SynGAP (Synaptic GTPase-Activating Protein) is enriched in the postsynaptic density, where it accelerates GTP hydrolysis by Ras- and Rap-GTPases. In forebrain, SynGAP is essential for synaptic plasticity and cognitive development, and de novo mutations in SYNGAP1 gene cause SynGAP-related disorder (SRD), characterized by intellectual disability, autism, and epilepsy. Beyond the nervous system, SynGAP regulates cell growth, differentiation, and cytoskeletal dynamics, and loss of its GAP activity has been implicated in tumorigenesis and cancer metastasis. Although classified as a RasGAP based on homology, SynGAP efficiently promotes GTP hydrolysis of both Ras and Rap1b. Here, a combination of modelling approaches, including in silico mutagenesis, free-energy calculations, and atomistic molecular dynamics (MD) simulations, were used to define the structural basis of this dual specificity. Cancer-associated and SRD-associated missense variants were analyzed to assess their effects on SynGAP interactions with Ras and Rap and, by extension, on GAP-mediated GTP hydrolysis. Modelling suggests that SynGAP regulates Ras through a canonical RasGAP mechanism, whereas Rap1b is controlled through a Plexin-like mechanism involving opening of Rap-Switch II and engagement of a previously uncharacterized region termed the SynGAP activation-segment. The modelling supports prior experimental evidence, including a stabilizing role for the C2-domain residue Arg401 in the open Rap-Switch II conformation, and suggests new testable hypotheses that some variants could differentially affect Ras and Rap. Enhanced GTP stabilization is proposed to explain the greater potency of SynGAP toward Rap1b. Overall, 33 missense variants, including eight ClinVar and 18 cancer-associated variants, were predicted to potentially impair SynGAP–GTPase complex formation. Author summary We study SynGAP, a protein essential for how brain cells communicate, learn, and develop. Changes in the SYNGAP1 gene are known to cause a neurodevelopmental disorder causing intellectual disability, autism, and epilepsy. SynGAP also helps regulate cell-growth pathways, and its dysfunction can contribute to cancer. In this work, we use computer-based approaches, including molecular dynamics simulations, to investigate how SynGAP interacts with the signaling GTPases Ras and Rap1b, which control key cellular processes. We show that SynGAP engages these proteins through two distinct interaction modes and model both at atom-level resolution. We also examine disease-associated missense variants and predict how they may disrupt these interactions. Our results help explain previously reported experimental findings and highlight regions of SynGAP that are critical for its function. By improving our understanding of how SynGAP regulates Ras and Rap1b, this work provides insight into SynGAP-related disorders and cancer as well as suggesting testable hypotheses for future experimental studies.
Parechovirus A1 (PeV A1) 3Dpol is an RNA-dependent RNA polymerase responsible for replication of the virus genome. We solved crystal structures of PeV A1 3Dpol structure in complex with GTP and in apo-state at 1.8-2.0 Å resolutions. In the 3Dpol-GTP complex, the conformation of the conserved motif B loop was stabilized by zinc ion coordination by cysteine residues. Apo-state structures of PeV A1 3Dpol showed significant conformational flexibility in the motif B loop, in the absence of zinc. While one of the conformational states of apo-3Dpol was similar to the 3Dpol-GTP complex structure, the alternative apo-3Dpol conformation showed a 4.3 Å movement of the motif B loop out of the active site cavity relative to the complex of 3Dpol with GTP. We propose that PeV A1 3Dpol activity is regulated by conformational stabilization of the motif B loop by zinc coordination.
Transcription factors (TFs) regulate gene expression by controlling the recruitment of transcriptional machinery to regulatory regions of the genome. Nearly 10% of the human genome encodes TFs, making them one of the largest protein families. Despite their central roles in gene regulation, TFs are historically considered challenging therapeutic targets due to their complex interactions with DNA, RNA and associated proteins. Although recent progress in studying TFs both at molecular and structural level excels our understanding on their function, yet a universal rule decoding their recognition process remains elusive. Here, we present a curated non-redundant dataset of TFs with 3570 sequences and 377 structures. We further characterize "unique interfaces" by quantifying interface identity across interacting chains in TF assemblies. Surprisingly, our data shows that the "unique interfaces" have optimal size ranging from 2000 Å2 to 4000 Å2 irrespective of their quaternary assembly. To understand the functional diversity, we integrate sequence motifs, structural domains, subcellular localization and functional enrichment of TFs. We have also catalogued association of TFs with various human diseases. Our dataset provides a comprehensive platform to perform large scale analysis of TF-assemblies and aid in computational methods for their prediction across domains of life.
AcrB is a member of the resistance-nodulation-cell division (RND) family of efflux transporters expressed in Escherichia coli, and it plays a crucial role in antimicrobial resistance. AcrB has been structurally characterized in a variety of states through observations using diverse methods, and by applying conditions that have not yet been explored, there is potential to reveal new structural features. In this study, we performed cryo-electron microscopy (cryo-EM) structural analysis of AcrB reconstituted into liposomes in the presence of the substrate doxorubicin (DOX). As a result, both the functional LTO state and the resting LLL state were observed on the same EM grid. In the LTO state, substrate binding was observed in the deep binding pocket of the T protomer, whereas the LLL state could be further classified into multiple subclasses. This study not only provides additional structural insight into AcrB but also highlights the importance of observing membrane proteins under a variety of conditions.
