Biology subjects

Gaspari, Z.

Publications and source records attributed to Gaspari, Z..

13 recordsLinked to original sources

bioRxiv2025

Modulation of the internal dynamics of the Homer1 EVH1 domain by putative autism-associated mutations

The EVH1 domain of the Homer1 scaffold protein interacts with the proline-rich region of Shank3, forming a key network within the postsynaptic density. Two mutations in EVH1, M65I and S97L, were suggested to be associated with autism spectrum disorder. Combining experimental and computational investigations, we show here that neither the overall structure nor the partner binding properties of the mutants exhibit substantial changes relative to the wild type. Compared to the S97L variant, the M65I mutant exhibits larger chemical shift perturbations both upon the mutation itself and during partner binding, and also shows signs of thermal destabilization. Integration of computational and NMR investigations suggests that both mutations perturb the s-ms time scale internal motions of the EVH1 domain.

molecular biology↗

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

Structural modelling and dynamics of the full-length Homer1 multimer

Abstract/SummaryHomer proteins are modular scaffold molecules that constitute an integral part of the protein network within the postsynaptic density. Full-length Homer1 forms a large homotetramer via a long coiled coil region, and can interact with proline-rich target sequences with its globular EVH1 domain. Here we report an atomistic model of the full-length Homer1 tetramer along with the NMR solution structure of its EVH1 domain. Compared to the already available EVH1 structures, our NMR ensemble exhibits subtle differences, mostly in and around its partner binding region, suggesting the presence of ligand-induced conformational transitions. Molecular dynamics simulations of the long coiled coil reveal distinct regions with different stability and flexibility, with the N-terminal part of the coiled coil exhibiting the largest motions. Interestingly, this segment is highly conserved, pointing to the functional relevance of the observed dynamical features. Our results indicate previously unexplored aspects of the flexibility of the full-length Homer1 tetramer that might contribute to the dynamic rearrangements of the postsynaptic protein network linked to its functional transitions.

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↗

bioRxiv2023

Simulated complexes formed from a set of postsynaptic proteins suggest a localised effect of a hypomorphic Shank mutation

The postsynaptic density is an elaborate protein network beneath the postsynaptic membrane built up from the same major proteins but differing between synapses in exact composition and organization. Mutations perturbing generally occurring protein:protein interactions in this network can lead to specific effects, and the translation of biochemical effects to the system level can be especially challenging. In this work we use systems biology-based modeling of protein complex distributions in a set of major postsynaptic proteins including either a wild-type or a hypomorphic PDZ mutant Shank protein. Our results suggest that the effect of such mutations is heavily dependent on the overall availability of the protein components of the whole network. We simulated protein complex formation based on experimental data of various cell types with diverse protein abundances. The simulations revealed that mutations interfering with a single protein interaction can lead to protein complex changes only in specific cell types and in specific complexes. Although our system is currently far from a comprehensive description of the PSD, the results suggest a way to interpret specific mutations within a complex framework. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC="FIGDIR/small/562557v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@fdf273org.highwire.dtl.DTLVardef@3891deorg.highwire.dtl.DTLVardef@1f0c37borg.highwire.dtl.DTLVardef@1b1e629_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIEffect of mutations affecting protein:protein interactions can be modeled in context C_LIO_LIChanges in emerging protein complexes upon mutations are dependent on the availability of the individual proteins C_LIO_LIOur model provides conceptual explanation of confided phenotypic effects of a mutation C_LI

systems biology↗

bioRxiv2021

Charged sequence motifs increase affinity towards liquid-liquid phase separation

Protein phase separation is a major governing factor in multiple cellular processes, such as those concerning RNA metabolism and RNA-binding proteins. Despite many key observations, the exact structural characteristics of proteins involved in the process are still not fully deciphered. In this work we show that proteins harbouring sequence regions with specific charged residue patterns are significantly associated with liquid-liquid phase separation. In particular, regions with repetitive arrays of alternating charges show the strongest association, whereas segments with generally high charge density and single -helices also show detectable but weaker connections.

bioinformatics↗

bioRxiv2021

PSINDB: A comprehensive database of postsynaptic protein-protein interactions

The postsynaptic region is the receiving part of the synapse comprising thousands of proteins forming an elaborate and dynamically changing network indispensable for the molecular mechanisms behind fundamental phenomena such as learning and memory. Despite the growing amount of information about individual protein-protein interactions in this network, these data are mostly scattered in the literature or are stored in generic databases that are not designed to display aspects which are fundamental to understanding postsynaptic functions. To overcome these limitations we collected postsynaptic protein-protein interactions (PPIs) together with a high amount of detailed structural and biological information and launched a freely available resource, the Postsynaptic Interaction Database (PSINDB) to make these data and annotations accessible. PSINDB includes tens of thousands of binding regions together with structural features mediating and regulating the formation of PPIs, annotated with detailed experimental information about each interaction. PSINDB is expected to be useful for numerous aspects of molecular neurobiology research, from experiment design to network and systems biology-based modeling and analysis of changes in the protein network upon various stimuli. PSINDB is available at http://psindb.itk.ppke.hu/.

bioinformatics↗

bioRxiv2021

A preliminary study on the cistrome of human postsynaptic density from an evolutionary and network-based perspective

The postsynaptic density (PSD) is a neuronal organelle that consists of thousands of protein complexes, having a role in signal transduction. The emergence of the complexes is dependent on the presence of proteins provided by gene expression. In this research we used Chip-seq data supported by protein level information. We developed a pipeline using data from five neuronal transcription factors, which reduces the false-positive hits of identified binding sites. In addition we found correlation between co-regulation and protein complex formation. The developed method paves the way for a future for large scale analysis utilizing a more comprehensive set of transcription factors.

bioinformatics↗

bioRxiv2020

Distribution of disease-causing germline mutations in coiled-coils suggests essential role of their N-terminal region

Next-generation sequencing resulted in the identification of a huge number of naturally occurring variations in human proteins. The correct interpretation of the functional effects of these variations necessitates the understanding of how they modulate protein structure. Coiled-coils are -helical structures responsible for a diverse range of functions, but most importantly, they facilitate the structural organization of macromolecular scaffolds via oligomerization. In this study, we analyzed a comprehensive set of disease-associated germline mutations in coiled-coil structures. Our results highlight the essential role of residues near the N-terminal part of coiled-coil regions, possibly critical for superhelix assembly and folding in some cases. We also show that coiled-coils of different oligomerization states exhibit characteristically distinct patterns of disease-causing mutations. Our study provides structural and functional explanations on how disease emerges through the mutation of these structural motifs.

bioinformatics↗

bioRxiv2019

Interdependence of intra- and inter-domain motions in the PSD-95 PDZ12 tandem

PSD-95 is the most abundant scaffold protein in the postsynaptic density of neurons. Its two N-terminal PDZ domains form an autonomous structural unit and their interdomain orientation and dynamics was shown to be dependent on binding to various partner proteins. To understand the mechanistic details of the effect of ligand binding on interdomain structure and dynamics, we generated conformational ensembles using experimentally determined NOE interatomic distances and S2 order parameters, available from the literature. In our approach no explicit restraints between the two domains were used and their fast dynamics was also treated independently. We found that intradomain structural changes induced by ligand binding have a profound effect on the interfaces where interdomain contacts can be formed, modulating the probability of the occurrence of specific domain-domain orientations. Our results suggest that the {beta}2-{beta}3 loop in the PDZ domains is a key regulatory region that, through interacting with the upstream residues of the C-terminal peptide ligand, influences both intradomain motions and supramodular rearrangement.

bioinformatics↗

bioRxiv2018

Multiple polyvalency provided by intrinsically disordered segments is a key feature of postsynaptic scaffold proteins

The human postsynaptic density is an elaborate network comprising thousands of proteins, playing a vital role in the molecular events of learning and the formation of memory. Despite our growing knowledge of specific proteins and their interactions, atomic-level details of their full three-dimensional structure and their rearrangements are mostly elusive. Advancements in structural bioinformatics enabled us to depict the characteristic features of proteins involved in different processes aiding neurotransmission. We show that postsynaptic protein-protein interactions are mediated through the delicate balance of intrinsically disordered regions and folded domains, and this duality is also imprinted in the amino acid sequence. We introduce Diversity of Potential Interactions (DPI), a structure and regulation based descriptor to assess the diversity of interactions. Our approach reveals that the postsynaptic proteome has its own characteristic features and these properties reliably discriminate them from other proteins of the human proteome. Our results suggest that postsynaptic proteins are especially susceptible to forming diverse interactions with each other, which might be key in the reorganization of the PSD in molecular processes related to learning and memory.

bioinformatics↗

bioRxiv2018

Structural ensembles based on NMR parameters suggest a complex pathway of ligand binding in human gastrotropin

Gastrotropin, the intracellular carrier of bile salts in the small intestine, binds two ligand molecules simultaneously in its internal cavity. The molecular rearrangements required for ligand entry are not yet fully clear. To improve our understanding of the binding process we combined molecular dynamics simulations with available structural and dynamic NMR parameters. The resulting ensembles reveal two distinct modes of barrel opening with one corresponding to the transition between the apo and holo states, whereas the other affecting different protein regions in both ligation states. Comparison of the calculated structures with NMR-derived parameters reporting on slow conformational exchange processes suggests that the protein undergoes partial unfolding along a path related to the second mode of the identified barrel opening motion.

bioinformatics↗