Anchoring of Surface Proteins to the Cell Wall of Staphylococcus aureus. III. Lipid II is an in vivo peptidoglycan substrate for sortase-catalyzed surface protein anchoring

Surface proteins of Staphylococcus aureus are anchored to the cell wall peptidoglycan by a mechanism requiring a C-terminal sorting signal with an LPXTG motif. Surface proteins are first synthesized in the bacterial cytoplasm and then transported across the cytoplasmic membrane. Cleavage of the N-terminal signal peptide of the cytoplasmic surface protein P1 precursor generates the extracellular P2 species, which is the substrate for the cell wall anchoring reaction. Sortase, a membrane-anchored transpeptidase, cleaves P2 between the threonine (T) and the glycine (G) of the LPXTG motif and catalyzes the formation of an amide bond between the carboxyl group of threonine and the amino group of cell wall cross-bridges. We have used metabolic labeling of staphylococcal cultures with [32P]phosphoric acid to reveal a P3 intermediate. The 32P-label of immunoprecipitated surface protein is removed by treatment with lysostaphin, a glycyl-glycine endopeptidase that separates the cell wall anchor structure. Furthermore, the appearance of P3 is prevented in the absence of sortase or by the inhibition of cell wall synthesis. 32P-Labeled cell wall anchor species bind to nisin, an antibiotic that is known to form a complex with lipid II. Thus, it appears that the P3 intermediate represents surface protein linked to the lipid II peptidoglycan precursor. The data support a model whereby lipid II-linked polypeptides are incorporated into the growing peptidoglycan via the transpeptidation and transglycosylation reactions of cell wall synthesis, generating mature cell wall-linked surface protein

Two-dimensional protein crystallization via metal-ion coordination by naturally occurring surface histidines

A powerful and potentially general approach to the targeting and crystallization of proteins on lipid interfaces through coordination of surface histidine residues to lipid-chelated divalent metal ions is presented. This approach, which should be applicable to the crystallization of a wide range of naturally occurring or engineered proteins, is illustrated here by the crystallization of streptavidin on a monolayer of an iminodiacetate-Cu(II) lipid spread at the air-water interface. This method allows control of the protein orientation at interfaces, which is significant for the facile production of highly ordered protein arrays and for electron density mapping in structural analysis of two-dimensional crystals. Binding of native streptavidin to the iminodiacetate-Cu lipids occurs via His-87, located on the protein surface near the biotin binding pocket. The two-dimensional streptavidin crystals show a previously undescribed microscopic shape that differs from that of crystals formed beneath biotinylated lipids

Філософія популізму як варіант сучасної філософії

We have previously reported on the functional interaction of Lipid II with human alpha-defensins, a class of antimicrobial peptides. Lipid II is an essential precursor for bacterial cell wall biosynthesis and an ideal and validated target for natural antibiotic compounds. Using a combination of structural, functional and in silico analyses, we present here the molecular basis for defensin-Lipid II binding. Based on the complex of Lipid II with Human Neutrophil peptide-1, we could identify and characterize chemically diverse low-molecular weight compounds that mimic the interactions between HNP-1 and Lipid II. Lead compound BAS00127538 was further characterized structurally and functionally; it specifically interacts with the N-acetyl muramic acid moiety and isoprenyl tail of Lipid II, targets cell wall synthesis and was protective in an in vivo model for sepsis. For the first time, we have identified and characterized low molecular weight synthetic compounds that target Lipid II with high specificity and affinity. Optimization of these compounds may allow for their development as novel, next generation therapeutic agents for the treatment of Gram-positive pathogenic infections

Pressure Induced Topological Phase Transitions in Membranes

Some highly unusual features of a lipid-water liquid crystal are revealed by high pressure x-ray diffraction, light scattering and dilatometric studies of the lamellar (bilayer $L_{\alpha}$) to nonlamellar inverse hexagonal ($H_{II}$) phase transition. (i) The size of the unit cell of the $H_{II}$ phase increases with increasing pressure. (ii) The transition volume, $\Delta V_{bh}$, decreases and appears to vanish as the pressure is increased. (iii) The intensity of scattered light increases as $\Delta V_{bh}$ decreases. Data are presented which suggest that this increase is due to the formation of an intermediate cubic phase, as predicted by recent theoretical suggestions of the underlying universal phase sequence.Comment: 12 pages, typed using REVTEX 2.

Kinetics and mechanism of the interconversion of inverse bicontinuous cubic mesophases

This paper describes time-resolved x-ray diffraction data monitoring the transformation of one inverse bicontinuous cubic mesophase into another, in a hydrated lipid system. The first section of the paper describes a mechanism for the transformation that conserves the topology of the bilayer, based on the work of Charvolin and Sadoc, Fogden and Hyde, and Benedicto and O'Brien in this area. We show a pictorial representation of this mechanism, in terms of both the water channels and the lipid bilayer. The second section describes the experimental results obtained. The system under investigation was 2:1 lauric acid: dilauroylphosphatidylcholine at a hydration of 50% water by weight. A pressure-jump was used to induce a phase transition from the gyroid (Q(II)(G)) to the diamond (Q(II)(D)) bicontinuous cubic mesophase, which was monitored by time-resolved x-ray diffraction. The lattice parameter of both mesophases was found to decrease slightly throughout the transformation, but at the stage where the Q(II)(D) phase first appeared, the ratio of lattice parameters of the two phases was found to be approximately constant for all pressure-jump experiments. The value is consistent with a topology-preserving mechanism. However, the polydomain nature of our sample prevents us from confirming that the specific pathway is that described in the first section of the paper. Our data also reveal signals from two different intermediate structures, one of which we have identified as the inverse hexagonal (H-II) mesophase. We suggest that it plays a role in the transfer of water during the transformation. The rate of the phase transition was found to increase with both temperature and pressure-jump amplitude, and its time scale varied from the order of seconds to minutes, depending on the conditions employed

Compatibility between shape equation and boundary conditions of lipid membranes with free edges

Only some special open surfaces satisfying the shape equation of lipid membranes can be compatible with the boundary conditions. As a result of this compatibility, the first integral of the shape equation should vanish for axisymmetric lipid membranes, from which two theorems of non-existence are verified: (i) There is no axisymmetric open membrane being a part of torus satisfying the shape equation; (ii) There is no axisymmetric open membrane being a part of a biconcave discodal surface satisfying the shape equation. Additionally, the shape equation is reduced to a second-order differential equation while the boundary conditions are reduced to two equations due to this compatibility. Numerical solutions to the reduced shape equation and boundary conditions agree well with the experimental data [A. Saitoh \emph{et al.}, Proc. Natl. Acad. Sci. USA \textbf{95}, 1026 (1998)].Comment: 6 journal pages, 4 figure

A Python based automated tracking routine for myosin II filaments

The study of motor protein dynamics within cytoskeletal networks is of high interest to physicists and biologists to understand how the dynamics and properties of individual motors lead to cooperative effects and control of overall network behaviour. Here, we report a method to detect and track muscular myosin II filaments within an actin network tethered to supported lipid bilayers. Based on the characteristic shape of myosin II filaments, this automated tracking routine allowed us to follow the position and orientation of myosin II filaments over time, and to reliably classify their dynamics into segments of diffusive and processive motion based on the analysis of displacements and angular changes between time steps. This automated, high throughput method will allow scientists to efficiently analyse motor dynamics in different conditions, and will grant access to more detailed information than provided by common tracking methods, without any need for time consuming manual tracking or generation of kymographs