Biology subjects

Kolodny, R.

Publications and source records attributed to Kolodny, R..

12 recordsLinked to original sources

bioRxiv2026

Nucleotide-binding motifs nucleated folding of the first enzymes

The earliest stages of protein evolution remain a mystery: the nature of the first protein forms, their roles in emergent biological systems, and the forces that shaped them are largely unknown. Here, we combine insights from metabolic modelling, the organization of protein structure space, and protein folding mechanisms to probe the emergence of two ubiquitous cofactor- binding folds: Rossmanns and P-loop NTPases. While both folds are essential for contemporary life, we show that Rossmanns catalyze reactions deeper within the metabolic core and are more central in structure space than P-loop NTPases. Folding mechanism analysis further reveals that, whereas P-loop NTPases may require non-local interactions to fold, Rossmann folding can be nucleated by a structural module at the heart of the fold that contains a nucleotide-binding motif. Because this motif also directly mediates biochemical activity, this result suggests how early proteins may have compactly satisfied both folding and biochemical activity. Our results imply that folding constraints favored early enzymatic forms with compact binding motifs and modest catalytic roles. We conclude that the early emergence of the Rossmann fold reflects the chemical and physical constraints of protein folding, explaining both its profound antiquity and sustained longevity.

biochemistry↗

bioRxiv2025

Contrastive learning unites sequence and structure in a global representation of protein space

Amino acid sequence dictates the three-dimensional structure and biological function of proteins. Yet, despite decades of research, our understanding of the interplay between sequence and structure is incomplete. To meet this challenge, we introduce Contrastive Learning Sequence-Structure (CLSS), an AI-based contrastive learning model trained to co-embed sequence and structure information in a self-supervised manner. We trained CLSS on large and diverse sets of protein building blocks called domains. CLSS represents both sequences and structures as vectors in the same high-dimensional space, where distance relates to sequence-structure similarity. Thus, CLSS provides a natural way to represent the protein universe, reflecting evolutionary relationships, as well as structural changes. We find that CLSS refines expert knowledge about the global organization of protein space, and highlights transitional forms that resist hierarchical classification. CLSS reveals linkage between domains of seemingly separate lineages, thereby significantly improving our understanding of evolutionary design.

bioinformatics↗

bioRxiv2025

Structural conservation and divergence across the Receptor Tyrosine Kinase superfamily

Members of the Receptor Tyrosine Kinase (RTK) superfamily are regulators of cellular signaling, playing essential roles in cellular growth, differentiation, and survival. Dysregulation of RTKs leads to diseases such as cancer, diabetes, and inflammatory disorders, making them important therapeutic targets. Despite extensive research on RTKs, the structural diversity and evolutionary relationships across the superfamily are not fully understood. Here, we systematically compared structural conservation and divergence among 245 extracellular domains from 54 RTKs, across 18 RTK families. Using experimentally-resolved structures and AlphaFold2 models, we conducted an all-versus-all structural alignment to explore domain architecture and quantify significant structural similarities within the RTK superfamily. We curated a comprehensive database encompassing PDB structures, 3D folds, ligand-binding properties, and sequence information of all RTK domains analyzed (https://fasslero.github.io/RTK-domains). Our analysis revealed numerous inter-family similarities and remote evolutionary connections, in particular among ligand-binding domains (LBDs), and distinct structural domain types and unexpected dissimilarities among domains previously classified as related. Our work highlights the intricate balance between structural conservation and divergence in RTKs and sheds light on the evolutionary mechanisms shaping this critical superfamily.

bioinformatics↗

bioRxiv2024

Structure-based mechanistic principles for the paradoxical effects of pathological RET mutations

The RET receptor is a transmembrane protein that belongs to the receptor tyrosine kinase family. RET activates signaling pathways regulating cell growth, differentiation, and survival in diverse tissues that include the thyroid and enteric nervous system. Mutations that reduce RET levels or disrupt function can cause Hirschsprungs disease (HSCR), characterized by abnormal distal colon innervation in the developing embryo. In contrast, mutations that constitutively activate RET can lead to tumorigenesis, most notably in multiple endocrine neoplasia type 2 (MEN2) syndromes usually involving the thyroid. Paradoxically, some RET mutations can both reduce RET activity in enteric ganglia, and increase signaling in other tissues, resulting in patients suffering from both HSCR and MEN2A. Although extensive research has been conducted on RET mutations, the structural and mechanistic bases underlying these paradoxical effects remain unclear. Here, we curated data on 70 positions in RET extracellular domains where point-mutations were associated with HSCR, MEN2A, or both. Taking a structure-based approach, we predict the potential effects of mutations in these positions on RET structure. Our analysis suggests that approximately 90% of positions associated with HSCR can, upon mutation, disrupt intramolecular interactions that stabilize RET tertiary structure: residues buried in the protein core, calcium-binding sites, or residues participating in stabilizing intramolecular electrostatic/covalent bonds. A smaller subset of mutations involves substitutions to/from glycines or prolines in key positions. Only a small minority of HSCR-associated positions affect protein-protein interactions needed for signal activation. On the other hand, our analysis showed that [~]75% of mutations in positions that cause MEN2A lead to an unpaired cysteine that can form an intermolecular disulfide bond between two RET monomers. Other mutations that cause MEN2A are also predicted to enhance RET homodimerization via extracellular domains that are proximal to the membrane. Importantly, substitutions that concurrently destabilizes RET tertiary structure and lead to an unpaired cysteine are predicted to cause the paradoxical co-occurrence of HSCR and MEN2A. Our findings suggest a mechanistic basis for almost all identified pathological mutations in RET and imply that therapeutic strategies for targeting RET activity in HSCR and MEN2A may need to be orthogonal.

bioinformatics↗

bioRxiv2024

Predicting gene sequences with AI to study evolutionarily selected codon usage patterns

Selective pressure acts on the codon use, optimizing multiple, overlapping signals that are only partially understood. We trained artificial intelligence (AI) models to predict the codons given their amino acid sequence in the eukaryotes Saccharomyces cerevisiae and Schizosaccharomyces pombe and the bacteria Escherichia coli and Bacillus subtilis, to study the extent to which we can learn patterns in naturally occurring codons to improve predictions. We trained our models on a subset of the proteins, and evaluated their predictions on large, separate sets of proteins of varying lengths and expression levels. Our models significantly outperformed naive frequency-based approaches, demonstrating that there are dependencies between codons that can be learned to better predict evolutionary-selected codon usage. The prediction accuracy advantage of our models is greater for highly expressed genes and it is greater in bacteria than eukaryotes, supporting the hypothesis that there is a monotonic relationship between selective pressure for complex codon patterns and effective population size. Also, in S. cerevisiae and bacteria, our models were more accurate for longer proteins, suggesting that the AI system may have learned patterns related to co-translational folding. Gene functionality and conservation were also important determinants that affect the performance of our models. Finally, we showed that using information encoded in homologous proteins has only a minor effect on prediction accuracy, perhaps due to complex codon-usage codes in genes undergoing rapid evolution. In summary, our study employing contemporary AI methods offers a new perspective on codon usage patterns and a novel tool to optimize codon usage in endogenous and heterologous proteins. Significance statementCan one predict codon sequences used by an organism to encode a given amino acid sequence? This is difficult, because there are exponentially many codon sequences that can encode the same amino acid sequence and evolution is stochastic. Indeed, codons frequencies vary, a phenomenon known as codon-bias, yet we improve upon frequency-based predictions using contemporary AI tools that learn complex patterns and capture interactions between codons. Because our predictions are tested fairly, on cases not seen during the training process, accurate predictions suggest that these learned patterns are not random, and may be related to the evolutionary process. Thus, studying where our predictions are more accurate, is expected to reveal novel insights related to the way evolution shapes coding regions.

evolutionary biology↗

bioRxiv2022

Proteins with multiple G protein-coupled receptor domains

Currently known G protein-coupled receptors (GPCRs) have a single transmembrane domain. Many GPCRs form dimers that have two transmembrane domains (one per protein), and there are indications that this dimeric interaction is functionally meaningful. Here, based on sequence analysis and structure predictions, we report the existence of 57 proteins with two, three, or four GPCR domains within the same protein chain. We analyze the structures of these multi-GPCRs and show that almost all have DRY/NPxxY motifs, a strong indication of signaling activity. By homology, most of the multi-GPCRs that we identified are olfactory-related; a few are chemokine-related. Multi-GPCR candidates are found in various Chordata species including fish, camel, marmite, Chinese hamster, and new world monkeys. The discovery of receptors with multiple transmembrane domains suggests the possibility for signal regulation and amplification within an individual receptor, revealing another step in GPCR evolution and a new layer of complexity in signal transduction.

bioinformatics↗

bioRxiv2021

Evidence for the Emergence of β-Trefoils by 'Peptide Budding' from an IgG-like β-Sandwich

As sequence and structure comparison algorithms gain sensitivity, the intrinsic interconnectedness of the protein universe has become increasingly apparent. Despite this general trend, {beta}-trefoils have emerged as an uncommon counterexample: They are an isolated protein lineage for which few, if any, sequence or structure associations to other lineages have been identified. If {beta}-trefoils are, in fact, remote islands in sequence-structure space, it implies that the oligomerizing peptide that founded the {beta}-trefoil lineage itself arose de novo. To better understand {beta}-trefoil evolution, and to probe the limits of fragment sharing across the protein universe, we identified both {beta}-trefoil bridging themes (evolutionarily-related sequence segments) and {beta}-trefoil-like motifs (structure motifs with a hallmark feature of the {beta}-trefoil architecture) in multiple, ostensibly unrelated, protein lineages. The success of the present approach stems, in part, from considering {beta}-trefoil sequence segments or structure motifs rather than the {beta}-trefoil architecture as a whole, as has been done previously. The newly uncovered inter-lineage connections presented here suggest a novel hypothesis about the origins of the {beta}-trefoil fold itself - namely, that it is a derived fold formed by budding from an Immunoglobulin-like {beta}-sandwich protein. These results demonstrate how the emergence of a folded domain from a peptide need not be a signature of antiquity and underpin an emerging truth: few protein lineages escape natures sewing table.

bioinformatics↗

bioRxiv2021

Gram-negative outer membrane proteins with multiple β-barrel domains

Outer membrane beta barrels (OMBBs) are found in the outer membrane of Gram-negative bacteria and eukaryotic organelles. OMBBs fold as antiparallel {beta}-sheets that close onto themselves, forming pores that traverse the membrane. Currently known structures include only one barrel, of 8-36 strands, per chain. The lack of multi-OMBB chains is surprising, as most OMBBs form oligomers and some function only in this state. Using a combination of sensitive sequence-comparison methods and co-evolutionary analysis tools, we identify many proteins combining multiple beta barrels within a single chain; combinations that include 8-stranded barrels prevail. These multi-barrels seem to be the result of independent, lineage-specific fusion and amplification events. The absence of multi-barrels that are universally conserved in bacteria with an outer membrane, coupled with their frequent de novo genesis suggests that their functions are not essential, but rather beneficial in specific environments. Adjacent barrels of complementary function within the same chain may allow for new functions beyond those of the individual barrels.

biophysics↗

bioRxiv2020

Bridging themes: short protein segments found in different architectures

The vast majority of theoretically possible polypeptide chains do not fold, let alone confer function. Hence, protein evolution from preexisting building blocks has clear potential advantages over ab initio emergence from random sequences. In support of this view, sequence similarities between different proteins is generally indicative of common ancestry, and we collectively refer to such homologous sequences as themes. At the domain level, sequence homology is routinely detected. However, short themes which are segments, or fragments of intact domains, are particularly interesting because they may provide hints about the emergence of domains, as opposed to divergence of preexisting domains, or their mixing-and-matching to form multi-domain proteins. Here we identified 525 representative short themes, comprising 20-to-80 residues, that are unexpectedly shared between domains considered to have emerged independently. Among these bridging themes are ones shared between the most ancient domains, e.g., Rossmann, P-loop NTPase, TIM-barrel, Flavodoxin, and Ferredoxin-like. We elaborate on several particularly interesting cases, where the bridging themes mediate ligand binding. Ligand binding may have contributed to the stability and the plasticity of these building blocks, and to their ability to invade preexisting domains or serve as starting points for completely new domains.

molecular biology↗

bioRxiv2020

On the Emergence of P-Loop NTPase and Rossmann Enzymes from a Beta-Alpha-Beta Ancestral Fragment

Dating back to the last universal common ancestor (LUCA), the P-loop NTPases and Rossmanns now comprise the most ubiquitous and diverse enzyme lineages. Intriguing similarities in their overall architecture and phosphate binding motifs suggest common ancestry; however, due to a lack of sequence identity and some fundamental structural differences, these families are considered independent emergences. To address this longstanding dichotomy, we systematically searched for bridge proteins with structure and sequence elements shared by both lineages. We detected homologous segments that span the first {beta}{beta} segment of both lineages and include two key functional motifs: (i) a phosphate binding loop - the Walker A motif of P-loop NTPases or the Rossmann equivalent, both residing at the N-terminus of 1; and (ii) an Asp at the tip of {beta}2. The latter comprises the Walker B aspartate that chelates the catalytic metal in P-loop NTPases, or the canonical Rossmann {beta}2-Asp that binds the cofactors ribose moiety. Tubulin, a Rossmann GTPase, demonstrates the potential of the {beta}2-Asp to take either one of these two roles. We conclude that common P-loops/Rossmann ancestry is plausible, although convergence cannot be completely ruled out. Regardless, both lineages most likely emerged from a polypeptide comprising a {beta}{beta} segment carrying the above two functional motifs, a segment that comprises the core of both enzyme families to this very day.

evolutionary biology↗

bioRxiv2018

Evolution of Environmentally-Enforced, Repeat Protein Topology in the Outer Membrane

Outer membrane beta barrels (OMBBs) are the proteins on the surface of Gram negative bacteria. These proteins have diverse functions but only a single topology, the beta barrel. It has been suggested that this common fold is a repeat protein with the repeating unit of a beta hairpin. By grouping structurally solved OMBBs by sequence, a detailed evolutionary story unfolds. A strand-number based pathway manifests with progression from a primordial 8-stranded barrel to 16-stranded and then to 18-stranded barrels. The transitions from 16- to 18-stranded barrels show mechanisms of strand number variation without domain duplication, such as a loop to hairpin transition. This indicates that repeat protein topology can be perpetuated without genetic duplication likely because the topology is being enforced by the membrane environment. Moreover, we find the evolutionary trace is particularly prominent in the C-terminal half of OMBBs which may be relevant to understanding OMBB folding pathways.

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↗