Biology subjects

Weeratunga, S.

Publications and source records attributed to Weeratunga, S..

4 recordsLinked to original sources

bioRxiv2023

VAMP2/Munc18-1 domain 3a interaction controls the nanoscale reorganization underpinning vesicular priming

SNARE-mediated secretory vesicle (SV) exocytosis underpins neuronal communication. Munc18-1 orchestrates SNARE complex formation by controlling the opening of syntaxin-1A. How the SV-plasma membrane interface becomes fusion-competent at the nanoscale level is poorly understood. Here, we propose that the interaction of Munc18-1 with VAMP2 during vesicular docking triggers nanoscale re-organization which renders the SV-plasma membrane interface fusion-competent. We identified and mutated key residues in Munc18-1 domain 3a (A297 and T304) hypothesised to impair its interaction with VAMP2. Munc18-1A297H, and to a lesser extent Munc18-1T304H, constrained SVs on the plasma membrane and reduced stimulated secretion, under re-expression conditions in Munc18-1/2 double knockout neurosecretory cells. Moreover, the de-clustering of Munc18-1 in response to activity was lost for both mutants. The interaction of VAMP2 with the Munc18-1 domain 3a therefore controls the re-organization of the nanoscale environment of the docked SV-plasma membrane interface, fostering syntaxin-1A opening and Munc18-1 release to ensure that SNARE assembly only occurs within the confinement of docked vesicles.

neuroscience↗

bioRxiv2023

Molecular basis for the interaction of Mint proteins with Munc18-1 and interactome analyses with AlphaFold2

Munc18-interacting proteins (Mints) are multi-domain adaptors that regulate neuronal membrane trafficking, signalling and neurotransmission. Mint1 and Mint2 are highly expressed in the brain with overlapping roles in the regulation of synaptic vesicle fusion required for neurotransmitter release by interacting with the essential synaptic protein Munc18-1. Here, we have used AlphaFold2 to identify and then validate the mechanisms that underpin both the specific interactions of neuronal Mint proteins with Munc18-1 as well as their wider interactome. We find a short acidic -helical motif (AHM) within Mint1 and Mint2 is necessary and sufficient for specific binding to Munc18-1 and binds a conserved surface on Munc18-1 domain3b. In Munc18-1/2 double knockout neurosecretory cells mutation of the Mint-binding site reduces the ability of Munc18-1 to rescue exocytosis, and although Munc18-1 can interact with Mint and Sx1a proteins simultaneously in vitro we find they have mutually reduced affinities, suggesting an allosteric coupling between the proteins. Using AlphaFold2 to then examine the entire cellular network of putative Mint interactors provides a structural model for their assembly with a variety of known and novel regulatory and cargo proteins including ARF3/ARF4 small GTPases, and the AP3 clathrin adaptor complex. Validation of Mint1 interaction with a new predicted binder TJAP1 provides experimental support that AlphaFold2 can correctly predict interactions across such large-scale datasets. Overall, our data provides insights into the diversity of interactions mediated by the Mint family and shows that Mints may help facilitate a key trigger point in SNARE complex assembly and vesicle fusion.

biochemistry↗

bioRxiv2022

ESCPE-1 Mediates Retrograde Endosomal Sorting of the SARS-CoV-2 Host Factor Neuropilin-1

Endosomal sorting maintains cellular homeostasis by recycling transmembrane proteins and associated proteins and lipids (termed cargoes) from the endosomal network to multiple subcellular destinations, including retrograde traffic to the trans-Golgi network (TGN). Viral and bacterial pathogens subvert retrograde trafficking machinery to facilitate infectivity. Here, we develop a proteomic screen to identify novel retrograde cargo proteins of the Endosomal SNX-BAR Sorting Complex Promoting Exit-1 (ESCPE-1). Using this methodology, we identify Neuropilin-1 (NRP1), a recently characterised host factor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as a cargo directly bound and trafficked by ESCPE-1. ESCPE-1 mediates retrograde trafficking of engineered nanoparticles functionalised with the NRP1-interacting peptide of the SARS-CoV-2 Spike protein. ESCPE-1 sorting of NRP1 may therefore play a role in the intracellular membrane trafficking of NRP1-interacting viruses such as SARS-CoV-2.

cell biology↗

bioRxiv2021

Experimental and in silico Alphafold2 derived structures of the SNX-RGS proteins suggest a new class of lipid transfer protein

Recent advances in protein structure prediction using machine learning such as AlphaFold2 and RosettaFold presage a revolution in structural biology. Genome-wide predictions of protein structures are providing unprecedented insights into their architecture and intradomain interactions, and applications have already progressed towards assessing protein complex formation. Here we present detailed analyses of the sorting nexin proteins that contain regulator of G-protein signalling domains (SNX-RGS proteins), providing a key example of the ability of AlphaFold2 to reveal novel structures with previously unsuspected biological functions. These large proteins are conserved in most eukaryotes and are known to associate with lipid droplets (LDs) and sites of LD-membrane contacts, with key roles in regulating lipid metabolism. They possess five domains, including an N-terminal transmembrane domain that anchors them to the endoplasmic reticulum, an RGS domain, a lipid interacting phox homology (PX) domain and two additional domains named the PXA and PXC domains of unknown structure and function. Here we report the crystal structure of the RGS domain of sorting nexin 25 (SNX25) and show that the AlphaFold2 prediction closely matches the experimental structure. Analysing the full-length SNX-RGS proteins across multiple homologues and species we find that the distant PXA and PXC domains in fact fold into a single unique structure that notably features a large and conserved hydrophobic pocket. The nature of this pocket strongly suggests a role in lipid or fatty acid binding, and we propose that these molecules represent a new class of conserved lipid transfer proteins.

biochemistry↗