Biology subjects

Fassler Bakhman, A.

Publications and source records attributed to Fassler Bakhman, A..

3 recordsLinked to original sources

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

Molecular determinants underlying differential recruitment of p115RhoGEF and PDZRhoGEF to activated Gα13

Heterotrimeric G proteins, particularly G12 and G13, are pivotal regulators of cellular signaling pathways. Their direct downstream effectors, which include p115RhoGEF and PDZRhoGEF, engage downstream signaling via Rho activation. Yet the molecular determinants that dictate their differential recruitment by G12/13 are not fully understood. Here, we combined quantitative computational residue-level analysis with site-directed mutagenesis and bioluminescence resonance energy transfer (BRET)-based assays to dissect G13 interactions with these RhoGEFs. We mapped the contributions of individual residues to binding and identified specific G13 residues in its helical domain, switch regions, and effector-binding site as key yet differential contributors to p115RhoGEF and PDZRhoGEF recruitment. Experimental validation with BRET confirmed that changes in many G13 residues impact p115RhoGEF more substantially than PDZRhoGEF, underscoring the specificity of G13 interactions with p115RhoGEF. Investigation of the p115RhoGEFs identified critical residues that contribute to interactions with G13 and G12. Our findings highlight residue-level differences in the molecular interactions of G13 with p115RhoGEF and PDZRhoGEF, providing insights into the specificity and regulation of G13-mediated signaling pathways. The resulting residue-level maps lay the groundwork for development of selective therapeutic strategies targeting G13-RhoGEF interactions.

biochemistry↗

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↗