Biology subjects

Slusky, J. S. G.

Publications and source records attributed to Slusky, J. S. G..

11 recordsLinked to original sources

bioRxiv2026

Human Metalloenzymes Are Disproportionately Linked to Disease: The HEAM Structural Annotation Resource

Catalytic metal-binding sites are established drug targets for many human diseases, yet systematic study of how metal function relates to disease is limited by the lack of a resource combining structural localization of human metal-binding sites with catalytic annotation at scale. We introduce HEAM (the Human Enzymatic Annotation of Metalloproteins), a structurally annotated dataset of 1,933 human metalloenzymes and 1,171 non-catalytic metalloproteins. HEAM incorporates annotated metals into protein structures at their corresponding binding sites and classifies each site as catalytic or non-catalytic using the machine learning classifier MAHOMES II. HEAM more than quadruples the human site-level coverage of the largest prior metalloenzyme database. Using HEAM, we show that human metalloproteins are significantly enriched in KEGG disease pathways relative to the human proteome, and that metalloenzymes specifically are significantly enriched in ClinVar pathogenic variants -- a distinction obscured in prior, catalytically unresolved metalloprotein datasets. These pathogenic variants are concentrated in metabolic disorders, indicating that metabolic dysfunction is a substantial contributor to metalloenzyme-linked disease. By pairing 3D structural annotation with catalytic classification, HEAM provides both a foundation for structure-guided drug design against metal-binding sites and a resource for determining how catalytic and non-catalytic metal function contribute to human disease.

biophysics↗

bioRxiv2026

COB: a comprehensive database of chloroplast outer envelope beta-barrel proteins

Despite their central role in metabolite exchange, lipid trafficking, and protein import, chloroplast outer envelope beta-barrel proteins lack a dedicated comprehensive sequence database spanning many plant proteomes. Here we present the database COB (chloroplast outer-envelope beta-barrel), consisting of 16,586 beta-barrel sequences organized across ten protein categories and an uncharacterized group. COB was constructed using a machine learning classifier that identifies chloroplast beta-barrels based on features derived from evolutionary protein contact maps. Analysis of COB reveals that Streptophyta have more barrels overall and use a greater variety of solute transporters than Chlorophyta. Furthermore, we find considerable structural diversity across OEP categories, including variation in beta-strand count and a high prevalence of open barrel conformations not observed in bacterial outer membrane proteins. Structure predictions for Arabidopsis thaliana outer envelope proteins identified candidate hybrid barrel assemblies, with TOC159 family members emerging as universal interaction partners. We also report single-chain multi-barrel domain architectures in the chloroplast outer envelope, a topology previously described only in Gram-negative bacteria. Finally, we find chloroplast membrane barrels have more open topologies and shorter strands than bacterial membrane barrels. COB provides a comprehensive sequence resource for chloroplast outer envelope beta-barrels and establishes a foundation for investigating the evolution, structure, and function of this essential protein in chloroplast.

plant biology↗

bioRxiv2023

Evolutionary engineering a larger porin using a loop-to-hairpin mechanism

In protein evolution, diversification is generally driven by genetic duplication. The hallmarks of this mechanism are visible in the repeating topology of various proteins. In outer membrane {beta}-barrels, duplication is visible with {beta}-hairpins as the repeating unit of the barrel. In contrast to the overall use of duplication in diversification, a computational study hypothesized evolutionary mechanisms other than hairpin duplications leading to increases in the number of strands in outer membrane {beta}-barrels. Specifically, the topology of some 16- and 18-stranded {beta}-barrels appear to have evolved through a loop to {beta}-hairpin transition. Here we test this novel evolutionary mechanism by creating a chimeric protein from an 18-stranded {beta}-barrel and an evolutionarily related 16-stranded {beta}-barrel. The chimeric combination of the two was created by replacing loop L3 of the 16-stranded barrel with the sequentially matched transmembrane {beta}-hairpin region of the 18-stranded barrel. We find the resulting chimeric protein is stable and has characteristics of increased strand number. This study provides the first experimental evidence supporting the evolution through a loop to {beta}-hairpin transition. HighlightsO_LIWe find evidence supporting a novel diversification mechanism in membrane {beta}-barrels C_LIO_LIThe mechanism is the conversion of an extracellular loop to transmembrane {beta}-hairpin C_LIO_LIA chimeric protein modeling this mechanism folds stably in the membrane C_LIO_LIThe chimera has more {beta}-structure and a larger pore, consistent with a loop-to-hairpin transition C_LI

biochemistry↗

bioRxiv2023

MAHOMES II: A webserver for predicting if a metal binding site is enzymatic

Recent advances have enabled high-quality computationally generated structures for proteins with no solved crystal structures. However, protein function data remains largely limited to experimental methods and homology mapping. Since structure determines function, it is natural that methods capable of using computationally generated structures for functional annotations need to be advanced. Our laboratory recently developed a method to distinguish between metalloenzyme and non-enzyme sites. Here we report improvements to this method by upgrading our physicochemical features to alleviate the need for structures with sub-angstrom precision and using machine learning to reduce training data labeling error. Our improved classifier identifies protein bound metal sites as enzymatic or non-enzymatic with 94% precision and 92% recall. We demonstrate that both adjustments increased predictive performance and reliability on sites with sub-angstrom variations. We constructed a set of predicted metalloprotein structures with no solved crystal structures and no detectable homology to our training data. Our model had an accuracy of 90 - 97.5% depending on the quality of the predicted structures included in our test. Finally, we found the physicochemical trends that drove this models successful performance were local protein density, second shell ionizable residue burial, and the pockets accessibility to the site. We anticipate that our models ability to correctly identify catalytic metal sites could enable identification of new enzymatic mechanisms and improve de novo metalloenzyme design success rates. Significance statementIdentification of enzyme active sites on proteins with unsolved crystallographic structures can accelerate discovery of novel biochemical reactions, which can impact healthcare, industrial processes, and environmental remediation. Our lab has developed an ML tool for predicting sites on computationally generated protein structures as enzymatic and non-enzymatic. We have made our tool available on a webserver, allowing the scientific community to rapidly search previously unknown protein function space.

bioinformatics↗

bioRxiv2022

Detergent headgroups control TolC folding in vitro

TolC is the trimeric outer membrane component of the efflux pump system in E. coli responsible for antibiotic efflux from bacterial cells. Over-expression of efflux pumps has been reported to decrease susceptibility to antibiotics in a variety of bacterial pathogens. Reliable production of membrane proteins allows for the biophysical and structural characterization needed to better understand efflux and for the development of therapeutics. Preparation of recombinant protein for biochemical/structural studies often involves the production of proteins as inclusion body aggregates from which bioactive proteins are recovered. Here we find that the in vitro folding of TolC into its functional trimeric state from inclusion bodies is dependent on the headgroup composition of detergent micelles used. Nonionic detergent favors the formation of functional trimeric TolC, whereas zwitterionic detergents induce the formation of a non-native trimeric TolC fold. We also find that nonionic detergents with shorter alkyl lengths facilitate TolC folding. It remains to be seen whether the charges in lipid headgroups have similar effects on membrane insertion and folding in biological systems.

biophysics↗

bioRxiv2021

Machine Learning Differentiates Enzymatic and Non-Enzymatic Metals in Proteins

Metalloenzymes are 40% of all enzymes and can perform all seven classes of enzyme reactions. Because of the physicochemical similarities between the active sites of metalloenzymes and inactive metal binding sites, it is challenging to differentiate between them. Yet distinguishing these two classes is critical for the identification of both native and designed enzymes. Because of similarities between these two types of metal binding sites, finding physicochemical features that distinguish active and inactive metal sites can indicate aspects that are critical to enzyme function. In this work, we develop the largest structural dataset of enzymatic and non-enzymatic metalloprotein sites to date. We then use a decision-tree ensemble machine learning model to classify metals bound to proteins as enzymatic or non-enzymatic with 92.2% precision and 90.1% recall. Our model scores electrostatic and pocket lining features as more important than pocket volume, despite the fact that volume is the most quantitatively different feature between enzyme and non-enzymatic sites. Finally, we find our model has overall better performance in a side-to-side comparison against other methods that differentiate enzymatic from non-enzymatic sequences. We anticipate that our models ability to correctly identify which metal sites are responsible for enzymatic activity could enable identification of new enzymatic mechanisms and de novo enzyme design.

biophysics↗

bioRxiv2021

Membrane barrels are taller, fatter, inside-out soluble barrels

Up-and-down {beta}-barrel topology exists in both the membrane and soluble environment. However, {beta}-barrels are virtually the only topology that exist in the outer membrane. By comparing features of these structurally similar proteins, we can determine what features are particular to the environment rather than the fold. Here we compare structures of membrane {beta}-barrels to soluble {beta}-barrels and evaluate their relative size, shape, amino acid composition, hydrophobicity, and periodicity. We find that membrane {beta}-barrels are generally larger than soluble {beta}-barrels in with more strands per barrel and more amino acids per strand, making them wider and taller. We also find that membrane {beta}-barrels are inside-out soluble {beta}-barrels. The inward region of membrane {beta}-barrels have similar hydrophobicity to the outward region of soluble {beta}-barrels, and the outward region of membrane {beta}-barrels has similar hydrophobicity to the inward region of the soluble {beta}-barrels. Moreover, even though both types of {beta}-barrel have been assumed to have strands with amino acids that alternate in direction and hydrophobicity, we find that the membrane {beta}-barrels have more regular alternation than soluble {beta}-barrels. These features give insight into how membrane barrels maintain their fold and function in the membrane.

biophysics↗

bioRxiv2020

Redesigning OmpA Loops Using Canonical Outer Membrane Protein Loop Structures

Outer membrane proteins are all beta barrels and these barrels have a variety of well-documented loop conformations. Here we test the effect of three different loop types on outer membrane protein A (OmpA) folding. We designed twelve 5-residue loops and experimentally tested the effect of replacing the long loops of outer membrane protein OmpA with the designed loops. Our studies succeeded in creating the smallest known outer membrane barrel. We find that significant changes in OmpA loops do not have a strong overall effect on OmpA folding. However, when decomposing folding into a fast rate and a slow rate we find that changes in loops strongly affect the slow rate of OmpA folding. Extracellular loop types with higher levels of hydrogen bonds had more instances of increasing the slow folding rate and extracellular loop types with low levels of hydrogen bonds had more instances of decreasing the slow folding rate. Having the slow rate affected by loop composition is consistent with the slow rate being associated with the insertion step of outer membrane protein folding.

biophysics↗

bioRxiv2020

High yield preparation of outer-membrane protein efflux pumps by in vitro refolding is concentration dependent

Overexpression of tripartite efflux pump systems in gram-negative bacteria are a principal component of antibiotic resistance. High-yield purification of the outer membrane component of these systems will enable biochemical and structural interrogation of their mechanisms of action and allow testing of compounds that target them. However, preparation of these proteins is typically hampered by low yields requiring laborious large-scale efforts. If refolding conditions can be found, refolding these proteins from inclusion bodies can lead to increased yields as compared to membrane isolations. Here, we develop a concentration-dependent folding protocol for refolding TolC, the outer membrane component of the antibiotic efflux pump from Escherichia coli. We show that by our method of re-folding, homotrimeric TolC remains folded in SDS-PAGE, retains binding to an endogenous ligand, and recapitulates the known crystal structure by single particle cryoEM analysis. We find that a key factor in successful re-folding is a concentration dependence of TolC oligomerization. We extended the scheme to CmeC, a homologous protein from Campylobacter jejuni, and find that concentration-dependent oligomerization is a general feature of these systems. Because outer-membrane efflux pump components are ubiquitous across gram-negative species, we anticipate that incorporating a concentration step in re-folding protocols will promote correct refolding allowing for reliable, high-yield preparation of this family of proteins.

biophysics↗

bioRxiv2019

Colicin E1 Fragments Potentiate Antibiotics by Plugging TolC

The double membrane architecture of Gram-negative bacteria forms a barrier that is effectively impermeable to extracellular threats. Bacteriocin proteins evolved to exploit the accessible, surface-exposed proteins embedded in the outer membrane to deliver cytotoxic cargo. Colicin E1 is a bacteriocin produced by, and lethal to, Escherichia coli that hijacks the outer membrane proteins TolC and BtuB to enter the cell. Here we capture the colicin E1 translocation domain inside its membrane receptor, TolC, by high-resolution cryoEM, the first reported structure of a bacteriocin bound to TolC. Colicin E1 binds stably to TolC as an open hinge through the TolC pore--an architectural rearrangement from colicin E1s unbound conformation. This binding is stable in live E. coli cells as indicated by single-molecule fluorescence microscopy. Finally, colicin E1 fragments binding to TolC plugs the channel, inhibiting its native efflux function as an antibiotic efflux pump and heightening susceptibility to three antibiotic classes. In addition to demonstrating that these protein fragments are useful starting points for developing novel antibiotic potentiators, this method could be expanded to other colicins to inhibit other outer membrane protein functions.

biophysics↗

bioRxiv2018

Efflux Pumps Represent Possible Evolutionary Convergence onto the Beta Barrel Fold

There are around 100 types of integral outer membrane proteins in each Gram negative bacteria. All of these proteins have the same fold--an up-down {beta}-barrel. It has been suggested that all membrane {beta}-barrels other than lysins are homologous. Here we suggest that {beta}-barrels of efflux pumps have converged on this fold as well. By grouping structurally-solved outer membrane {beta}-barrels (OMBBs) by sequence we find evidence that the membrane environment may have led to convergent evolution of the barrel fold. Specifically, the lack of sequence linkage to other barrels coupled with distinctive structural differences, such as differences in strand tilt and barrel radius, suggest that efflux pumps have evolutionarily converged on the barrel. Finally, we find a possible ancestor for the OMBB efflux pumps as they are related to periplasmic components of the same pumps.

biophysics↗