Biology subjects

Gulsevin, A.

Publications and source records attributed to Gulsevin, A..

9 recordsLinked to original sources

bioRxiv2024

VUStruct: a compute pipeline for high throughput and personalized structural biology

Effective diagnosis and treatment of rare genetic disorders requires the interpretation of a patients genetic variants of unknown significance (VUSs). Today, clinical decision-making is primarily guided by gene-phenotype association databases and DNA-based scoring methods. Our web-accessible variant analysis pipeline, VUStruct, supplements these established approaches by deeply analyzing the downstream molecular impact of variation in context of 3D protein structure. VUStructs growing impact is fueled by the co-proliferation of protein 3D structural models, gene sequencing, compute power, and artificial intelligence. Contextualizing VUSs in protein 3D structural models also illuminates longitudinal genomics studies and biochemical bench research focused on VUS, and we created VUStruct for clinicians and researchers alike. We now introduce VUStruct to the broad scientific community as a mature, web-facing, extensible, High-Performance Computing (HPC) software pipeline. VUStruct maps missense variants onto automatically selected protein structures and launches a broad range of analyses. These include energy-based assessments of protein folding and stability, pathogenicity prediction through spatial clustering analysis, and machine learning (ML) predictors of binding surface disruptions and nearby post-translational modification sites. The pipeline also considers the entire input set of VUS and identifies genes potentially involved in digenic disease. VUStructs utility in clinical rare disease genome interpretation has been demonstrated through its analysis of over 175 Undiagnosed Disease Network (UDN) Patient cases. VUStruct-leveraged hypotheses have often informed clinicians in their consideration of additional patient testing, and we report here details from two cases where VUStruct was key to their solution. We also note successes with academic research collaborators, for whom VUStruct has informed research directions in both computational genomics and wet lab studies.

biochemistry↗

bioRxiv2022

Design principles of caveolins across metazoa and beyond

Caveolins are a unique family of membrane-remodeling proteins present broadly across animals (Metazoa), and in vertebrates form flask-shaped invaginations known as caveolae. While human caveolin-1 assembles into an amphipathic disc composed of 11 spirally packed protomers, the structural basis underlying caveolin function across animals remains elusive. Here, we predicted structures for 73 caveolins spanning animal diversity, as well as a newly identified choanoflagellate caveolin from Salpingoeca rosetta, a unicellular relative to animals. This analysis revealed seven conserved structural elements and a propensity to assemble into amphipathic discs. Despite extreme sequence variability, new cryo-EM structures of caveolins from the choanoflagellate and the purple sea urchin Strongylocentrotus purpuratus exhibit striking structural similarities to human caveolin-1, validating the structural predictions. Lastly, tracing the chromosomal evolutionary history of caveolins revealed evolutionary branches where caveolins translocated and expanded, including a parahoxozoan ancestral chromosome as the origin of most caveolin diversity. These results show that caveolins possess an ancient structural framework predating Metazoa and provide a new structural paradigm to explore the molecular basis of caveolin function across diverse evolutionary lineages.

biophysics↗

bioRxiv2022

Template-free prediction of a new protein fold and oligomeric assembly by Alphafold2

AlphaFold2 (AF2) has revolutionized the field of protein structural prediction. Here, we test its ability to predict the tertiary and quaternary structure of a previously undescribed scaffold with new folds and unusual architecture, the monotopic membrane protein caveolin-1 (CAV1). CAV1 assembles into a disc-shaped oligomer composed of 11 symmetrically arranged protomers, each assuming an identical new fold, and contains the largest parallel {beta}-barrel known to exist in nature. Remarkably, AF2 predicts both the fold of the protomers and interfaces between them. It also assembles between 7 and 15 copies of CAV1 into disc-shaped complexes. However, the predicted multimers are energetically strained, especially the parallel {beta}-barrel. These findings highlight the ability of AF2 to correctly predict new protein folds and oligomeric assemblies at a granular level while missing some elements of higher order complexes, thus positing a new direction for the continued development of deep learning protein structure prediction approaches.

biophysics↗

bioRxiv2022

Structural characterization of a breast cancer-associated mutation in caveolin-1

Caveolin-1 (CAV1) is a membrane sculpting protein that oligomerizes to generate flask-shaped invaginations of the plasma membrane known as caveolae. Mutations in CAV1 have been linked to multiple diseases in humans. Such mutations often interfere with oligomerization and the intracellular trafficking processes required for successful caveolae assembly, but the molecular mechanisms underlying these defects have not been structurally explained. Here, we investigate how a breast cancer-associated mutation in one of the most highly conserved residues in CAV1, P132L, affects CAV1 structure and oligomerization. We show that P132 is positioned at a major site of protomer-protomer interactions within the CAV1 complex, providing a structural explanation for why the mutant protein fails to homo-oligomerize correctly. Using a combination of computational, structural, biochemical, and cell biological approaches, we find that despite its homo-oligomerization defects P132L is capable of forming mixed hetero-oligomeric complexes with wild type CAV1 and that these complexes can be incorporated into caveolae. These findings provide insights into the fundamental mechanisms that control the formation of homo- and hetero-oligomers of caveolins that are essential for caveolae biogenesis, as well as how these processes are disrupted in human disease.

biophysics↗

bioRxiv2022

First crystal structure of a non-canonical amino acid linked to a paramagnetic lanthanide tag facilitates protein structure determination using NMR-derived restraints

Site-directed spin labeling of proteins via non-canonical amino acids (ncAAs) is a non-traditional method for the measurement of pseudocontact shifts (PCSs) by nuclear magnetic resonance (NMR) spectroscopy. PCSs provide long-range distance and orientational information between a paramagnetic center and protein nuclei that can be used as restraints for computational structural modeling techniques. Here, we present the first experimental structure of an ncAA chemically linked to a lanthanide tag conjugated to the protein, T4-Lysozyme (T4L). T4L was crystallized with a cyclen-based C3 tag coordinated to the paramagnetic ion terbium (Tb3+). The paramagnetic C3-lanthanide tag generated PCSs measured at four different ncAA sites. We show that the addition of these restraints improves structure prediction protocols for T4L using the RosettaNMR framework. Generated models provide insight into T4L conformational flexibility sampled in solution. This integrative modeling protocol is readily transferable to larger proteins. Methods to predict protein structures are advancing into an exciting arena such that reliable experimental data will play important roles for evaluating the biophysical relevance of predicted structural models. Our contribution here caters to the growing interest in using ncAAs for a range of biophysical studies, and these methods can be readily transferred to larger protein systems of interest.

biophysics↗

bioRxiv2022

Molecular architecture of the human caveolin-1 complex

Membrane sculpting proteins shape the morphology of cell membranes and facilitate remodeling in response to physiological and environmental cues. Complexes of the monotopic membrane protein caveolin function as essential curvature-generating components of caveolae, flask-shaped invaginations that sense and respond to plasma membrane tension. However, the structural basis for caveolins membrane remodeling activity is currently unknown. Here, we show, using cryo-electron microscopy, that the human caveolin-1 complex is composed of 11 protomers organized into a tightly packed disc with a flat membrane-embedded surface. The structural insights suggest a new mechanism for how membrane sculpting proteins interact with membranes and reveal how key regions of caveolin-1, including its scaffolding, oligomerization, and intramembrane domains, contribute to its function. One-Sentence SummaryCryo-electron microscopy reveals that Caveolin-1 oligomerizes into a tightly packed disc with a flat membrane-binding surface.

biophysics↗

bioRxiv2022

Benchmarking Peptide Structure Prediction with AlphaFold2

AlphaFold2 (AF2) is a computational tool developed for the determination of protein structures with high accuracy. AF2 has been used for the modeling of many soluble and membrane proteins, but its performance in modeling peptide structures has not been systematically investigated so far. We benchmarked the accuracy of AF2 in predicting peptide structures between 16 - 60 amino acids using experimentally-determined peptide structures as reference. Our results show that while AF2 can predict the structures of certain peptide scaffolds with RMSD values below 3 [A], it is less successful in predicting the structures of peptides that have kinks, turns, or have extended flexible regions. Further, AF2 had several shortcomings in predicting rotamer recoveries, disulfide bonds, and the lowest RMSD structures based on pLDDT values. In summary, AF2 can be a powerful tool to determine peptide structures, but additional steps may be necessary to analyze and validate the results.

biophysics↗

bioRxiv2021

The allosteric activation of α7 nAChR by α-conotoxin MrIC is modified by mutations at the vestibular site

-conotoxins are 13-19 amino acid toxin peptides that bind various nicotinic acetylcholine receptor (nAChR) subtypes. -conotoxin Mr1.7c (MrIC) is a 17 amino acid peptide that targets 7 nAChR. Although MrIC has no activating effect on 7 nAChR when applied by itself, it evokes a large response when co-applied with the type II positive allosteric modulator PNU-120596, which potentiates 7 nAChR response by recovering it from a desensitized state. Lack of standalone activity despite activation upon co-application with a positive allosteric modulator was previously observed for molecules that bind to an extracellular domain allosteric activation (AA) site at the vestibule of the receptor. We hypothesized that MrIC may activate 7 nAChR allosterically through this site. We ran voltage-clamp electrophysiology experiments and in silico peptide docking calculations to gather evidence in support of 7 nAChR activation by MrIC through the AA site. The experiments with the wild-type 7 nAChR supported an allosteric mode of action, which was confirmed by the increased MrIC + PNU-120596 responses of three 7 nAChR AA site mutants that were designed in silico to improve MrIC binding. Overall, our results shed light on allosteric activation of 7 nAChR by MrIC and suggest involvement of the AA site. Significance Statement-conotoxin MrIC (MrIC) is an allosteric agonist of the 7 nicotinic acetylcholine receptor (nAChR). This mode of action is unique among -conotoxins since these peptides typically act as orthosteric antagonists of nAChR. However, the mechanism of 7 nAChR activation by MrIC has been elusive so far. This work demonstrates that activation by MrIC is independent of the 7 nAChR orthosteric site and is related to a vestibular allosteric activation site at the extracellular domain of the receptor. Our experimental and computational studies identified the residues that play a role in allosteric activation and confirmed the utility of ensemble docking methods in understanding peptide - nAChR interactions, thus providing a basis for the design of peptides for allosteric modulation of nAChR.

biophysics↗

bioRxiv2020

Prediction of amphipathic helix - membrane interactions with Rosetta

Amphipathic helices have hydrophobic and hydrophilic/charged residues situated on opposite faces of the helix. They can anchor peripheral membrane proteins to the membrane, be attached to integral membrane proteins, or exist as independent peptides. Despite the widespread presence of membrane-interacting amphipathic helices, there is no computational tool within Rosetta to model their interactions with membranes. In order to address this need, we developed the AmphiScan protocol with PyRosetta, which runs a grid search to find the most favorable position of an amphipathic helix with respect to the membrane. The performance of the algorithm was tested in benchmarks with the RosettaMembrane, ref2015_memb, and franklin2019 score functions on six engineered and 44 naturally-occurring amphipathic helices using membrane coordinates from the OPM and PDBTM databases, OREMPRO server, and MD simulations for comparison. The AmphiScan protocol predicted the coordinates of amphipathic helices within less than 3[A] of the reference structures and identified membrane-embedded residues with a Matthews Correlation Constant (MCC) of up to 0.57. Overall, AmphiScan stands as fast, accurate, and highly-customizable protocol that can be pipelined with other Rosetta and Python applications.

biophysics↗