Zoltán Gáspári

The EVH1 domain of the Homer1 scaffold protein interacts with the proline‐rich region of Shank3, forming a key network within the postsynaptic density. While two mutations (M65I and S97L) in the EVH1 domain have been suggested to be associated with autism spectrum disorder, our results show that neither mutation has a substantial effect on the overall structure or the partner binding properties of Homer1. Compared to the S97L variant, the M65I mutant exhibits larger chemical shift perturbations both upon the mutation itself and during partner binding, while also showing signs of thermal destabilization. Finally, integration of computational and NMR data suggests that both mutations perturb the μs‐ms timescale internal motions of the EVH1 domain.

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 μs-ms time scale internal motions of the EVH1 domain.

Homer 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 Homer1 coiled coil region along with the NMR solution structure and backbone dynamics of its EVH1 domain, with implications for the organization of the full‐length tetramer. 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.

Guanylate kinase-associated protein, GKAP, is a largely disordered scaffold protein with multiple interaction partners, playing a role in the organization of the postsynaptic protein network. Its C-terminus contains a binding motif for the PDZ domain of Shank proteins, another class of postsynaptic scaffolds. Based on predictions, this motif is preceded by a ~ 40-residue disordered segment with no known additional binding sites or established functional role. Here we report the expression, purification, and ¹ H, ¹³ C and ¹⁵ N resonance assignment of the GKAP Ct43 construct containing the C-terminal 43 residues. This region is functionally intact, having a binding affinity to Shank1 PDZ in the micromolar range. Chemical shifts indicate that this region is indeed disordered but displays helical propensity in two regions. The short, slightly helical segment immediately before a PDZ-binding motif is somewhat comparable to the structural organization observed at the C-terminus of the E6 and RSK1 proteins, but its significance in GKAP is yet to be explored.

The function of our brain is the result of the balanced interplay between billions of neurons forming a network of enormous complexity. However, the neurons themselves are also immensely complex entities, with many specialized macromolecular structures orchestrating signal processing and propagation. The postsynaptic density is an elaborate network of interconnected proteins, a dynamic yet highly organized molecular assembly beneath the dendritic membrane, and plays a pivotal role in learning, memory formation, and the development of a number of cognitive disorders. In this review, we argue that with the recent blooming of AI-assisted computational tools in structural biology, we might be able to get closer to understanding the molecular-level mechanistic aspects of this machinery. Nevertheless, we have to use these methods with caution as they are not yet capable of solving all the questions that arise for such a complex macromolecular system. First, we focus on the unique features of the postsynaptic protein network, highlighting those that pose particular challenges for such a modeling task, and put these in the light of the currently available deep learning-based approaches. We highlight the aspects that need specific attention and the areas where future developments could facilitate the detailed description of neural function at the molecular level.

The postsynaptic density (PSD) is a complex, multilayered protein network largely situated on the internal surface of the postsynaptic membrane. It is the first processing unit for incoming synaptic signals, 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 labeling technique that does not disrupt the function of labeled 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.

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 two GKAP molecules and two LC8 dimers. Using nuclear magnetic resonance (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, and that the two LC8 dimers might interact with each other. 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.

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 β2-β3 loop, which is likely to play an important role in ligand selectivity. 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 mutations in general weaken most interactions, the R736Q mutant, 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. Statement: The Shank1 protein plays a possible role in autism spectrum disorders and also cancer, two conditions that affect many lives. Our paper provides a more-detailed-than-ever model of Shank1 PDZ - including disease-associated mutants - bound to its ligands, and we highlight the importance of flexibility and dynamics, which appear to be the main driving force behind its ligand selectivity, and reveal remarkable complexity hiding within this small protein domain.

/Summary: Homer 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.

Single alpha-helices (SAHs) are protein regions with unique mechanical properties, forming long, stable, monomeric helical structures in solution. To date, only a few naturally occurring SAH regions have been extensively characterized, primarily from myosins, leaving the structural and dynamic variability of SAH regions largely unexplored. Drebrin (developmentally regulated brain protein) contains a predicted SAH segment with unique sequence characteristics, including aromatic residues within the SAH region and a preference for arginine over lysine in its C-terminal half. Using circular dichroism (CD) and NMR spectroscopy, combined with small-angle X-ray scattering (SAXS) measurements, we demonstrate that the Drebrin-SAH is helical and monomeric in solution. NMR resonance assignment required specific 4D techniques to resolve severe signal overlap resulting from the low complexity and largely helical conformation of the sequence. To further characterize its structure, we generated a structural ensemble consistent with Cα, Cβ chemical shifts and SAXS data, revealing a primarily extended structure with non-uniform helicity. Our results suggest that dynamic rearrangement of salt bridges and potential transient cation-π interactions contribute to the formation and stabilization of both helical and non-helical local conformational states.