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

BACKGROUND: The postsynaptic density is an elaborate protein network beneath the postsynaptic membrane involved in the molecular processes underlying learning and memory. The postsynaptic density is built up from the same major proteins but its exact composition and organization differs between synapses. Mutations perturbing protein: protein interactions generally occurring in this network might lead to effects specific for cell types or processes, the understanding of which can be especially challenging. RESULTS: In this work we use systems biology-based modeling of protein complex distributions in a simplified set of major postsynaptic proteins to investigate the effect of a hypomorphic Shank mutation perturbing a single well-defined interaction. We use data sets with widely variable abundances of the constituent proteins. Our results suggest that the effect of the mutation is heavily dependent on the overall availability of all the protein components of the whole network and no trivial correspondence between the expression level of the directly affected proteins and overall complex distribution can be observed. CONCLUSIONS: Our results stress the importance of context-dependent interpretation of mutations. Even the weakening of a generally occurring protein: protein interaction might have well-defined effects, and these can not easily be predicted based only on the abundance of the proteins directly affected. Our results provide insight on how cell-specific effects can be exerted by a mutation perturbing a generally occurring interaction even when the wider interaction network is largely similar.

Directed Evolution-Driven Increase of Structural Plasticity Is a Prerequisite for Binding the Complement Lectin Pathway Blocking MASP-Inhibitor Peptides.

MASP-1 and MASP-2 are key activator proteases of the complement lectin pathway. The first specific mannose-binding lectin-associated serine protease (MASP) inhibitors had been developed from the 14-amino-acid sunflower trypsin inhibitor (SFTI) peptide by phage display, yielding SFTI-based MASP inhibitors, SFMIs. Here, we present the crystal structure of the MASP-1/SFMI1 complex that we analyzed in comparison to other existing MASP-1/2 structures. Rigidified backbone structure has long been accepted as a structural prerequisite for peptide inhibitors of proteases. We found that a hydrophobic cluster organized around the P2 Thr residue is essential for the structural stability of wild-type SFTI. We also found that the same P2 Thr prevents binding of the rigid SFTI-like peptides to the substrate-binding cleft of both MASPs as the cleft is partially blocked by large gatekeeper enzyme loops. Directed evolution removed this obstacle by replacing the P2 Thr with a Ser, providing the SFMIs with high-degree structural plasticity, which proved to be essential for MASP inhibition. To gain more insight into the structural criteria for SFMI-based MASP-2 inhibition, we systematically modified MASP-2-specific SFMI2 by capping its two termini and by replacing its disulfide bridge with varying length thioether linkers. By doing so, we also aimed to generate a versatile scaffold that is resistant to reducing environment and has increased stability in exopeptidase-containing biological environments. We found that the reduction-resistant disulfide-substituted l-2,3-diaminopropionic acid (Dap) variant possessed near-native potency. As MASP-2 is involved in the life-threatening thrombosis in COVID-19 patients, our synthetic, selective MASP-2 inhibitors could be relevant coronavirus drug candidates.

PSINDB: the postsynaptic protein-protein interaction database.

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 (PPIs) in this network, these data are mostly scattered in the literature or stored in generic databases that are not designed to display aspects that are fundamental to the understanding of postsynaptic functions. To overcome these limitations, we collected postsynaptic PPIs complemented by 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 various aspects of molecular neurobiology research, from experimental design to network and systems biology-based modeling and analysis of changes in the protein network upon various stimuli. Database URL https://psindb.itk.ppke.hu/.

Diversity of synaptic protein complexes as a function of the abundance of their constituent proteins: A modeling approach.

The postsynaptic density (PSD) is a dense protein network playing a key role in information processing during learning and memory, and is also indicated in a number of neurological disorders. Efforts to characterize its detailed molecular organization are encumbered by the large variability of the abundance of its constituent proteins both spatially, in different brain areas, and temporally, during development, circadian rhythm, and also in response to various stimuli. In this study we ran large-scale stochastic simulations of protein binding events to predict the presence and distribution of PSD complexes. We simulated the interactions of seven major PSD proteins (NMDAR, AMPAR, PSD-95, SynGAP, GKAP, Shank3, Homer1) based on previously published, experimentally determined protein abundance data from 22 different brain areas and 42 patients (altogether 524 different simulations). Our results demonstrate that the relative ratio of the emerging protein complexes can be sensitive to even subtle changes in protein abundances and thus explicit simulations are invaluable to understand the relationships between protein availability and complex formation. Our observations are compatible with a scenario where larger supercomplexes are formed from available smaller binary and ternary associations of PSD proteins. Specifically, Homer1 and Shank3 self-association reactions substantially promote the emergence of very large protein complexes. The described simulations represent a first approximation to assess PSD complex abundance, and as such, use significant simplifications. Therefore, their direct biological relevance might be limited but we believe that the major qualitative findings can contribute to the understanding of the molecular features of the postsynapse.

Charged sequence motifs increase the propensity towards liquid-liquid phase separation.

Protein phase separation is a major governing factor in multiple cellular processes, such as RNA metabolism and those involving RNA-binding proteins. Despite many key observations, the exact structural characteristics of proteins involved in the phase separation 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 α-helices also show detectable but weaker connections.

Resonance assignment of the Shank1 PDZ domain.

Shank proteins are among the most abundant and well-studied postsynaptic scaffold proteins. Their PDZ domain has unique characteristics as one of its loop regions flanking the ligand-binding site is uniquely long and has also been implicated in the formation of PDZ dimers. Here we report the initial characterization of the Shank1 PDZ domain by solution NMR spectroscopy. The assigned chemical shifts are largely consistent with the common features of PDZ domains in general and the available Shank PDZ crystal structures in particular. Our analysis suggests that under the conditions investigated, the domain is monomeric and the unique loop harbors a short helical segment, observed in only one of the known X-ray structures so far. Our work stresses the importance of solution-state investigations to fully decipher the functional relevance of the structural and dynamical features unique to Shank PDZ domains.

DIPEND: An Open-Source Pipeline to Generate Ensembles of Disordered Segments Using Neighbor-Dependent Backbone Preferences.

Ensemble-based structural modeling of flexible protein segments such as intrinsically disordered regions is a complex task often solved by selection of conformers from an initial pool based on their conformity to experimental data. However, the properties of the conformational pool are crucial, as the sampling of the conformational space should be sufficient and, in the optimal case, relatively uniform. In other words, the ideal sampling is both efficient and exhaustive. To achieve this, specialized tools are usually necessary, which might not be maintained in the long term, available on all platforms or flexible enough to be tweaked to individual needs. Here, we present an open-source and extendable pipeline to generate initial protein structure pools for use with selection-based tools to obtain ensemble models of flexible protein segments. Our method is implemented in Python and uses ChimeraX, Scwrl4, Gromacs and neighbor-dependent backbone distributions compiled and published previously by the Dunbrack lab. All these tools and data are publicly available and maintained. Our basic premise is that by using residue-specific, neighbor-dependent Ramachandran distributions, we can enhance the efficient exploration of the relevant region of the conformational space. We have also provided a straightforward way to bias the sampling towards specific conformations for selected residues by combining different conformational distributions. This allows the consideration of a priori known conformational preferences such as in the case of preformed structural elements. The open-source and modular nature of the pipeline allows easy adaptation for specific problems. We tested the pipeline on an intrinsically disordered segment of the protein Cd3ϵ and also a single-alpha helical (SAH) region by generating conformational pools and selecting ensembles matching experimental data using the CoNSEnsX+ server.

Ensemble-Based Analysis of the Dynamic Allostery in the PSD-95 PDZ3 Domain in Relation to the General Variability of PDZ Structures.

PDZ domains are abundant interaction hubs found in a number of different proteins and they exhibit characteristic differences in their structure and ligand specificity. Their internal dynamics have been proposed to contribute to their biological activity via changes in conformational entropy upon ligand binding and allosteric modulation. Here we investigate dynamic structural ensembles of PDZ3 of the postsynaptic protein PSD-95, calculated based on previously published backbone and side-chain S2 order parameters. We show that there are distinct but interdependent structural rearrangements in PDZ3 upon ligand binding and the presence of the intramolecular allosteric modulator helix α3. We have also compared these rearrangements in PDZ1-2 of PSD-95 and the conformational diversity of an extended set of PDZ domains available in the PDB database. We conclude that although the opening-closing rearrangement, occurring upon ligand binding, is likely a general feature for all PDZ domains, the conformer redistribution upon ligand binding along this mode is domain-dependent. Our findings suggest that the structural and functional diversity of PDZ domains is accompanied by a diversity of internal motional modes and their interdependence.

Distribution of disease-causing germline mutations in coiled-coils implies an important 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 α-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 suggest an important 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.

MAP Kinase-Mediated Activation of RSK1 and MK2 Substrate Kinases.

Mitogen-activated protein kinases (MAPKs) control essential eukaryotic signaling pathways. While much has been learned about MAPK activation, much less is known about substrate recruitment and specificity. MAPK substrates may be other kinases that are crucial to promote a further diversification of the signaling outcomes. Here, we used a variety of molecular and cellular tools to investigate the recruitment of two substrate kinases, RSK1 and MK2, to three MAPKs (ERK2, p38α, and ERK5). Unexpectedly, we identified that kinase heterodimers form structurally and functionally distinct complexes depending on the activation state of the MAPK. These may be incompatible with downstream signaling, but naturally they may also form structures that are compatible with the phosphorylation of the downstream kinase at the activation loop, or alternatively at other allosteric sites. Furthermore, we show that small-molecule inhibitors may affect the quaternary arrangement of kinase heterodimers and thus influence downstream signaling in a specific manner.