Biology subjects

Shkumatov, A. V.

Publications and source records attributed to Shkumatov, A. V..

5 recordsLinked to original sources

bioRxiv2025

Stereoselectivity and functional plasticity of a common ligand-binding pocket in TRPM3

The transient receptor potential melastatin 3 (TRPM3) channel is a key mediator of peripheral pain signaling, and pathogenic mutations in TRPM3 are linked to neurodevelopmental delay and epilepsy. Despite the therapeutic promise of TRPM3 modulators, the molecular mechanisms by which ligands modulate channel gating remain poorly understood. Here, we combine cryo-electron microscopy (cryo-EM) with functional analyses to characterize a promiscuous ligand-binding pocket formed by transmembrane helices S1-S4. This pocket accommodates several chemically diverse plant-derived and synthetic agonists and antagonists. We uncover unanticipated stereoselectivity of TRPM3 for the non-natural enantiomer of the flavonoid antagonist isosakuranetin and the (R)-enantiomer of the synthetic agonist CIM0216. Mutations within this pocket--including newly identified patient variants--alter ligand affinity and, in some cases, invert the functional outcome of ligand binding. These findings reveal the stereoselectivity and functional plasticity of the TRPM3 ligand-binding pocket, highlighting how subtle changes in the molecular interactions can produce divergent effects on channel gating, with important ramifications for TRPM3-targeted drug development and therapy.

biochemistry↗

bioRxiv2022

AFM-based force spectroscopy unravels the stepwise-formation of a DNA transposition complex driving multi-drug resistance dissemination

Transposon Tn4430 belongs to a widespread family of bacterial transposons, the Tn3 family, which plays a prevalent role in the dissemination of antibiotic resistance among pathogens. So far, the molecular mechanisms underlying the replicative transposition of these elements are still poorly understood. Here, we use force-distance curve-based atomic force microscopy to probe the binding of the TnpA transposase of Tn4430 to DNA molecules containing one or two transposon ends and to extract the thermodynamic and kinetic parameters of transposition complex assembly. Comparing wild-type TnpA with previously isolated deregulated TnpA mutants supports a stepwise pathway for transposition complex formation and activation during which TnpA first binds to a single transposon end and then undergoes a structural transition that enables it to bind the second end co-operatively and to become activated for transposition catalysis. Our study thus provides an unprecedented approach to probe the dynamic of a complex DNA processing machinery at the single-particle level.

biophysics↗

bioRxiv2022

Metamorphism of catalytic domain controls transposition in Tn3 family transposases

Transposons account for a remarkable diversity of mobile genetic elements that play the role of genome architects in all domains of life. Tn3 is a family of widespread and among first identified bacterial transposons notorious for their contribution to dissemination of antibiotic resistance. Transposition within this family is mediated by a large TnpA transposase facilitating both transposition and target immunity. The structural framework for understanding the mechanism of the TnpA transposition is however absent. Here, we describe the cryo-EM structures of TnpA from Tn4430 in apo form and paired with transposon ends. We show that TnpA has a unique architecture and exhibits a family-specific regulation mechanism involving metamorphic refolding of the RNase H-like catalytic domain. The TnpA structure, constrained by a double dimerization interface, creates a unique topology that suggests a specific role for target DNA in transpososome assembly, activation, and target immunity.

molecular biology↗

bioRxiv2020

Structural basis of SALM3 dimerization and synaptic adhesion complex formation with PTPσ

Synaptic adhesion molecules play an important role in the formation, maintenance and refinement of neuronal connectivity. Recently, several leucine rich repeat (LRR) domain containing neuronal adhesion molecules have been characterized including netrin G-ligands, SLITRKs and the synaptic adhesion-like molecules (SALMs). Dysregulation of these adhesion molecules have been genetically and functionally linked to various neurological disorders. Here we investigated the molecular structure and mechanism of ligand interactions for the postsynaptic SALM3 adhesion protein with its presynaptic ligand, receptor protein tyrosine phosphatase {sigma} (PTP{sigma}). We solved the crystal structure of the dimerized leucine rich repeat (LRR) domain of SALM3, revealing the conserved structural features and mechanism of dimerization. Furthermore, we determined the complex structure of SALM3 with PTP{sigma} using small angle X-ray scattering, revealing a 2:2 complex similar to that observed for SALM5. Solution studies unraveled additional flexibility for the complex structure, but validated the uniform mode of action for SALM3 and SALM5 to promote synapse formation. The relevance of the key interface residues was further confirmed by mutational analysis with cellular binding assays and artificial synapse formation assays. Collectively, our results suggest that SALM3 dimerization is a pre-requisite for the SALM3-PTP{sigma} complex to exert synaptogenic activity.

biochemistry↗

bioRxiv2017

The structure of SALM5 suggests a dimeric assembly for the presynaptic RPTP ligand recognition

Synaptic adhesion molecules play a crucial role in the regulation of synapse development and maintenance. Recently several families of leucine rich repeat domain containing neuronal adhesion molecules have been characterized, including netrin G-ligands, LRRTMs, and the SALM family proteins. Most of these are expressed at the excitatory glutamatergic synapses, and dysfunctions of these genes are genetically linked with cognitive disorders, such as autism spectrum disorders and schizophrenia. The SALM family proteins SALM3 and SALM5, similar to SLITRKs, have been shown to bind to the presynaptic receptor protein tyrosine phosphatase (RPTP) family ligands. Here we present the 3 [A] crystal structure of the SALM5 LRR-Ig domain construct, and biophysical studies that verify the crystallographic results. We show that both SALM3 and SALM5 extracellular domains form similar dimeric structures, in which the LRR domains form the dimer interface. Both proteins bind to the RPTP lg-domains with micromolar affinity. SALM3 shows a clear preference for RPTP-ligands with the meB splice insert. This is in accordance with previous results showing that the LRR domain is also required for the ligand binding. Our structural studies and sequence conservation analysis suggests a ligand binding site and mechanism for RPTP binding via the dimeric LRR domain region.

biochemistry↗