Role of membrane environment and membrane-spanning protein regions in assembly and function of the Class II Major Histocompatibility complex

Class II Major Histocompatibility complex (MHC-II) is a polymorphic heterodimer that binds antigen-derived peptides and presents them on the surface of antigen presenting cells. This mechanism of antigen presentation leads to recognition by CD4 T-cells and T-cell activation, making it a critical element of adaptive immune response. For this reason, the structural determinants of MHC-II function have been of great interest for the past 30 years, resulting in a robust structural understanding of the extracellular regions of the complex. However, the membrane-localized regions have also been strongly implicated in protein-protein and protein-lipid interactions that facilitate Class II assembly, transport and function, and it is these regions that are the focus of this review. Here we describe studies that reveal the strong and selective interactions between the transmembrane domains of the MHC α, and invariant chains which, when altered, have broad reaching impacts on antigen presentation and Class II function. We also summarize work that clearly demonstrates the link between membrane lipid composition (particularly the presence of cholesterol) and MHC-II conformation, subsequent peptide binding, and downstream T-cell activation. We have integrated these studies into a comprehensive view of Class II transmembrane domain biology. [Abstract copyright: Copyright © 2018. Published by Elsevier Inc.

Bacterial expression, purification and biophysical characterization of the smallest plant reticulon isoform, RTNLB13

Reticulons are a large family of integral membrane proteins that are ubiquitous in eukaryotes and play a key role in functional remodelling of the endoplasmic reticulum membrane. The reticulon family is especially large in plants, with the Arabidopsis thaliana genome containing twenty-one isoforms. Reticulons vary in length but all contain a conserved C-terminal reticulon homology domain (RHD) that associates with membranes. An understanding of the structure and membrane interactions of RHDs is key to unlocking their mechanism of function, however no three-dimensional structure has been solved. We believe that this is, in part, due to difficulties in obtaining reticulon proteins in yields sufficient for structural study. To address this, we report here the first bacterial overexpression, purification, and biophysical investigation of a reticulon protein from plants, the RTNLB13 protein from A. thaliana. RTNLB13 is the smallest plant reticulon and is made up of a single RHD. We used circular dichroism, SDS-PAGE and analytical ultracentrifugation to reveal that RTNLB13 is 45% α-helical in a number of detergent environments, monomeric at low concentrations, and capable of self-association at higher concentrations. We used solution-state NMR to screen the effect of detergent type on the fold of isotopically-enriched RTNLB13, and found that ∼60% of the expected protein peaks were broadened due to slow dynamics. This broadening points toward a large network of protein-membrane interactions throughout the sequence. We have interpreted our results in light of current literature and suggest a preliminary description of RTNLB13 structure and topology

Characterisation of the carboxypeptidase G2 catalytic site and design of new inhibitors for cancer therapy

The enzyme carboxypeptidase G2 (CPG2) is used in antibody‐directed enzyme prodrug therapy (ADEPT) to catalyse the formation of an active drug from an inert prodrug. Free CPG2 in the bloodstream must be inhibited before administration of the prodrug in order to avoid a systemic reaction in the patient. Although a few small‐molecule CPG2 inhibitors have been reported, none has been taken forward thus far. This lack of progress is due in part to a lack of structural understanding of the CPG2 active site as well as the absence of small molecules that can block the active site whilst targeting the complex for clearance. The work described here aimed to address both areas. We report the structural/functional impact of extensive point mutation across the putative CPG2 catalytic site and adjacent regions for the first time, revealing that residues outside the catalytic region (K208A, S210A and T357A) are crucial to enzyme activity. We also describe novel molecules that inhibit CPG2 whilst maintaining the accessibility of galactosylated moieties aimed at targeting the enzyme for clearance. This work acts as a platform for the future development of high‐affinity CPG2 inhibitors that occupy new chemical space and will advance the safe application of ADEPT in cancer treatment

Revealing the mechanism of protein-lipid interactions for a putative membrane curvature sensor in plant endoplasmic reticulum

Membrane curvature sensing via helical protein domains, such as those identified in Amphiphysin and ArfGAP1, have been linked to a diverse range of cellular processes. However, these regions can vary significantly between different protein families and thus remain challenging to identify from sequence alone. Greater insight into the protein-lipid interactions that drive this behavior could lead to production of therapeutics that specifically target highly curved membranes. Here we demonstrate the curvature-dependence of membrane binding for an amphipathic helix (APH) in a plant reticulon, namely RTNLB13 from A. thaliana. We utilize solution-state nuclear magnetic resonance spectroscopy to establish the exact location of the APH and map the residues involved in protein-membrane interactions at atomic resolution. We find that the hydrophobic residues making up the membrane binding site are conserved throughout all A. thaliana reticulons. Our results also provide mechanistic insight that leads us to propose that membrane binding by this APH may act as a feedback element, only forming when ER tubules reach a critical size and adding stabilization to these structures without disrupting the bilayer. A shallow hydrophobic binding interface appears to be a feature shared more broadly across helical curvature sensors and would automatically restrict the penetration depth of these structures into the membrane. We also suggest this APH is highly tuned to the composition of the membrane in which it resides, and that this property may be universal in curvature sensors thus rationalizing the variety of mechanisms reported for these functional elements. [Abstract copyright: Copyright © 2019. Published by Elsevier B.V.

The cell edit : looking at and beyond non-structural proteins to understand membrane rearrangement in coronaviruses

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-stranded RNA virus that sits at the centre of the recent global pandemic. As a member of the coronaviridae family of viruses, it shares features such as a very large genome (>30 kb) that is replicated in a purpose-built replication organelle. Biogenesis of the replication organelle requires significant and concerted rearrangement of the endoplasmic reticulum membrane, a job that is carried out by a group of integral membrane non-structural proteins (NSP3, 4 and 6) expressed by the virus along with a host of viral replication enzymes and other factors that support transcription and replication. The primary sites for RNA replication within the replication organelle are double membrane vesicles (DMVs). The small size of DMVs requires generation of high membrane curvature, as well as stabilization of a double-membrane arrangement, but the mechanisms that underlie DMV formation remain elusive. In this review, we discuss recent breakthroughs in our understanding of the molecular basis for membrane rearrangements by coronaviruses. We incorporate established models of NSP3-4 protein-protein interactions to drive double membrane formation, and recent data highlighting the roles of lipid composition and host factor proteins (e.g. reticulons) that influence membrane curvature, to propose a revised model for DMV formation in SARS-CoV-2. [Abstract copyright: Copyright © 2023. Published by Elsevier Inc.

The extracellular domain of two-component system sensor kinase VanS from streptomyces coelicolor binds Vancomycin at a newly identified binding site

The glycopeptide antibiotic vancomycin has been widely used to treat infections of Gram-positive bacteria including Clostridium difficile and methicillin-resistant Staphylococcus aureus. However, since its introduction, high level vancomycin resistance has emerged. The genes responsible require the action of the two-component regulatory system VanSR to induce expression of resistance genes. The mechanism of detection of vancomycin by this two-component system has yet to be elucidated. Diverging evidence in the literature supports activation models in which the VanS protein binds either vancomycin, or Lipid II, to induce resistance. Here we investigated the interaction between vancomycin and VanS from Streptomyces coelicolor (VanSSC), a model Actinomycete. We demonstrate a direct interaction between vancomycin and purified VanSSC, and traced these interactions to the extracellular region of the protein, which we reveal adopts a predominantly α-helical conformation. The VanSSC-binding epitope within vancomycin was mapped to the N-terminus of the peptide chain, distinct from the binding site for Lipid II. In targeting a separate site on vancomycin, the effective VanS ligand concentration includes both free and lipid-bound molecules, facilitating VanS activation. This is the first molecular description of the VanS binding site within vancomycin, and could direct engineering of future therapeutics

Modulation of transmembrane domain interactions in neu receptor tyrosine kinase by membrane fluidity and cholesterol

The activation mechanism of the ErbB family of receptors is of considerable medical interest as they are linked to a number of human cancers, including an aggressive form of breast cancer. In the rat analogue of the human ErbB2 receptor, referred to as Neu, a point mutation in the transmembrane domain (V664E) has been shown to trigger oncogenic transformation. While the structural impact of this mutation has been widely studied in the past to yield models for the active state of the Neu receptor, little is known about the impact of cholesterol on its structure. Given previous reports of the influence of cholesterol on other receptor tyrosine kinases (RTKs), as well as the modulation of lipid composition in cancer cells, we wished to investigate how cholesterol content impacts the structure of the Neu transmembrane domain. We utilized high-resolution magic angle spinning solid-state NMR to measure 13C–13C coupling of selectively labelled probe residues in the Neu transmembrane domain in lipid bilayers containing cholesterol. We observe inter-helical coupling between residues that support helix–helix interactions on both dimerization motifs reported in the literature (A661-XXX-G665 and I659-XXX-V663). We further explore how changes in cholesterol concentration alter transmembrane domain interactions and the properties and mechanics of the bilayer. We interpret our results in light of previous studies relating RTK activity to cholesterol enrichment and/or depletion, and propose a novel model to explain our data that includes the recognition and binding of cholesterol by the Neu transmembrane domain through a putative cholesterol-recognition/interaction amino acid consensus sequence

Characterization of interactions within the Igα/Igβ transmembrane domains of the human B-cell receptor provides insights into receptor assembly

The B-cell receptor (BCR), a complex comprised of a membrane-associated immunoglobulin (mIg) and the Ig/ heterodimer, is one of the most important immune receptors in humans and controls B-cell development, activity, selection, and death. BCR signaling plays key roles in autoimmune diseases and lymphoproliferative disorders yet, despite the clinical significance of this protein complex, key regions (i.e. the transmembrane domains) have yet to be structurally characterized. The mechanism for BCR signaling also remains unclear, and has been variously described by the mutually exclusive crosslinking and dissociation activation models. Common to these models is the significance of local plasma membrane composition, which implies that interactions between BCR transmembrane domains (TMDs) play a role in receptor functionality. Here we used an in vivo assay of TMD oligomerization called GALLEX alongside spectroscopic and computational methods to characterize the structures and interactions of human Ig and Ig TMDs in detergent micelles and natural membranes. We observed weak self-association of the Igb TMD and strong self-association of the Iga TMD, which scanning mutagenesis revealed was entirely stabilized by an E-X10-P motif. We also demonstrated strong heterotypic interactions between the Iga and Igb TMDs both in vitro and in vivo, which scanning mutagenesis and computational models suggest is multiconfigurational but can accommodate distinct interaction sites for self- and heterotypic interactions of the Iga TMD. Taken together, these results demonstrate that the TMDs of the human B-cell receptor are sites of strong protein-protein interactions that may direct BCR assembly, ER retention, and immune signaling

Curvature sensing amphipathic helix in the C-terminus of RTNLB13 is conserved in all endoplasmic reticulum shaping reticulons in Arabidopsis thaliana

The reticulon family of integral membrane proteins are conserved across all eukaryotes and typically localize to the endoplasmic reticulum (ER), where they are involved in generating highly-curved tubules. We recently demonstrated that Reticulon-like protein B13 (RTNLB13) from Arabidopsis thaliana contains a curvature-responsive amphipathic helix (APH) important for the proteins’ ability to induce curvature in the ER membrane, but incapable of generating curvature by itself. We suggested it acts as a feedback element, only folding/binding once a sufficient degree of curvature has been achieved, and stabilizes curvature without disrupting the bilayer. However, it remains unclear whether this is unique to RTNLB13 or is conserved across all reticulons—to date, experimental evidence has only been reported for two reticulons. Here we used biophysical methods to characterize a minimal library of putative APH peptides from across the 21 A. thaliana isoforms. We found that reticulons with the closest evolutionary relationship to RTNLB13 contain curvature-sensing APHs in the same location with sequence conservation. Our data reveal that a more distantly-related branch of reticulons developed a ~ 20-residue linker between the transmembrane domain and APH. This may facilitate functional flexibility as previous studies have linked these isoforms not only to ER remodeling but other cellular activities

Plants Survive Rapid Decompression: Implications for Bioregenerative Life Support

No abstract availabl