216 research outputs found
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
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<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
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Structure of the SecY channel during initiation of protein translocation
Many secretory proteins are targeted by signal sequences to a protein-conducting channel, formed by prokaryotic SecY- or eukaryotic Sec61-complexes, and are translocated across the membrane during their synthesis1,2. Crystal structures of the inactive channel show that the SecY subunit of the heterotrimeric complex consists of two halves that form an hourglass-shaped pore with a constriction in the middle of the membrane and a lateral gate that faces the lipid phase3-5. The closed channel has an empty cytoplasmic funnel and an extracellular funnel that is filled with a small helical domain, called the plug. During initiation of translocation, a ribosome–nascent chain complex binds to the SecY/Sec61 complex, resulting in insertion of the nascent chain. However, the mechanism of channel opening during translocation is unclear. Here, we have addressed this question by determining structures of inactive and active ribosome–channel complexes with cryo-electron microscopy. Non-translating ribosome–SecY channel complexes derived from Methanococcus jannaschii or Escherichia coli show the channel in its closed state, and indicate that ribosome binding per se causes only minor changes. The structure of an active E. coli ribosome–channel complex demonstrates that the nascent chain opens the channel, causing mostly rigid body movements of the N- and C-terminal halves of SecY. In this early translocation intermediate, the polypeptide inserts as a loop into the SecY channel with the hydrophobic signal sequence intercalated into the open lateral gate. The nascent chain also forms a loop on the cytoplasmic surface of SecY rather than directly entering the channel
Coarse-grained simulations of bacterial cell wall growth reveal that local coordination alone can be sufficient to maintain rod shape
Bacteria are surrounded by a peptidoglycan (PG) cell wall that must be remodeled to allow cell growth. While many structural details and properties of PG and the individual enzymes involved are known, how the process is coordinated to maintain cell integrity and rod shape is not understood. We have developed a coarse-grained method to simulate how individual transglycosylases, transpeptidases, and endopeptidases could introduce new material into an existing unilayer PG network. We find that a simple model with no enzyme coordination fails to maintain cell wall integrity and rod shape. We then iteratively analyze failure modes and explore different mechanistic hypotheses about how each problem might be overcome by the macromolecules involved. In contrast to a current theory, which posits that long MreB filaments are needed to coordinate PG insertion sites, we find that local coordination of enzyme activities in individual complexes can be sufficient to maintain cell integrity and rod shape. We also present possible molecular explanations for the existence of monofunctional transpeptidases and glycosidases (glycoside hydrolases), trimeric peptide crosslinks, cell twisting during growth, and synthesis of new strands in pairs
The mobility of two kinase domains in the Escherichia coli chemoreceptor array varies with signalling state
Motile bacteria sense their physical and chemical environment
through highly cooperative, ordered arrays of
chemoreceptors. These signalling complexes phosphorylate
a response regulator which in turn governs
flagellar motor reversals, driving cells towards favourable
environments. The structural changes that translate
chemoeffector binding into the appropriate kinase
output are not known. Here, we apply high-resolution
electron cryotomography to visualize mutant chemoreceptor
signalling arrays in well-defined kinase activity
states. The arrays were well ordered in all signalling
states, with no discernible differences in receptor
conformation at 2–3 nm resolution. Differences were
observed, however, in a keel-like density that we identify
here as CheA kinase domains P1 and P2, the
phosphorylation site domain and the binding domain
for response regulator target proteins. Mutant receptor
arrays with high kinase activities all exhibited small
keels and high proteolysis susceptibility, indicative of
mobile P1 and P2 domains. In contrast, arrays in
kinase-off signalling states exhibited a range of keel
sizes. These findings confirm that chemoreceptor
arrays do not undergo large structural changes during
signalling, and suggest instead that kinase activity is
modulated at least in part by changes in the mobility of
key domains
Variational Methods for Biomolecular Modeling
Structure, function and dynamics of many biomolecular systems can be
characterized by the energetic variational principle and the corresponding
systems of partial differential equations (PDEs). This principle allows us to
focus on the identification of essential energetic components, the optimal
parametrization of energies, and the efficient computational implementation of
energy variation or minimization. Given the fact that complex biomolecular
systems are structurally non-uniform and their interactions occur through
contact interfaces, their free energies are associated with various interfaces
as well, such as solute-solvent interface, molecular binding interface, lipid
domain interface, and membrane surfaces. This fact motivates the inclusion of
interface geometry, particular its curvatures, to the parametrization of free
energies. Applications of such interface geometry based energetic variational
principles are illustrated through three concrete topics: the multiscale
modeling of biomolecular electrostatics and solvation that includes the
curvature energy of the molecular surface, the formation of microdomains on
lipid membrane due to the geometric and molecular mechanics at the lipid
interface, and the mean curvature driven protein localization on membrane
surfaces. By further implicitly representing the interface using a phase field
function over the entire domain, one can simulate the dynamics of the interface
and the corresponding energy variation by evolving the phase field function,
achieving significant reduction of the number of degrees of freedom and
computational complexity. Strategies for improving the efficiency of
computational implementations and for extending applications to coarse-graining
or multiscale molecular simulations are outlined.Comment: 36 page
Transmembrane but not soluble helices fold inside the ribosome tunnel
Integral membrane proteins are assembled into the ER membrane via a continuous ribosome-translocon channel. The hydrophobicity and thickness of the core of the membrane bilayer leads to the expectation that transmembrane (TM) segments minimize the cost of harbouring polar polypeptide backbones by adopting a regular pattern of hydrogen bonds to form α-helices before integration. Co-translational folding of nascent chains into an α-helical conformation in the ribosomal tunnel has been demonstrated previously, but the features governing this folding are not well understood. In particular, little is known about what features influence the propensity to acquire α-helical structure in the ribosome. Using in vitro translation of truncated nascent chains trapped within the ribosome tunnel and molecular dynamics simulations, we show that folding in the ribosome is attained for TM helices but not for soluble helices, presumably facilitating SRP (signal recognition particle) recognition and/or a favourable conformation for membrane integration upon translocon entry
T-Analyst: a program for efficient analysis of protein conformational changes by torsion angles
T-Analyst is a user-friendly computer program for analyzing trajectories from molecular modeling. Instead of using Cartesian coordinates for protein conformational analysis, T-Analyst is based on internal bond-angle-torsion coordinates in which internal torsion angle movements, such as side-chain rotations, can be easily detected. The program computes entropy and automatically detects and corrects angle periodicity to produce accurate rotameric states of dihedrals. It also clusters multiple conformations and detects dihedral rotations that contribute hinge-like motions. Correlated motions between selected dihedrals can also be observed from the correlation map. T-Analyst focuses on showing changes in protein flexibility between different states and selecting representative protein conformations for molecular docking studies. The program is provided with instructions and full source code in Perl
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