family of transcription factors and illustrates the value of 3D structural predictions to annotate protein domains and interpret their functions.

Bacterial effector kinases and strategies to identify their target host substrates.

Post-translational modifications (PTMs) are critical in regulating protein function by altering chemical characteristics of proteins. Phosphorylation is an integral PTM, catalyzed by kinases and reversibly removed by phosphatases, that modulates many cellular processes in response to stimuli in all living organisms. Consequently, bacterial pathogens have evolved to secrete effectors capable of manipulating host phosphorylation pathways as a common infection strategy. Given the importance of protein phosphorylation in infection, recent advances in sequence and structural homology search have significantly expanded the discovery of a multitude of bacterial effectors with kinase activity in pathogenic bacteria. Although challenges exist due to complexity of phosphorylation networks in host cells and transient interactions between kinases and substrates, approaches are continuously being developed and applied to identify bacterial effector kinases and their host substrates. In this review, we illustrate the importance of exploiting phosphorylation in host cells by bacterial pathogens via the action of effector kinases and how these effector kinases contribute to virulence through the manipulation of diverse host signaling pathways. We also highlight recent developments in the identification of bacterial effector kinases and a variety of techniques to characterize kinase-substrate interactions in host cells. Identification of host substrates provides new insights for regulation of host signaling during microbial infection and may serve as foundation for developing interventions to treat infection by blocking the activity of secreted effector kinases.

Crystal structure of the C-terminal domain of the plant-specific microtubule-associated protein Spiral2.

Plant cells form microtubule arrays, called `cortical microtubules', beneath the plasma membrane which are critical for cell-wall organization and directional cell growth. Cortical microtubules are nucleated independently of centrosomes. Spiral2 is a land-plant-specific microtubule minus-end-targeting protein that stabilizes the minus ends by inhibiting depolymerization of the filament. Spiral2 possesses an N-terminal microtubule-binding domain and a conserved C-terminal domain whose function is unknown. In this study, the crystal structure of the conserved C-terminal domain of Spiral2 was determined using the single-wavelength anomalous dispersion method. Refinement of the model to a resolution of 2.2 Å revealed a helix-turn-helix fold with seven α-helices. The protein crystallized as a dimer, but SEC-MALS analysis showed the protein to be monomeric. A structural homology search revealed that the protein has similarity to the C-terminal domain of the katanin regulatory subunit p80. The structure presented here suggests that the C-terminal domain of Spiral2 represents a new class of microtubule dynamics modulator across the kingdom.

Structure of the hypothetical protein TTHA1873 from Thermus thermophilus.

The crystal structure of an uncharacterized hypothetical protein, TTHA1873 from Thermus thermophilus, has been determined by X-ray crystallography to a resolution of 1.78 Å using the single-wavelength anomalous dispersion method. The protein crystallized as a dimer in two space groups: P43212 and P6122. Structural analysis of the hypothetical protein revealed that the overall fold of TTHA1873 has a β-sandwich jelly-roll topology with nine β-strands. TTHA1873 is a dimeric metal-binding protein that binds to two Ca2+ ions per chain, with one on the surface and the other stabilizing the dimeric interface of the two chains. A structural homology search indicates that the protein has moderate structural similarity to one domain of cell-surface proteins or agglutinin receptor proteins. Red blood cells showed visible agglutination at high concentrations of the hypothetical protein.

Cloacal virome of an ancient host lineage – The tuatara (Sphenodon punctatus) – Reveals abundant and diverse diet-related viruses

Tuatara (Sphenodon punctatus) are one of the most phylogenetically isolated vertebrate species and provide a unique host system to study virus evolution. While the tuatara genome, sequenced in 2020, revealed many endogenous viral elements, we know little of the exogenous viruses that infect tuatara. We performed a metatranscriptomics study of tuatara cloaca samples from a wild population on Takapourewa (Stephens Island), Aotearoa New Zealand. From these data we identified 49 potentially novel viral species that spanned 19 RNA viral families and/or orders, the vast majority (48) of which were likely dietary-related. Notably, using a protein structure homology search, we identified a highly divergent novel virus within the Picornaviridae which may directly infect tuatara. Additionally, two endogenous tuatara adintoviruses were characterised that exhibited long-term viral-host co-divergence. Overall, our results indicate that the tuatara cloacal virome is highly diverse, likely due to a large number of dietary-related viruses.

SHAPE-guided RNA structure homology search and motif discovery

The rapidly growing popularity of RNA structure probing methods is leading to increasingly large amounts of available RNA structure information. This demands the development of efficient tools for the identification of RNAs sharing regions of structural similarity by direct comparison of their reactivity profiles, hence enabling the discovery of conserved structural features. We here introduce SHAPEwarp, a largely sequence-agnostic SHAPE-guided algorithm for the identification of structurally-similar regions in RNA molecules. Analysis of Dengue, Zika and coronavirus genomes recapitulates known regulatory RNA structures and identifies novel highly-conserved structural elements. This work represents a preliminary step towards the model-free search and identification of shared and conserved RNA structural features within transcriptomes.

Pathogenic Leptospira Evolved a Unique Gene Family Comprised of Ricin B-Like Lectin Domain-Containing Cytotoxins.

Leptospirosis is a globally important neglected zoonotic disease. Previous data suggest that a family of virulence-modifying (VM) proteins (PF07598) is a distinctive feature of group I pathogenic Leptospira that evolved as important virulence determinants. Here, we show that one such VM protein, LA3490 (also known as Q8F0K3), is expressed by Leptospira interrogans serovar Lai, as a secreted genotoxin that is potently cytotoxic to human cells. Structural homology searches using Phyre2 suggested that VM proteins are novel R-type lectins containing tandem N-terminal ricin B-chain-like β-trefoil domains. Recombinant LA3490 (rLA3490) and an N-terminal fragment, t3490, containing only the predicted ricin B domain, bound to the terminal galactose and N-acetyl-galactosamine residues, asialofetuin, and directly competed for asialofetuin-binding sites with recombinant ricin B chain. t3490 alone was sufficient for binding, both to immobilized asialofetuin and to the HeLa cell surface but was neither internalized nor cytotoxic. Treatment of HeLa cells with rLA3490 led to cytoskeleton disassembly, caspase-3 activation, and nuclear fragmentation, and was rapidly cytolethal. rLA3490 had DNase activity on mammalian and bacterial plasmid DNA. The combination of cell surface binding, internalization, nuclear translocation, and DNase functions indicate that LA3490 and other VM proteins evolved as novel forms of the bacterial AB domain-containing toxin paradigm.

Cannabinoid receptor interacting protein 1a interacts with myristoylated Gαi N terminus via a unique gapped β-barrel structure

Cannabinoid receptor interacting protein 1a (CRIP1a) modulates CB1 cannabinoid receptor G-protein coupling in part by altering the selectivity for Gαi subtype activation, but the molecular basis for this function of CRIP1a is not known. We report herein the first structure of CRIP1a at a resolution of 1.55 Å. CRIP1a exhibits a 10-stranded and antiparallel β-barrel with an interior comprised of conserved hydrophobic residues and loops at the bottom and a short helical cap at the top to exclude solvent. The β-barrel has a gap between strands β8 and β10, which deviates from β-sandwich fatty acid–binding proteins that carry endocannabinoid compounds and the Rho-guanine nucleotide dissociation inhibitor predicted by computational threading algorithms. The structural homology search program DALI identified CRIP1a as homologous to a family of lipidated-protein carriers that includes phosphodiesterase 6 delta subunit and Unc119. Comparison with these proteins suggests that CRIP1a may carry two possible types of cargo: either (i) like phosphodiesterase 6 delta subunit, cargo with a farnesyl moiety that enters from the top of the β-barrel to occupy the hydrophobic interior or (ii) like Unc119, cargo with a palmitoyl or a myristoyl moiety that enters from the side where the missing β-strand creates an opening to the hydrophobic pocket. Fluorescence polarization analysis demonstrated CRIP1a binding of an N-terminally myristoylated 9-mer peptide mimicking the Gαi N terminus. However, CRIP1a could not bind the nonmyristolyated Gαi peptide or cargo of homologs. Thus, binding of CRIP1a to Gαi proteins represents a novel mechanism to regulate cell signaling initiated by the CB1 receptor.

3DFI: a pipeline to infer protein function using structural homology.

Inferring protein function is an integral part of genome annotation and analysis. This process is usually performed in silico, and most in silico inferences are based on sequence homology approaches, which can fail when in presence of divergent sequences. However, because protein structures and their biological roles are intertwined, protein function can also be inferred by searching for structural homology. Many excellent tools have been released in recent years with regards to protein structure prediction, structural homology searches and protein visualization. Unfortunately, these tools are disconnected from each other and often use a web server-based approach that is ill-suited to high-throughput genome-wide analyses. To help assist genome annotation, we built a structural homology-based pipeline called 3DFI (for tridimensional functional inference) leveraging some of the best structural homology tools. This pipeline was built with simplicity of use in mind and enables genome-wide structural homology inferences. 3DFI is available on GitHub https://github.com/PombertLab/3DFI under the permissive MIT license. The pipeline is written in Perl and Python.

Overexpression of the Bacteriophage T4 motB Gene Alters H-NS Dependent Repression of Specific Host DNA.

The bacteriophage T4 early gene product MotB binds tightly but nonspecifically to DNA, copurifies with the host Nucleoid Associated Protein (NAP) H-NS in the presence of DNA and improves T4 fitness. However, the T4 transcriptome is not significantly affected by a motB knockdown. Here we have investigated the phylogeny of MotB and its predicted domains, how MotB and H-NS together interact with DNA, and how heterologous overexpression of motB impacts host gene expression. We find that motB is highly conserved among Tevenvirinae. Although the MotB sequence has no homology to proteins of known function, predicted structure homology searches suggest that MotB is composed of an N-terminal Kyprides-Onzonis-Woese (KOW) motif and a C-terminal DNA-binding domain of oligonucleotide/oligosaccharide (OB)-fold; either of which could provide MotB’s ability to bind DNA. DNase I footprinting demonstrates that MotB dramatically alters the interaction of H-NS with DNA in vitro. RNA-seq analyses indicate that expression of plasmid-borne motB up-regulates 75 host genes; no host genes are down-regulated. Approximately 1/3 of the up-regulated genes have previously been shown to be part of the H-NS regulon. Our results indicate that MotB provides a conserved function for Tevenvirinae and suggest a model in which MotB functions to alter the host transcriptome, possibly by changing the association of H-NS with the host DNA, which then leads to conditions that are more favorable for infection.

Remote homology search with hidden Potts models.

Most methods for biological sequence homology search and alignment work with primary sequence alone, neglecting higher-order correlations. Recently, statistical physics models called Potts models have been used to infer all-by-all pairwise correlations between sites in deep multiple sequence alignments, and these pairwise couplings have improved 3D structure predictions. Here we extend the use of Potts models from structure prediction to sequence alignment and homology search by developing what we call a hidden Potts model (HPM) that merges a Potts emission process to a generative probability model of insertion and deletion. Because an HPM is incompatible with efficient dynamic programming alignment algorithms, we develop an approximate algorithm based on importance sampling, using simpler probabilistic models as proposal distributions. We test an HPM implementation on RNA structure homology search benchmarks, where we can compare directly to exact alignment methods that capture nested RNA base-pairing correlations (stochastic context-free grammars). HPMs perform promisingly in these proof of principle experiments.

Structure of the RECK CC domain, an evolutionary anomaly

Five small protein domains, the CC-domains, at the N terminus of the RECK protein, play essential roles in signaling by WNT7A and WNT7B in the context of central nervous system angiogenesis and blood-brain barrier formation and maintenance. We have determined the structure of CC domain 4 (CC4) at 1.65-A resolution and find that it folds into a compact four-helix bundle with three disulfide bonds. The CC4 structure, together with homology modeling of CC1, reveals the surface locations of critical residues that were shown in previous mutagenesis studies to mediate GPR124 binding and WNT7A/WNT7B recognition and signaling. Surprisingly, sequence and structural homology searches reveal no other cell-surface or secreted domains in vertebrates that resemble the CC domain, a pattern that is in striking contrast to other ancient and similarly sized domains, such as Epidermal Growth Factor, Fibronectin Type 3, Immunoglobulin, and Thrombospondin type 1 domains, which are collectively present in hundreds of proteins.

Structure of the RECK CC domain, an evolutionary anomaly

Five small protein domains, the CC-domains, at the N terminus of the RECK protein, play essential roles in signaling by WNT7A and WNT7B in the context of central nervous system angiogenesis and blood-brain barrier formation and maintenance. We have determined the structure of CC domain 4 (CC4) at 1.65-Å resolution and find that it folds into a compact four-helix bundle with three disulfide bonds. The CC4 structure, together with homology modeling of CC1, reveals the surface locations of critical residues that were shown in previous mutagenesis studies to mediate GPR124 binding and WNT7A/WNT7B recognition and signaling. Surprisingly, sequence and structural homology searches reveal no other cell-surface or secreted domains in vertebrates that resemble the CC domain, a pattern that is in striking contrast to other ancient and similarly sized domains, such as Epidermal Growth Factor, Fibronectin Type 3, Immunoglobulin, and Thrombospondin type 1 domains, which are collectively present in hundreds of proteins.

SSIPe: accurately estimating protein-protein binding affinity change upon mutations using evolutionary profiles in combination with an optimized physical energy function.

Most proteins perform their biological functions through interactions with other proteins in cells. Amino acid mutations, especially those occurring at protein interfaces, can change the stability of protein-protein interactions (PPIs) and impact their functions, which may cause various human diseases. Quantitative estimation of the binding affinity changes (ΔΔGbind) caused by mutations can provide critical information for protein function annotation and genetic disease diagnoses. We present SSIPe, which combines protein interface profiles, collected from structural and sequence homology searches, with a physics-based energy function for accurate ΔΔGbind estimation. To offset the statistical limits of the PPI structure and sequence databases, amino acid-specific pseudocounts were introduced to enhance the profile accuracy. SSIPe was evaluated on large-scale experimental data containing 2204 mutations from 177 proteins, where training and test datasets were stringently separated with the sequence identity between proteins from the two datasets below 30%. The Pearson correlation coefficient between estimated and experimental ΔΔGbind was 0.61 with a root-mean-square-error of 1.93 kcal/mol, which was significantly better than the other methods. Detailed data analyses revealed that the major advantage of SSIPe over other traditional approaches lies in the novel combination of the physical energy function with the new knowledge-based interface profile. SSIPe also considerably outperformed a former profile-based method (BindProfX) due to the newly introduced sequence profiles and optimized pseudocount technique that allows for consideration of amino acid-specific prior mutation probabilities. Web-server/standalone program, source code and datasets are freely available at https://zhanglab.ccmb.med.umich.edu/SSIPe and https://github.com/tommyhuangthu/SSIPe. Supplementary data are available at Bioinformatics online.

2590. Streptolysin O Enhances Binding of the Group A Streptococcal NAD+-Glycohydrolase Toxin to Oropharyngeal Keratinocytes

BackgroundStreptolysin O (SLO) and the NAD+-glycohydrolase (NADase) are co-toxins secreted by group A Streptococcus (GAS) that play a significant role in virulence. NADase requires SLO for translocation into the host cell cytoplasm, a process termed cytolysin-mediated translocation (CMT). Recently, we noted that interaction of the two toxins mutually increased their stability. Although NADase is predicted to bind to the host cell surface, this interaction is incompletely understood. Here, we investigate potential mechanisms by which NADase binds to oropharyngeal keratinocytes.MethodsThe amino terminal region of NADase has been implicated in CMT, but the structure of the putative translocation domain has not been characterized. We determined the solution structure of this domain by NMR spectroscopy. We used flow-cytometry and confocal microscopy to investigate whether NADase could interact directly with oropharyngeal keratinocytes. Finally, since we expect that NADase and SLO are co-expressed from the same operon, are secreted in a coordinated fashion, and interact in solution, we tested whether SLO affects NADase binding to host cells.ResultsThe solution structure of the NADase translocation domain revealed a β-sandwich fold with an elongated N-terminal intrinsically disordered region. Structural homology searches (DALI) identified a potential carbohydrate binding module, suggesting the translocation domain could play a role in glycan binding. We also demonstrated by flow-cytometry that purified recombinant NADase toxin is able to independently interact with the cell surface of oropharyngeal keratinocytes. Importantly, interaction with SLO significantly enhanced the association of NADase with the cell surface, resulting in a 5-fold increase of the geometric mean fluorescence intensity.ConclusionThe structure of the NADase translocation domain reveals a potential carbohydrate binding module, which may mediate binding of the toxin to a cell-surface glycan. Binding of NADase to host cells is markedly enhanced by its interaction with SLO. We conclude that interaction of the two toxins contributes to the CMT process by functionally increasing the local concentration of NADase at the cell surface.Disclosures All authors: No reported disclosures.

Crystal structure of L-aspartate aminotransferase from Schizosaccharomyces pombe.

L-aspartate aminotransferase is a pyridoxal 5ʹ-phosphate-dependent transaminase that catalyzes reversible transfer of an α-amino group from aspartate to α-ketoglutarate or from glutamate to oxaloacetate. L-aspartate aminotransferase not only mediates amino acid and carbohydrate metabolism but also regulates the cellular level of amino acids by catalyzing amino acid degradation and biosynthesis. To expand our structural information, we determined the crystal structure of L-aspartate aminotransferase from Schizosaccharomyces pombe at 2.1 Å resolution. A structural comparison between two yeast L-aspartate aminotransferases revealed conserved enzymatic mechanism mediated by the open–closed conformational change. Compared with higher eukaryotic species, L-aspartate aminotransferases showed distinguishable inter-subunit interaction between the N-terminal arm and a large domain of the opposite subunit. Interestingly, structural homology search showed varied conformation of the N-terminal arm among 71 structures of the family. Therefore, we classified pyridoxal 5ʹ-phosphate-dependent enzymes into eight subclasses based on the structural feature of N-terminal arms. In addition, structure and sequence comparisons showed strong relationships among the eight subclasses. Our results may provide insights into structure-based evolutionary aspects of pyridoxal 5ʹ-phosphate-dependent enzymes.

Crystal Structure of the Type VI Secretion System Accessory Protein TagF from Pseudomonas Aeruginosa.

Type VI Secretion System (T6SS) has been found in approximately onequarter of the gram-negative bacterial species, and its structural characteristics appear to slightly differ from species to species. The genes encoding T6SS are designated as type six secretion A-M (tssA-M). The expression of the tss gene cluster is regulated by various accessory genes, designated as type VI-associated genes A-P (tagA-P). Tag family proteins have been commonly found in bacteria expressing T6SS but not in all bacterial species. For instance, the tag gene cluster is well-conserved in Pseudomonas aeruginosa (Pa). The PaTagF protein has large homology with ImpM in Rhizobium leguminosarum and SciT in Salmonella enterica. The overexpression of PaTagF represses T6SS complex accumulation and suppresses T6SS antibacterial activity. Thus, the functions of TagF are mediated through direct interactions with the forkhead-associated protein Fha, as evident from the results of the yeast-two hybrid assays. Fha is involved in recruiting a membrane-associated complex either in threonine phosphorylation pathway-dependent or - independent manner. However, functional reports of the tag gene cluster are still limited. In this article, our motivation is to understand the molecular mechanism underlying the regulation of expression of the type VI secretion system complex. In this article, we start with obtaining the gene encoding PaTagF protein by polymerase chain reaction (PCR). Subsequently, the cloned gene is applied to overexpress of PaTagF protein in Escherichia coli, then purify the recombinant PaTagF protein. Thereafter, the protein is crystallized in a condition of 2.5 M NaCl, 0.1 M imidazole (pH 8.0), 3.2 M NaCl, 0.1 M BIS-TRIS propane (pH 7.0) and diffraction datasets of the PaTagF crystals are collected at the Pohang Accelerator Laboratory (PAL). The molecular structure of PaTagF protein is determined by molecular replacement using the uncharacterized protein PA0076 (PDB code:2QNU) as an initial search model by PHENIX crystallographic software package. Model building of PaTagF structure is performed using Coot program. Finally, the structural model is validated using phenix.refine program. PaTagF exists as a tetramer in the asymmetric unit, and the overall fold of each monomer is composed of continuous beta-sheets wrapped by alpha-helices. Each monomer has variable conformations and lengths of both the N- and C-termini. Twelve residues, including the His6 tag from the N-terminus of a symmetry-related molecule, have been found in two of the tetrameric PaTagF structures. A structural homology search revealed that PaTagF was similar to the α-β-α sandwichlike structure of the longin domain on the differentially expressed in normal and neoplastic (DENN) superfamily, which is commonly found in proteins related to trafficking. The tetrameric structure of PaTagF comprises varied N- and C-terminal regions in each subunit and may be stabilized by a symmetry-related molecule. This feature was also shown in the TssL structure from V. cholerae. Furthermore, our study showed that the overall fold of PaTagF is homologous to the longin domain of the DENN family. Therefore, further studies are warranted to elucidate the structure-based evolutionary relationship between protein transport systems from the bacteria and eukaryotic cells.

Computational Prediction of Carbohydrate-Binding Proteins and Binding Sites.

Protein-carbohydrate interaction is essential for biological systems, and carbohydrate-binding proteins (CBPs) are important targets when designing antiviral and anticancer drugs. Due to the high cost and difficulty associated with experimental approaches, many computational methods have been developed as complementary approaches to predict CBPs or carbohydrate-binding sites. However, most of these computational methods are not publicly available. Here, we provide a comprehensive review of related studies and demonstrate our two recently developed bioinformatics methods. The method SPOT-CBP is a template-based method for detecting CBPs based on structure through structural homology search combined with a knowledge-based scoring function. This method can yield model complex structure in addition to accurate prediction of CBPs. Furthermore, it has been observed that similarly accurate predictions can be made using structures from homology modeling, which has significantly expanded its applicability. The other method, SPRINT-CBH, is a de novo approach that predicts binding residues directly from protein sequences by using sequence information and predicted structural properties. This approach does not need structurally similar templates and thus is not limited by the current database of known protein-carbohydrate complex structures. These two complementary methods are available at https://sparks-lab.org. © 2018 by John Wiley & Sons, Inc.

Caffeic acid phenethyl ester (CAPE) possesses pro-hypoxia and anti-stress activities: bioinformatics and experimental evidences.

Honeybee propolis and its bioactive component, caffeic acid phenethyl ester (CAPE), are known for a variety of therapeutic potentials. By recruiting a cell-based reporter assay for screening of hypoxia-modulating natural drugs, we identified CAPE as a pro-hypoxia factor. In silico studies were used to probe the capacity of CAPE to interact with potential hypoxia-responsive proteins. CAPE could not dock into hypoxia inducing factor (HIF-1), the master regulator of hypoxia response pathway. On the other hand, it was predicted to bind to factor inhibiting HIF (FIH-1). The active site residue (Asp201) of FIH-1α was involved in hydrogen bond formation with CAPE and its analogue, caffeic acid methyl ester (CAME), especially in the presence of Fe and 2-oxoglutaric acid (OGA). We provide experimental evidence that the low doses of CAPE, that did not cause cytotoxicity or anti-migratory effect, activated HIF-1α and inhibited stress-induced protein aggregation, a common cause of age-related pathologies. Furthermore, by structural homology search, we explored and found candidate compounds that possess stronger FIH-1 binding capacity. These compounds could be promising candidates for modulating therapeutic potential of CAPE, and its recruitment in treatment of protein aggregation-based disorders.

APOL1 variants change C-terminal conformational dynamics and binding to SNARE protein VAMP8.

APOL1 variants in African populations mediate resistance to trypanosomal infection but increase risk for kidney diseases through unknown mechanisms. APOL1 is expressed in glomerular podocytes and does not vary with underlying kidney disease diagnoses or APOL1 genotypes, suggesting that the kidney disease-associated variants dysregulate its function rather than its localization or abundance. Structural homology searches identified vesicle-associated membrane protein 8 (VAMP8) as an APOL1 protein interactor. VAMP8 colocalizes with APOL1 in the podocyte, and the APOL1:VAMP8 interaction was confirmed biochemically and with surface plasmon resonance. APOL1 variants attenuate this interaction. Computational modeling of APOL1's 3-dimensional structure, followed by molecular dynamics simulations, revealed increased motion of the C-terminal domain of reference APOL1 compared with either variant, suggesting that the variants stabilize a closed or autoinhibited state that diminishes protein interactions with VAMP8. Changes in ellipticity with increasing urea concentrations, as assessed by circular dichroism spectroscopy, showed higher conformational stability of the C-terminal helix of the variants compared with the reference protein. These results suggest that reference APOL1 interacts with VAMP8-coated vesicles, a process attenuated by variant-induced reduction in local dynamics of the C-terminal. Disordered vesicular trafficking in the podocyte may cause injury and progressive chronic kidney diseases in susceptible African Americans subjects.