The Roles of Pore Ring and Plug in the SecY Protein-conducting Channel

The protein-conducting channel, or translocon, is an evolutionarily conserved complex that allows nascent proteins to cross a cellular membrane or integrate into it. The crystal structure of an archaeal translocon, the SecY complex, revealed that two elements contribute to sealing the channel: a small “plug” domain blocking the periplasmic region of the channel, and a pore ring composed of six hydrophobic residues acting as a constriction point at the channel's center. To determine the independent functions of these two elements, we have performed molecular dynamics simulations of the native channel as well as of two recently structurally resolved mutants in which portions of their plugs were deleted. We find that in the mutants, the instability in the plug region leads to a concomitant increase in flexibility of the pore ring. The instability is quantified by the rate of water permeation in each system as well as by the force required for oligopeptide translocation. Through a novel simulation in which the interactions between the plug and water were independently controlled, we find that the role of the plug in stabilizing the pore ring is significantly more important than its role as a purely steric barrier

Coupling of Ca2+ and Substrate Binding in the Outer Membrane Transporter BtuB

Aprovat per la Gerència del Consorci el 22-12-201

Architecture and assembly of the Gram-positive cell wall

The bacterial cell wall is a mesh polymer of peptidoglycan – linear glycan strands cross-linked by flexible peptides – that determines cell shape and provides physical protection. While the glycan strands in thin ‘Gram-negative’ peptidoglycan are known to run circumferentially around the cell, the architecture of the thicker ‘Gram-positive’ form remains unclear. Using electron cryotomography, here we show that Bacillus subtilis peptidoglycan is a uniformly dense layer with a textured surface. We further show it rips circumferentially, curls and thickens at free edges, and extends longitudinally when denatured. Molecular dynamics simulations show that only atomic models based on the circumferential topology recapitulate the observed curling and thickening, in support of an ‘inside-to-outside’ assembly process. We conclude that instead of being perpendicular to the cell surface or wrapped in coiled cables (two alternative models), the glycan strands in Gram-positive cell walls run circumferentially around the cell just as they do in Gram-negative cells. Together with providing insights into the architecture of the ultimate determinant of cell shape, this study is important because Gram-positive peptidoglycan is an antibiotic target crucial to the viability of several important rod-shaped pathogens including Bacillus anthracis, Listeria monocytogenes, and Clostridium difficile

Assembly and Architecture of Gram-Positive and -Negative Cell Walls

The cell wall, a porous mesh-like structure, provides shape and physical protection for bacteria. At the atomic level, it is composed of peptidoglycan (PG), a polymer of stiff glycan strands cross-linked by short, flexible peptides. However, at the mesoscale, multiple models for the organization of PG have been put forth, distinguished by glycan strands parallel to the cell surface (the so-called "layered'' model) or perpendicular (the “scaffold” model). To test these models, and to resolve the mechanical properties of PG, we have built and simulated at an atomic scale patches of both Gram-positive and negative cell walls in different organizations up to 50 nanometers in size. In the case of Gram-positive PG, molecular dynamics simulations of the layered model are found to elucidate the mechanisms behind a distinct curling effect observed in three-dimensional electron cryo-tomography images of fragmented cell walls. For Gram-negative PG, simulations of patches with different average-glycan-strand lengths reveal an anisotropic elasticity, in good agreement with atomic-force microscopy experiments. Insights from the simulations reveal how mesoscopic and macroscopic properties of a ubiquitous bacterial ultrastructure arise from its atomic-scale interactions and organization

<i>Escherichia coli</i> Peptidoglycan Structure and Mechanics as Predicted by Atomic-Scale Simulations

Bacteria face the challenging requirement to maintain their shape and avoid rupture due to the high internal turgor pressure, but simultaneously permit the import and export of nutrients, chemical signals, and virulence factors. The bacterial cell wall, a mesh-like structure composed of cross-linked strands of peptidoglycan, fulfills both needs by being semi-rigid, yet sufficiently porous to allow diffusion through it. How the mechanical properties of the cell wall are determined by the molecular features and the spatial arrangement of the relatively thin strands in the larger cellular-scale structure is not known. To examine this issue, we have developed and simulated atomic-scale models of Escherichia coli cell walls in a disordered circumferential arrangement. The cell-wall models are found to possess an anisotropic elasticity, as known experimentally, arising from the orthogonal orientation of the glycan strands and of the peptide cross-links. Other features such as thickness, pore size, and disorder are also found to generally agree with experiments, further supporting the disordered circumferential model of peptidoglycan. The validated constructs illustrate how mesoscopic structure and behavior emerge naturally from the underlying atomic-scale properties and, furthermore, demonstrate the ability of all-atom simulations to reproduce a range of macroscopic observables for extended polymer meshes.</p

Non-detergent isolation of a cyanobacterial photosystem I using styrene maleic acid alternating copolymers

Photosystem I (PSI) from the thermophilic cyanobacterium Thermosynechococcus elongatus (Te) is the largest membrane protein complex to have had its structure solved by X-ray diffraction. This trimeric complex has 36 protein subunits, over 380 non-covalently bound cofactors and a molecular weight of ∼1.2 MDa. Previously, it has been isolated and characterized in a detergent micelle using the non-ionic detergent n-dodecyl-β-D-maltoside (DDM). We have now succeeded in isolating this complex without the use of detergents, using styrene–maleic acid (SMA) alternating copolymer. Intriguingly, a partially esterified copolymer formulation (SMA 1440, Cray Valley) was found to be most efficient in cyanobacterial thylakoid membranes. A host of biochemical, biophysical and functional assays have been applied to characterize this non-detergent form of PSI, referred to as a SMA Lipid Particle (SMALP). The PSI-SMALP has a lower sedimentation coefficient compared to PSI-DDM, suggesting decreased density or a more extended particle shape. We show the 77 K fluorescence maximum for PSI is red shifted in PSI-SMALP compared to PSI-DDM, suggesting a more native orientation of PsaA/B associated chlorophyll. We report that PSI-SMALPs are functional despite the selective loss of one transmembrane subunit, PsaF. This loss may reflect a more labile interaction of the PSI core and PsaF, or a selective displacement during copolymer insertion and/or assembly. PSI-SMALP exhibited decreased reduction kinetics with native recombinant cytochromes c6, while non-native horse heart cytochrome c shows faster reduction of PSI-SMALP compared to PSI-DDM. This is the largest membrane protein isolated using SMA copolymers, and this study expands the potential use of this approach for the isolation and characterization of large supramolecular complexes

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