The structure suggests that the ribosome alone, even without a nascent chain, is sufficient for lateral opening of Sec61 in a lipid environment, independent of its functional state.
Biological membranes are usually asymmetric lipid bilayers that possess an oligosaccharide coat on the outside into which loops of transmembrane proteins extend. Structurally, the protein-bilayer interface is not well explored. As a paradigmatic example, we examine the structure of the bacterial outer membrane protein G (OmpG) expressed into E. coli outer membranes (OM). We investigate two strands of the β-sheet and loops 6 and 7 that extend into the lipopolysaccharide (LPS) layer and which are involved in pore opening (pH > 6.0) and closing (pH < 6.0). Previous x-ray crystallography studies described these loops as well-ordered, whereas solid-state NMR described them as fully disordered, largely due to a lack of signal. Here, solid-state NMR measurements of OmpG in OM vesicles at 1.2 GHz 1H frequency now yield extended sets of cross peaks for His and Trp residues. Two alternative conformations are observed for two His residues, which are likely H231 and H261. Trp sidechain cross peak intensities in cross polarization spectra are in agreement with an increase of conformational freedom toward the surface. The oligosaccharide layer appears to protect the loops while promoting flexibility and multiple conformations. Our investigation highlights the importance of structural investigations in a quasi-native environment.
The Sec61 complex plays a critical role in protein translocation into the endoplasmic reticulum (ER), enabling the passage of soluble proteins into the lumen and the lateral insertion of membrane proteins. The Sec complex, comprising Sec61, Sec63, Sec62, and other auxiliary proteins, is especially important for the translocation of precursor proteins with inefficient signal sequences (SSs) that require additional assistance to trigger the opening of the Sec61 channel. In this study, we investigated how the hydrophobicity of SSs affects the interaction with the Sec complex at the single-molecule level using optical tweezers. We analyzed three SSs representing low, moderate, and high hydrophobicity in the h-regions. Our results show that the highly hydrophobic SS from DPAP-B pαF forms a less stable bond with the Sec complex, evidenced by shorter interaction time and reduced energy barrier to the unbound state. In contrast, SSs with lower hydrophobicity establish more stable interactions, characterized by extended interaction times, and elevated energy barriers to reach the unbound state. These findings suggest that the Sec complex may selectively recognize and interact with SSs based on their hydrophobicity, offering insights into the mechanisms by which defects in SS translocation contribute to ER-associated diseases.
W. Sánchez, Luka Robeson, Hilda M. Alfaro-Valdés et al.· Biochemical and Biophysical...· 0 citations
Regarded as one of the most complex membrane protein folds, CLCs form a large family of membrane proteins that function as anion channels and secondary active anion/proton transporters. Despite low sequence similarity, available structures are remarkably similar, maintaining the same inverted topology and fold, with subunits assembled as dimers in wild-type structures. Because of these strong structural features, CLC-ec1, a prokaryotic homolog from E. coli, has become a highly valuable model system for studying membrane protein folding and oligomerization assembly. Associating via a membrane-embedded dimerization interface, the subunits participate in a dynamic equilibrium between monomers and dimers in lipid bilayers, enabling investigation of the physical driving forces underlying the formation of stable membrane complexes. Like soluble protein assembly, studies indicate that CLC-ec1 dimerization is driven by a solvophobic force arising from the free energy gained by burying lipid bilayer defects. Dimerization stability is influenced by lipid composition and pH, which, in turn, provide a mechanism for functional regulation through oligomerization. In this review, I provide an overview of how the exploration of CLC folding and oligomerization advances our understanding of membrane protein self-assembly in general and the role of oligomerization in regulating function.
Janice L. Robertson· Chemical Reviews· 0 citations
Almost all membrane proteins are inserted or translocated across membranes by the universally conserved Sec translocon. Despite its central role, experimental access to Sec function has remained limited. Here, we present ProSecCO (Protein Secretion in Cell-free via synthetic Operons), which is a cell-free protein synthesis platform that inserts SecYEG into synthetic vesicles, enabling direct testing of Sec in real-time and high-throughput, circumventing longstanding viability constraints. Screening 300 Sec variants in a single experiment, we consolidate three decades of Sec research, while vastly expanding mutant diversity for structure-function insights. Mapping over 30 functionally critical regions that modulate Sec activity across three orders of magnitude, we uncover dozens of super-active translocation variants and one variant of improved insertion activity. We further leverage ProSecCO to increase membrane protein quality and nanobody export, highlighting the potential of our system for advancing applications in synthetic biology and biotechnology.
M. Meier, Scott A. Scholz, Leo von Bank et al.· Science Advances· 2 citations
Solute carrier proteins (SLCs) are essential membrane protein transporters of small solutes. Among them, the SLC12 family is known to facilitate transport of ions. Members of the SLC12 family ensure cell homeostasis by co-transporting chloride alongside sodium and/or potassium across the plasma membrane. The majority of SLC12 proteins are well described, and a recent surge in structural studies facilitated by cryo-electron microscopy revealed molecular details of their function. These include multiple conformations of the transporters, covering a range of functional states and providing a window into the ion transport mechanism. Yet, only limited knowledge exists regarding their dimerization or higher order oligomerization and its role in regulation, despite SLC12 proteins consistently operating as dimers. In this review, we highlight the structural knowledgebase established in recent years and summarize the varying dimerization mechanisms. Altogether, it is becoming increasingly clear that large conformational changes in dimeric arrangements deserve attention, alongside other understudied areas like lipid interactions, nucleotide or N-terminal binding to the dimerization domains, and potential roles of the less described member SLC12A9.
Alexandra Náplavová, Poul Nissen, R. K. Flygaard· Journal of Structural Biolog...· 0 citations
P4-ATPases are lipid flippases that maintain membrane phospholipid asymmetry by transporting specific phospholipids from the exoplasmic to the cytosolic leaflet, an essential process for membrane integrity, trafficking and signaling. Several P4-ATPases are tightly regulated by autoinhibitory N- and C-terminal extensions, yet the molecular basis of this regulation remains incompletely understood. Here, we investigated the autoinhibition mechanism of the human flippase ATP8B1 using trans-inhibition assays with synthetic peptides derived from its C-terminal tail. Using purified C-terminally truncated ATP8B1-CDC50A, we systematically assessed the inhibitory properties of peptides corresponding to distinct segments of the C-terminus. We show that the distal disordered region of the C-terminal tail significantly contributes to autoinhibition, likely through transient interactions with the cytosolic domains. We further identify a critical interaction between R1228 in the C-terminal tail and E219 in the A-domain, whose disruption markedly reduces inhibitory potency. In addition, we demonstrate that a minimal peptide spanning residues 1216-1228, which bridges the A- and N-domains in the autoinhibited conformation, is sufficient to inhibit ATPase activity. Together, these results refine the molecular description of ATP8B1 autoinhibition, open the way for structure-based activation strategies and provide insight into conserved regulatory mechanisms among P4-ATPases.
Michelle Juknaviciute Laursen, Mathilde Roth, Poul Nissen et al.· Journal of Membrane Biology· 0 citations
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