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ABSTRACT Diacylglycerol O‐acyltransferase 1 (DGAT1) is an integral membrane protein that uses acyl‐coenzyme A (acyl‐CoA) and diacylglycerol (DAG) to catalyze the formation of triacylglycerides (TAGs). The acyl transfer reaction occurs between the activated carboxylate group of the fatty acid and the free hydroxyl group on the glycerol backbone of DAG. However, how the two substrates enter DGAT1's catalytic reaction chamber and interact with DGAT1 remains elusive. This study aims to explore the structural basis of DGAT1's substrate recognition by investigating each substrate's pathway to the reaction chamber. Using a human DGAT1 cryo‐EM structure in complex with an oleoyl‐CoA substrate, we designed two different all‐atom molecular dynamics (MD) simulation systems: DGAT1^away(both acyl‐CoA and DAG away from the reaction chamber) and DGAT1^bound(acyl‐CoA bound in and DAG away from the reaction chamber). Our DGAT1^awaysimulations reveal that acyl‐CoA approaches the reaction chamber via interactions with positively charged residues in transmembrane helix 7. DGAT1^boundsimulations show DAGs entering into the reaction chamber from the cytosol leaflet. The bound acyl‐CoA's fatty acid lines up with the headgroup of DAG, which appears to be competent to TAG formation. We then converted them into TAG and coenzyme (CoA) and used adaptive biasing force (ABF) simulations to explore the egress pathways of the products. We identify their escape routes, which are aligned with their respective entry pathways. Visualization of the substrate and product pathways and their interactions with DGAT1 is expected to guide future experimental design to better understand DGAT1 structure and function. 

Abstract Structural and mechanistic studies on human odorant receptors (ORs), key in olfactory signaling, are challenging because of their low surface expression in heterologous cells. The recent structure of OR51E2 bound to propionate provided molecular insight into odorant recognition, but the lack of an inactive OR structure limited understanding of the activation mechanism of ORs upon odorant binding. Here, we determined the cryo-electron microscopy structures of consensus OR52 (OR52_cs), a representative of the OR52 family, in the ligand-free (apo) and octanoate-bound states. The apo structure of OR52_csreveals a large opening between transmembrane helices (TMs) 5 and 6. A comparison between the apo and active structures of OR52_csdemonstrates the inward and outward movements of the extracellular and intracellular segments of TM6, respectively. These results, combined with molecular dynamics simulations and signaling assays, shed light on the molecular mechanisms of odorant binding and activation of the OR52 family. 

Abstract Caveolin‐1 is an integral membrane protein that is known to acquire a number of posttranslational modifications upon trafficking to the plasma membrane. In particular, caveolin‐1 is palmitoylated at three cysteine residues (C133, C143, and C156) located within theC‐terminal domain of the protein which could have structural and topological implications. Herein, a reliable preparation of full‐lengthS‐alkylated caveolin‐1, which closely mimics the palmitoylation observed in vivo, is described. HPLC and ESI‐LC‐MS analyses verified the addition of the C16 alkyl groups to caveolin‐1 constructs containing one (C133), two (C133 and C143), and three (C133, C143, and C156) cysteine residues. Circular dichroism spectroscopy analysis of the constructs revealed thatS‐alkylation does not significantly affect theglobalhelicity of the protein; however, molecular dynamics simulations revealed that there werelocalregions where the helicity was altered positively or negatively byS‐alkylation. In addition, the simulations showed that lipidation tames the topological promiscuity of theC‐terminal domain, resulting in a disposition within the bilayer characterized by increased depth. 

Abstract G-protein coupled receptors (GPCRs) and ion channels serve as key molecular switches through which extracellular stimuli are transformed into intracellular effects, and it has long been postulated that ion channels are direct effector molecules of the alpha subunit of G-proteins (Gα). However, no complete structural evidence supporting the direct interaction between Gα and ion channels is available. Here, we present the cryo-electron microscopy structures of the human transient receptor potential canonical 5 (TRPC5)-Gα_i3complexes with a 4:4 stoichiometry in lipid nanodiscs. Remarkably, Gα_i3binds to the ankyrin repeat edge of TRPC5 ~ 50 Å away from the cell membrane. Electrophysiological analysis shows that Gα_i3increases the sensitivity of TRPC5 to phosphatidylinositol 4,5-bisphosphate (PIP₂), thereby rendering TRPC5 more easily opened in the cell membrane, where the concentration of PIP₂is physiologically regulated. Our results demonstrate that ion channels are one of the direct effector molecules of Gα proteins triggered by GPCR activation–providing a structural framework for unraveling the crosstalk between two major classes of transmembrane proteins: GPCRs and ion channels. 

Abstract Cell signaling by receptor protein tyrosine kinases (RTKs) is tightly controlled by the counterbalancing actions of receptor protein tyrosine phosphatases (RPTPs). Due to their role in attenuating the signal‐initiating potency of RTKs, RPTPs have long been viewed as therapeutic targets. However, the development of activators of RPTPs has remained limited. We previously reported that the homodimerization of a representative member of the RPTP family (protein tyrosine phosphatase receptor J or PTPRJ) is regulated by specific transmembrane (TM) residues. Disrupting this interaction by single point mutations promotes PTPRJ access to its RTK substrates (e.g., EGFR and FLT3), reduces RTK's phosphorylation and downstream signaling, and ultimately antagonizes RTK‐driven cell phenotypes. Here, we designed and tested a series of first‐in‐class pH‐responsive TM peptide agonists of PTPRJ that are soluble in aqueous solution but insert as a helical TM domain in lipid membranes when the pH is lowered to match that of the acidic microenvironment of tumors. The most promising peptide reduced EGFR's phosphorylation and inhibited cancer cell EGFR‐driven migration and proliferation, similar to the PTPRJ's TM point mutations. Developing tumor‐selective and TM‐targeting peptide binders of critical RPTPs could afford a potentially transformative approach to studying RPTP's selectivity mechanism without requiring less specific inhibitors and represent a novel class of therapeutics against RTK‐driven cancers. 

Abstract This study was undertaken to identify and characterize the first ligands capable of selectively identifying nicotinic acetylcholine receptors containing α7 and β2 subunits (α7β2‐nAChR subtype). Basal forebrain cholinergic neurons express α7β2‐nAChR. Here, they appear to mediate neuronal dysfunction induced by the elevated levels of oligomeric amyloid‐β associated with early Alzheimer's disease. Additional work indicates that α7β2‐nAChR are expressed across several further critically important cholinergic and GABAergic neuronal circuits within the central nervous system. Further studies, however, are significantly hindered by the inability of currently available ligands to distinguish heteromeric α7β2‐nAChR from the closely related and more widespread homomeric α7‐only‐nAChR subtype. Functional screening using two‐electrode voltage‐clamp electrophysiology identified a family of α7β2‐nAChR‐selective analogs of α‐conotoxin PnIC (α‐CtxPnIC). A combined electrophysiology, functional kinetics, site‐directed mutagenesis, and molecular dynamics approach was used to further characterize the α7β2‐nAChR selectivity and site of action of these α‐CtxPnIC analogs. We determined that α7β2‐nAChR selectivity of α‐CtxPnIC analogs arises from interactions at a site distinct from the orthosteric agonist‐binding site shared between α7β2‐ and α7‐only‐nAChR. As numerous previously identified α‐Ctx ligands are competitive antagonists of orthosteric agonist‐binding sites, this study profoundly expands the scope of use of α‐Ctx ligands (which have already provided important nAChR research and translational breakthroughs). More immediately, analogs of α‐CtxPnIC promise to enable, for the first time, both comprehensive mapping of the distribution of α7β2‐nAChR and detailed investigations of their physiological roles. 

Abstract Transient receptor potential vanilloid (TRPV) channels play various important roles in human physiology. As membrane proteins, these channels are modulated by their endogenous lipid environment as the recent wealth of structural studies has revealed functional and structural lipid binding sites. Additionally, it has been shown that exogenous ligands can exchange with some of these lipids to alter channel gating. Here, we used molecular dynamics simulations to examine how one member of the TRPV family, TRPV2, interacts with endogenous lipids and the pharmacological modulator cannabidiol (CBD). By computationally reconstituting TRPV2 into a typical plasma membrane environment, which includes phospholipids, cholesterol, and phosphatidylinositol (PIP) in the inner leaflet, we showed that most of the interacting surface lipids are phospholipids without strong specificity for headgroup types. Intriguingly, we observed that the C‐terminal membrane proximal region of the channel binds preferentially to PIP lipids. We also modelled two structural lipids in the simulation: one in the vanilloid pocket and the other in the voltage sensor‐like domain (VSLD) pocket. The simulation shows that the VSLD lipid dampens the fluctuation of the VSLD residues, while the vanilloid lipid exhibits heterogeneity both in its binding pose and in its influence on protein dynamics. Addition of CBD to our simulation system led to an open selectivity filter and a structural rearrangement that includes a clockwise rotation of the ankyrin repeat domains, TRP helix, and VSLD. Together, these results reveal the interplay between endogenous lipids and an exogenous ligand and their effect on TRPV2 stability and channel gating. 

Abstract Neuropeptide Y (NPY) is highly abundant in the brain and involved in various physiological processes related to food intake and anxiety, as well as human diseases such as obesity and cancer. However, the molecular details of the interactions between NPY and its receptors are poorly understood. Here, we report a cryo-electron microscopy structure of the NPY-bound neuropeptide Y1 receptor (Y₁R) in complex with G_i1protein. The NPY C-terminal segment forming the extended conformation binds deep into the Y₁R transmembrane core, where the amidated C-terminal residue Y36 of NPY is located at the base of the ligand-binding pocket. Furthermore, the helical region and two N-terminal residues of NPY interact with Y₁R extracellular loops, contributing to the high affinity of NPY for Y₁R. The structural analysis of NPY-bound Y₁R and mutagenesis studies provide molecular insights into the activation mechanism of Y₁R upon NPY binding. 

Abstract Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), the virus causing COVID‐19, has continued to mutate and spread worldwide despite global vaccination efforts. In particular, the Omicron variant, first identified in South Africa in late November 2021, has become the dominant strain worldwide. Compared to the original strain identified in Wuhan, Omicron features 50 genetic mutations, with 15 mutations in the receptor‐binding domain (RBD) of the spike protein, which binds to the human angiotensin‐converting enzyme 2 (ACE2) receptor for viral entry. However, it is not completely understood how these mutations alter the interaction and binding strength between the Omicron RBD and ACE2. In this study, we used a combined steered molecular dynamics (SMD) simulation and experimental microscale thermophoresis (MST) approach to quantify the interaction between Omicron RBD and ACE2. We report that the Omicron brings an enhanced RBD‐ACE2 interface through N501Y, Q498R, and T478K mutations; the changes further lead to unique interaction patterns, reminiscing the features of previously dominated variants, Alpha (N501Y) and Delta (L452R and T478K). Among the Q493K and Q493R, we report that Q493R shows stronger binding to ACE2 than Q493K due to increased interactions. Our MST data confirmed that the Omicron mutations in RBD are associated with a five‐fold higher binding affinity to ACE2 compared to the RBD of the original strain. In conclusion, our results could help explain the Omicron variant's prevalence in human populations, as higher interaction forces or affinity for ACE2 likely promote greater viral binding and internalization, leading to increased infectivity.