Transmembrane Helices Are an Over-Presented and Evolutionarily Conserved Source of Major Histocompatibility Complex Class I and II Epitopes

Cytolytic T cell responses are predicted to be biased towards membrane proteins. The peptide-binding grooves of most alleles of histocompatibility complex class I (MHC-I) are relatively hydrophobic, therefore peptide fragments derived from human transmembrane helices (TMHs) are predicted to be presented more often as would be expected based on their abundance in the proteome. However, the physiological reason of why membrane proteins might be over-presented is unclear. In this study, we show that the predicted over-presentation of TMH-derived peptides is general, as it is predicted for bacteria and viruses and for both MHC-I and MHC-II, and confirmed by re-analysis of epitope databases. Moreover, we show that TMHs are evolutionarily more conserved, because single nucleotide polymorphisms (SNPs) are present relatively less frequently in TMH-coding chromosomal regions compared to regions coding for extracellular and cytoplasmic protein regions. Thus, our findings suggest that both cytolytic and helper T cells are more tuned to respond to membrane proteins, because these are evolutionary more conserved. We speculate that TMHs are less prone to mutations that enable pathogens to evade T cell responses.

Novel biallelic variants expand the SLC5A6-related phenotypic spectrum

The sodium (Na+):multivitamin transporter (SMVT), encoded by SLC5A6, belongs to the sodium:solute symporter family and is required for the Na+-dependent uptake of biotin (vitamin B7), pantothenic acid (vitamin B5), the vitamin-like substance α-lipoic acid, and iodide. Compound heterozygous SLC5A6 variants have been reported in individuals with variable multisystemic disorder, including failure to thrive, developmental delay, seizures, cerebral palsy, brain atrophy, gastrointestinal problems, immunodeficiency, and/or osteopenia. We expand the phenotypic spectrum associated with biallelic SLC5A6 variants affecting function by reporting five individuals from three families with motor neuropathies. We identified the homozygous variant c.1285 A > G [p.(Ser429Gly)] in three affected siblings and a simplex patient and the maternally inherited c.280 C > T [p.(Arg94*)] variant and the paternally inherited c.485 A > G [p.(Tyr162Cys)] variant in the simplex patient of the third family. Both missense variants were predicted to affect function by in silico tools. 3D homology modeling of the human SMVT revealed 13 transmembrane helices (TMs) and Tyr162 and Ser429 to be located at the cytoplasmic facing region of TM4 and within TM11, respectively. The SLC5A6 missense variants p.(Tyr162Cys) and p.(Ser429Gly) did not affect plasma membrane localization of the ectopically expressed multivitamin transporter suggesting reduced but not abolished function, such as lower catalytic activity. Targeted therapeutic intervention yielded clinical improvement in four of the five patients. Early molecular diagnosis by exome sequencing is essential for timely replacement therapy in affected individuals.

Bias in FGFR1 signaling in response to FGF4, FGF8, and FGF9

FGFR1 signals differently in response to the FGF ligands FGF4, FGF8 and FGF9, but the mechanism behind the differential ligand recognition is poorly understood. Here, we use biophysical tools to quantify multiple aspects of FGFR1 signaling in response to the three FGFs: potency, efficacy, ligand-induced oligomerization and downregulation, and conformation of the active FGFR1 dimers. We show that FGF4, FGF8, and FGF9 are biased ligands, and that bias can explain differences in FGF8 and FGF9-mediated cellular responses. Our data suggest that ligand bias arises due to structural differences in the ligand-bound FGFR1 dimers, which impact the interactions of the FGFR1 transmembrane helices, leading to differential recruitment and activation of the downstream signaling adaptor FRS2. This study expands the mechanistic understanding of FGF signaling during development and brings the poorly understood concept of receptor tyrosine kinase ligand bias into the spotlight.

Cryo-EM structure of human somatostatin receptor 2 complex with its agonist somatostatin delineates the ligand binding specificity

Somatostatin is a peptide hormone regulating endocrine systems through binding to G-protein-coupled somatostatin receptors. somatostatin receptor 2 (SSTR2) is one of the human somatostatin receptors and highly implicated in cancers and neurological disorders. Here, we report the high resolution cryo-EM structure of full-length human SSTR2 bound to the agonist somatostatin (SST-14) complex with inhibitory G (Gi) proteins. Our structure shows that seven transmembrane helices form a deep pocket for ligand binding and that the highly conserved Trp-Lys motif of SST-14 positions at the bottom of the pocket. Furthermore, our sequence analysis combined with AlphaFold modeled structures of other SSTR isoforms provide how SSTR family proteins specifically interact with their cognate ligands. This work provides the first glimpse into the molecular recognition of somatostatin receptor and crucial resource to develop therapeutics targeting somatostatin receptors.

Mechanical gating of the auditory transduction channel TMC1 involves the fourth and sixth transmembrane helices

The transmembrane channel-like (TMC) 1 and 2 proteins play a central role in auditory transduction, forming ion channels that convert sound into electrical signals. However, the molecular mechanism of their gating remains unknown. Here, using predicted structural models as a guide, we probed the effects of twelve mutations on the mechanical gating of the transduction currents in native hair cells of Tmc1/2-null mice expressing virally introduced TMC1 variants. Whole-cell electrophysiological recordings revealed that mutations within the pore-lining transmembrane (TM) helices 4 and 6 modified gating, reducing the force sensitivity or shifting the open probability of the channels, or both. For some of the mutants, these changes were accompanied by a change in single-channel conductance. Our observations are in line with a model wherein conformational changes in the TM4 and TM6 helices are involved in the mechanical gating of the transduction channel.

Upstream charged and hydrophobic residues impact the timing of membrane insertion of transmembrane helices

During SecYEG-mediated cotranslational insertion of membrane proteins, transmembrane helices (TMHs) first make contact with the membrane when their N-terminal end is ~45 residues away from the peptidyl transferase center. However, we recently uncovered instances where the first contact is delayed by up to ~10 residues. Here, we recapitulate these effects using a model TMH fused to two short segments from the BtuC protein: a positively charged loop and a re-entrant loop. We show that the critical residues are two Arg residues in the positively charged loop and four hydrophobic residues in the re-entrant loop. Thus, both electrostatic and hydrophobic interactions involving sequence elements that are not part of a TMH can impact the way the latter behaves during membrane insertion.

Fusion protein strategies for cryo-EM study of G protein-coupled receptors

Single particle cryogenic-electron microscopy (cryo-EM) is used extensively to determine structures of activated G protein-coupled receptors (GPCRs) in complex with G proteins or arrestins. However, applying it to GPCRs without signaling proteins remains challenging because most receptors lack structural features in their soluble domains to facilitate image alignment. In GPCR crystallography, inserting a fusion protein between transmembrane helices 5 and 6 is a highly successful strategy for crystallization. Although the similar strategy has the potential to broadly facilitate cryo-EM structure determination of GPCRs alone without signaling protein, the critical determinants that make this approach successful are not yet clear. Here, we address this shortcoming by exploring different fusion protein designs, which led to structures of antagonist bound A2A adenosine receptor at 3.4&Aring resolution and unliganded Smoothened at 3.7&Aring resolution. The fusion strategies explored here are likely applicable to cryo-EM interrogation of other GPCRs and small integral membrane proteins.

The structure of HiSiaQM defines the architecture of tripartite ATP-independent periplasmic (TRAP) transporters

SummaryTripartite ATP-independent periplasmic (TRAP) transporters are widespread in bacteria and archaea and provide important uptake routes for many metabolites 1–3. They consist of three structural domains, a soluble substrate-binding protein (P-domain), and two transmembrane domains (Q- and M-domains) that form a functional unit 4. While the structures of the P-domains are well-known, an experimental structure of any QM-domain has been elusive. HiSiaPQM is a TRAP transporter for the monocarboxylate sialic acid, which plays a key role in the virulence of pathogenic bacteria 5. Here, we present the first cryo-electron microscopy structure of the membrane domains of HiSiaPQM reconstituted in lipid nanodiscs. The reconstruction reveals that TRAP transporters consist of 15 transmembrane helices and are structurally related to elevator-type transporters, such as GltPh and VcINDY 6, 7. Whereas the latter proteins function as multimers, the idiosyncratic Q-domain of TRAP transporters enables the formation of a monomeric elevator architecture. Structural and mutational analyses together with an AlphaFold 8 model of the tripartite (PQM) complex reveal the structural and conformational coupling of the substrate-binding protein to the transporter domains. Furthermore, we characterize high-affinity VHHs that bind to the periplasmic side of HiSiaQM and inhibit sialic acid uptake in vivo. Thereby, they also confirm the orientation of the protein in the membrane. Our study provides the first structure of any binding-protein dependent secondary transporter and provides starting points for the development of specific inhibitors.

The SPICA Coarse-Grained Force Field for Proteins and Peptides

ABSTRACTA coarse-grained (CG) model for peptides and proteins was developed as an extension of the SPICA (Surface Property fItting Coarse grAined) force field (FF). The model was designed to examine membrane proteins that are fully compatible with the lipid membranes of the SPICA FF. A preliminary version of this protein model was created using thermodynamic properties, including the surface tension and density in the SPICA (formerly called SDK) FF. In this study, we improved the CG protein model to facilitate molecular dynamics (MD) simulation with a reproduction of multiple properties from both experiments and all-atom (AA) simulations. The side chain analogs reproduced the transfer free energy profiles across the lipid membrane and demonstrated reasonable dimerization free energies in water compared to those from AA-MD. A series of peptides/proteins adsorbed or penetrated into the membrane simulated by the CG-MD correctly predicted the penetration depths and tilt angles of peripheral and transmembrane peptides/proteins comparable to those in the orientation of protein in membrane (OPM) database. In addition, the dimerization free energies of several transmembrane helices within a lipid bilayer were comparable to those from experimental estimation. Application studies on a series of membrane protein assemblies, scramblases, and poliovirus capsids demonstrated a good performance of the SPICA FF.

Folding and Insertion of Transmembrane Helices at the ER

In eukaryotic cells, the endoplasmic reticulum (ER) is the entry point for newly synthesized proteins that are subsequently distributed to organelles of the endomembrane system. Some of these proteins are completely translocated into the lumen of the ER while others integrate stretches of amino acids into the greasy 30 Å wide interior of the ER membrane bilayer. It is generally accepted that to exist in this non-aqueous environment the majority of membrane integrated amino acids are primarily non-polar/hydrophobic and adopt an α-helical conformation. These stretches are typically around 20 amino acids long and are known as transmembrane (TM) helices. In this review, we will consider how transmembrane helices achieve membrane integration. We will address questions such as: Where do the stretches of amino acids fold into a helical conformation? What is/are the route/routes that these stretches take from synthesis at the ribosome to integration through the ER translocon? How do these stretches ‘know’ to integrate and in which orientation? How do marginally hydrophobic stretches of amino acids integrate and survive as transmembrane helices?