Biology subjects

Nagy-Kanta, E.

Publications and source records attributed to Nagy-Kanta, E..

3 recordsLinked to original sources

bioRxiv2025

Redistribution of sidechain-sidechain interactions govern ligand-specific binding affinity changes in missense Shank1 PDZ mutants

Shank proteins represent a family of abundant scaffolds in the postsynaptic density. Their dysfunctions had been identified as possible causes behind autism spectrum disorders and various types of cancer. The remarkably promiscuous PDZ domain of the Shank family is highly conserved through isoforms, and contains a unique dynamic segment, the {beta}2-{beta}3 loop, located close to the binding site. We used the Shank1 PDZ as a model system to analyze the perturbing effects of five disease-associated missense mutations on the binding of different partner peptides. Using experimental methods and molecular dynamics simulations, we characterized the interactions in detail, focusing on their dynamic aspect. While the investigated variants in general weaken most interactions, the R736Q variant, unique in having increased thermal stability, also binds the GKAP peptide with higher affinity than the wild type. Overall, our results show that the perturbing effect of mutations is highly partner-specific and depends on the dynamic rearrangements of both uniformly occurring and ligand-specific residue-residue interactions.

molecular biology↗

bioRxiv2025

Diffusion-based size determination of solute particles: a method adapted for PSD proteins

The postsynaptic density (PSD) is a complex, multi-layered protein network largely situated on the internal surface of the postsynaptic membrane. It is the first processing unit for incoming synaptic transmissions, and changes in its internal structure are associated with synaptic strength and plasticity. These structural changes are largely governed by multivalent interactions between its components. The in vitro characterization of such complexes requires unbiased methods that can be used to estimate the size of the emerging assemblies for systems with multiple possible stoichiometries. Here we present an experimental method for detecting specific PSD proteins as well as their complexes based on their diffusion in a microfluidic environment. The method requires a fluorescent labelling technique that does not disrupt the function of labelled proteins, a microfluidic device that can maintain laminar flow for protein solutions, a microscope that can record the fluorescent signal emitted by these solutions, and an analytic software package that can process the collected experimental data and convert them into approximate particle sizes. We demonstrate the applicability of our method on protein constructs of various postsynaptic proteins, including the multivalent assembly between GKAP and LC8.

molecular biology↗

bioRxiv2024

Residual flexibility in the topologically constrained multivalent complex between the GKAP scaffold and LC8 hub proteins

Guanylate kinase-associated protein (GKAP) is a large postsynaptic scaffold protein bearing two closely spaced noncanonical binding sites for the bivalent dynein light chain LC8 hub protein. This might allow the formation of heterogeneous complexes with different sizes and topologies. Here, we show that a well-defined hexameric complex is formed, composed of 2 GKAP molecules and 2 LC8 dimers. Using NMR spectroscopy, we demonstrate that the LC8-binding segment of GKAP is intrinsically disordered and the flexibility of the linker region is largely retained even in the complex form. Molecular dynamics calculations suggest that besides the tightly bound residues, the hexamer also exhibits several dynamically interchanging interactions. The flanking regions of the two binding sites on GKAP exhibit different interaction patterns, hinting at additional contacts that might explain the fixed stoichiometry of the assembly. Our results demonstrate that constrained stoichiometry can coexist with substantial flexibility in a multivalent system.

molecular biology↗