Antimicrobial properties of distinctin in an experimental model of MRSA-infected wounds.

The aim of this study was to evaluate the efficacy of distinctin in the management of cutaneous methicillin-resistant Staphylococcus aureus (MRSA) wound infections in an experimental mouse model. Wounds, made in the panniculus carnosus of BALB/c mice, were inoculated with 5 × 10(7) colony-forming units (CFU) of MRSA. Mice were treated with topical distinctin (1 mg/kg of body weight), topical teicoplanin (7 mg/kg of body weight), intraperitoneal teicoplanin (7 mg/kg of body weight); topical teicoplanin and daily intraperitoneal teicoplanin; topical distinctin and daily intraperitoneal teicoplanin. Bacterial cultures of excised tissues and histological examination of microvessel density and of vascular endothelial growth factor (VEGF) expression were studied. It was found that topical distinctin combined with parenteral teicoplanin inhibited bacterial growth to levels comparable with those observed in uninfected animals. Wounded areas of animals treated with distinctin were characterized by a more mature granulation tissue, with a more organized and denser type of connective tissue, compared to mice treated only with teicoplanin. Treatment with topical distinctin had a significant impact on VEGF expression and microvessel density. The combined use of distinctin with teicoplanin may be useful in the management of infected wounds by significantly inhibiting bacterial growth and accelerating the repair process.

Structural dynamics and topology of phospholamban in oriented lipid bilayers using multidimensional solid-state NMR.

Phospholamban (PLN), a single-pass membrane protein, regulates heart muscle contraction and relaxation by reversible inhibition of the sarco(endo)plasmic reticulum Ca-ATPase (SERCA). Studies in detergent micelles and oriented lipid bilayers have shown that in its monomeric form PLN adopts a dynamic L shape (bent or T state) that is in conformational equilibrium with a more dynamic R state. In this paper, we use solid-state NMR on both uniformly and selectively labeled PLN to refine our initial studies, describing the topology and dynamics of PLN in oriented lipid bilayers. Two-dimensional PISEMA (polarization inversion spin exchange at the magic angle) experiments carried out in DOPC/DOPE mixed lipid bilayers reveal a tilt angle of the transmembrane domain with respect to the static magnetic field, of 21 +/- 2 degrees and, at the same time, map the rotation angle of the transmembrane domain with respect to the bilayer. PISEMA spectra obtained with selectively labeled samples show that the cytoplasmic domain of PLN is helical and makes an angle of 93 +/- 6 degrees with respect to the bilayer normal. In addition, using samples tilted by 90 degrees , we find that the transmembrane domain of PLN undergoes fast long-axial rotational diffusion about the bilayer normal with the cytoplasmic domain undergoing this motion and other complex dynamics, scaling the values of chemical shift anisotropy. While this dynamic was anticipated by previous solution NMR relaxation studies in micelles, these measurements in the anisotropic lipid environment reveal new dynamic and conformational features encoded in the free protein that might be crucial for SERCA recognition and subsequent inhibition.

Effects of Ser16 phosphorylation on the allosteric transitions of phospholamban/Ca(2+)-ATPase complex.

Phosphorylation by protein kinase A and dephosphorylation by protein phosphatase 1 modulate the inhibitory activity of phospholamban (PLN), the endogenous regulator of the sarco(endo)plasmic reticulum calcium Ca(2+) ATPase (SERCA). This cyclic mechanism constitutes the driving force for calcium reuptake from the cytoplasm into the myocite lumen, regulating cardiac contractility. PLN undergoes a conformational transition between a relaxed (R) and tense (T) state, an equilibrium perturbed by the addition of SERCA. Here, we show that the single phosphoryl transfer at Ser16 induces a more pronounced conformational switch to the R state in phosphorylated PLN (pPLN). The binding affinity of PLN to SERCA is not affected (K(d) values for the transmembrane domains of pPLN and PLN are approximately 60 microM), supporting the hypothesis that phosphorylation at Ser16 does not dissociate PLN from SERCA. However, the binding surface and dynamics in domain Ib (residues 22-31) change substantially upon phosphorylation. Since PLN can be singly or doubly phosphorylated at Ser16 and Thr17, we propose that these sites remotely control the conformation of domain Ib. These findings constitute a paradigm for how post-translational modifications such as phosphorylation in the cytoplasmic portion of membrane proteins control intramembrane protein-protein interactions.

Functional and structural properties of stannin: roles in cellular growth, selective toxicity, and mitochondrial responses to injury.

Stannin (Snn) was discovered using subtractive hybridization methodology designed to find gene products related to selective organotin toxicity and apoptosis. The cDNAs for Snn were first isolated from brain tissues sensitive to trimethyltin, and were subsequently used to localize, characterize, and identify genomic DNA, and other gene products of Snn. Snn is a highly conserved, 88 amino acid protein found primarily in vertebrates. There is a minor divergence in the C-terminal sequence between amphibians and primates, but a nearly complete conservation of the first 60 residues in all vertebrates sequenced to date. Snn is a membrane-bound protein and is localized, in part, to the mitochondria and other vesicular organelles, suggesting that both localization and conservation are significant for the overall function of the protein. The structure of Snn in a micellar environment and its architecture in lipid bilayers have been determined using a combination of solution and solid-state NMR, respectively. Snn structure comprised a single transmembrane domain (residues 10-33), a 28-residue linker region from residues 34-60 that contains a conserved CXC metal binding motif and a putative 14-3-3xi binding region, and a cytoplasmic helix (residues 61-79), which is partially embedded into the membrane. Of primary interest is understanding how this highly-conserved peptide with an interesting structure and cellular localization transmits both normal and potentially toxic signals within the cell. Evidence to date suggests that organotins such as trimethyltin interact with the CXC region of Snn, which is vicinal to the putative 14-3-3 binding site. In vitro transfection analyses and microarray experiments have inferred a possible role of Snn in several key signaling systems, including activation of the p38-ERK cascade, p53-dependent pathways, and 14-3-3xi protein-mediated processes. TNFalpha can induce Snn mRNA expression in endothelial cells in a PKC-epsilon dependent manner. Studies with Snn siRNA suggest that this protein may be involved in growth regulation, since inhibition of Snn expression alone leads to reduced endothelial cells growth and induction of COP-1, a negative regulator of p53 function. A key piece of the puzzle, however, is how and why such a highly-conserved protein, localized to mitochondria, interacts with other regulatory proteins to alter growth and apoptosis. By knowing the structure, location, and possible signaling pathways involved, we propose that Snn constitutes an important sensor of mitochondrial damage, and plays a key role in the mediation of cross-talk between mitochondrial and nuclear compartments in specific cell types.

Mapping the interaction surface of a membrane protein: unveiling the conformational switch of phospholamban in calcium pump regulation.

We have used magnetic resonance to map the interaction surface of an integral membrane protein for its regulatory target, an integral membrane enzyme. Phospholamban (PLN) regulates cardiac contractility via its modulation of sarco(endo)plasmic reticulum calcium ATPase (SERCA) activity. Impairment of this regulatory process causes heart failure. To map the molecular details of the PLN/SERCA interaction, we have functionally reconstituted SERCA with labeled PLN in dodecylphosphocholine micelles for high-resolution NMR spectroscopy and in both micelles and lipid bilayers for EPR spectroscopy. Differential perturbations in NMR linewidths and chemical shifts, measured as a function of position in the PLN sequence, provide a vivid picture of extensive SERCA contacts in both cytoplasmic and transmembrane domains of PLN and provide structural insight into previously reported functional mutagenesis data. NMR and EPR data show clear and complementary evidence for a dynamic (micros-to-ms) equilibrium between two conformational states in the cytoplasmic domain of PLN. These results support the hypothesis that SERCA attracts the cytoplasmic domain of PLN away from the lipid surface, shifting the preexisting equilibrium of PLN conformers toward a structure that is poised to interact with the regulatory target. EPR shows that this conformational switch behaves similarly in micelles and lipid membranes. Based on structural and dynamics data, we propose a model in which PLN undergoes allosteric activation upon encountering SERCA.

Structure and topology of a peptide segment of the 6th transmembrane domain of the Saccharomyces cerevisae alpha-factor receptor in phospholipid bilayers.

A detailed analysis of the structure of an 18-residue peptide AQSLLVPSIIFILAYSLK [M6(252-269, C252A)] in 1,2-dimyristoyl-sn-glycero-phosphocholine bilayers was carried out using solid state NMR and attenuated total reflection Fourier transform infrared spectroscopy. The peptide corresponds to a portion of the 6th transmembrane domain of the alpha-factor receptor of Saccharomyces cerevisiae. Ten homologs of M6(252-269, C252A) were synthesized in which individual residues were labeled with (15)N. One- and two-dimensional solid state NMR experiments were used to determine the chemical shifts and (1)H-(15)N dipolar coupling constants for the (15)N-labeled peptides in oriented dimyristoylphosphatidylcholine bilayers on stacked glass plates. These parameters were used to calculate the structure and orientation of M6(252-269, C252A) in the bilayers. The results indicate that the carboxyl terminal residues (9-14) are alpha-helical and oriented with an angle of about 8 degrees with respect to the bilayer normal. Independently, an attenuated total reflection Fourier transform infrared spectroscopy analysis on M6(252-269, C252A) in a 1,2-dimyristoyl-sn-glycero-phosphocholine bilayer concluded that the helix tilt angle was about 12.5 degrees. The results on the structure of M6(252-269, C252A) in bilayers are in good agreement with the structure determined in trifluoroethanol/water solutions (B. Arshava et al. Biopolymers, 1998, Vol. 46, pp. 343-357). The present study shows that solid state NMR spectroscopy can provide high resolution information on the structure of transmembrane domains of a G protein-coupled receptor.

A synthetic peptide corresponding to the 550-585 region of alpha-dystroglycan binds beta-dystroglycan as revealed by NMR spectroscopy.

We have probed the binding of a synthetic peptide corresponding to the region 550-585 of the alpha subunit of dystroglycan with a recombinant protein fragment corresponding to the N-terminal extracellular region of beta-dystroglycan (654-750), using NMR in solution. In a 30:1 molar ratio, the peptide binds to the recombinant protein fragment in the fast/intermediate exchange regime. By monitoring the peptide intra-residue HN-Halpha peak volumes of the 2D TOCSY NMR spectra, both in the absence and in the presence of the recombinant fragment, we determined the differential binding affinities of each amino acid. We found that the residues in the region 550-565 (SWVQFNSNSQLMYGLP) are more influenced by the presence of the protein, whereas the C-terminal portion is marginally involved. These NMR results have been confirmed by solid-phase binding assays.

High-resolution structure and conformational dynamics of rigid, cofacially aligned porphyrin-bridge-quinone systems as determined by NMR spectroscopy and ab initio simulated annealing calculations.

The high-resolution solution structure and dynamics of a cofacially aligned porphyrin--phenylene--quinone compound have been determined using (1)H NMR spectroscopy and simulated annealing calculations. Members of this class of pi-stacked assemblies feature a 1,8-naphthyl pillaring motif that enforces sub van der Waals interplanar separations between juxtaposed porphyryl, aromatic bridge, and quinonyl components of the donor--spacer--acceptor compound; this structural motif gives rise to a comprehensive set of structurally significant NOE signatures that can be used as constraints in quantitative structural calculations. Examination of such data using ab initio simulated annealing analytical methods shows that 5-[8'-(4' '-[8' "-(2' " ',5' "-benzoquinonyl)-1' "-naphthyl]-1' '-phenyl)-1'-naphthyl]-10,20-diphenylporphyrin displays an unusual degree of conformational homogeneity in the condensed phase, and represents a rare example where such an analysis determines unequivocally a single unique structure in solution.

Solution conformation of the Pseudomonas syringae MSU 16H phytotoxic lipodepsipeptide Pseudomycin A determined by computer simulations using distance geometry and molecular dynamics from NMR data.

Pseudomycin A is a cyclic lipodepsinonapeptide phytotoxin produced by a strain of the plant pathogenic bacterium Pseudomonas syringae. Like other members of this family of bacterial metabolites, it is characterised by a fatty acylated cyclic peptide with mixed chirality and lactonic closure. Several biological activities of Pseudomycin A are lower than those found for some of its congeners, a difference which might depend on the diverse number and distribution of charged residues in the peptide moiety. Hence, it was of interest to investigate its conformation in solution. After the complete interpretation of the two-dimensional NMR spectra, NOE data were obtained and the structure was determined by computer simulations, applying distance geometry and molecular dynamics procedures. The conformation of the large ring of Pseudomycin A in solution includes three rigid structural regions interrupted by three short flexible regions that act as hinges. The overall three-dimensional structure of the cyclic moiety is similar to that of previously studied bioactive lipodepsinonapeptides produced by other pseudomonads.

1H NMR studies on the interaction of beta-carboline derivatives with human serum albumin.

1H NMR studies were performed on two beta-carboline derivatives interacting with human serum albumin. The spin-lattice relaxation rates of the two derivatives, having side chains of different length and polarity, were used to demonstrate a diverse motional behavior in solution together with slightly different relaxation pathways. Single- and double-selective excitation made it possible to evaluate dynamics in the free and protein-bound states. Occurrence of a relatively long hydrophilic chain interacting with the proton-acceptor nitrogen of the beta-carboline moiety was shown to yield lower association constants, slower dissociation rates, and diverse interacting modes with the indole hydrophobic site of the protein.

Conformational study of [Met5]enkephalin-Arg-Phe in the presence of phosphatidylserine vesicles.

The interaction of [Met5]enkephalin-Arg.Phe with phosphatidylserine (PtdSer) was studied by circular dichroism (CD), two-dimensional nuclear magnetic resonance spectroscopy, hybrid distance geometry simulated annealing (DG-SA) and molecular dynamics (MD) calculations. The very low solubility of [Met5]enkephalin-Arg-Phe and the instability of the solution containing PtdSer vesicles at low pH values did not allow us to observe the amide proton resonances in the usual two-dimensional NMR work. NOESY cross-peaks of protons of side chains from two-dimensional NMR were converted into distances which were used as restraints for modelling with DG-SA and MD. Our results indicate that, in aqueous solutions at pH 7.68 [Met5]enkephalin-Arg-Phe exists in the absence of PtdSer as a random distribution of conformers, whereas in the presence of PtdSer it adopts conformations containing a common orientation of the bonds of C alpha 2, C alpha 3, C alpha 4, and C alpha 5, although different orientations of the peptide planes are consistent with the results. Two of the reported conformers from MD simulations are characterized by the presence of a 2<--4 gamma and inverse gamma turns centered on Gly3. A gradual decline of order was observed when moving from the central moiety of the peptide to both the N-terminus and C-terminus. Finally, the DG-SA and MD calculations resulted in a structure such that the orientation of the Phe4 and Met5 side chains favours hydrophobic interactions with the apolar portion of the PtdSer vesicle to form a hydrophobic cluster. These data support the hypothesis of a role of lipids to modify the conformation of [Met5]enkephalin-Arg-Phe to permit the interactions with the receptor site.