The high resolution structure of tyrocidine A reveals an amphipathic dimer

AbstractTyrocidine A, one of the first antibiotics ever to be discovered, is a cyclic decapeptide that binds to membranes of target bacteria, disrupting their integrity. It is active against a broad spectrum of Gram-positive organisms, and has recently engendered interest as a potential scaffold for the development of new drugs to combat antibiotic-resistant pathogens. We present here the X-ray crystal structure of tyrocidine A at a resolution of 0.95Å. The structure reveals that tyrocidine forms an intimate and highly amphipathic homodimer made up of four beta strands that associate into a single, highly curved antiparallel beta sheet. We used surface plasmon resonance and potassium efflux assays to demonstrate that tyrocidine binds tightly to mimetics of bacterial membranes with an apparent dissociation constant (KD) of 10μM, and efficiently permeabilizes bacterial cells at concentrations equal to and below the KD. Using variant forms of tyrocidine in which the fluorescent probe p-cyano-phenylalanine had been inserted on either the polar or apolar face of the molecule, we performed fluorescence quenching experiments, using both water-soluble and membrane-embedded quenchers. The quenching results, together with the structure, strongly support a membrane association model in which the convex, apolar face of tyrocidine's beta sheet is oriented toward the membrane interior, while the concave, polar face is presented to the aqueous phase

Quantizing Horava-Lifshitz Gravity via Causal Dynamical Triangulations

We extend the discrete Regge action of causal dynamical triangulations to include discrete versions of the curvature squared terms appearing in the continuum action of (2+1)-dimensional projectable Horava-Lifshitz gravity. Focusing on an ensemble of spacetimes whose spacelike hypersurfaces are 2-spheres, we employ Markov chain Monte Carlo simulations to study the path integral defined by this extended discrete action. We demonstrate the existence of known and novel macroscopic phases of spacetime geometry, and we present preliminary evidence for the consistency of these phases with solutions to the equations of motion of classical Horava-Lifshitz gravity. Apparently, the phase diagram contains a phase transition between a time-dependent de Sitter-like phase and a time-independent phase. We speculate that this phase transition may be understood in terms of deconfinement of the global gravitational Hamiltonian integrated over a spatial 2-sphere.Comment: 24 pages; 10 figure

Structural Basis for Calmodulin as a Dynamic Calcium Sensor

Calmodulin is a prototypical and versatile Ca2+ sensor with EF-hands as its high-affinity Ca2+ binding domains. Calmodulin is present in all eukaryotic cells, mediating Ca2+-dependent signaling. Upon binding Ca2+, calmodulin changes its conformation to form complexes with a diverse array of target proteins. Despite a wealth of knowledge on calmodulin, little is known on how target proteins regulate calmodulin’s ability to bind Ca2+. Here, we take advantage of two splice variants of SK2 channels, which are activated by Ca2+-bound calmodulin, but show different sensitivity to Ca2+ for their activation. Protein crystal structures and other experiments show that depending on which SK2 splice variant it binds to calmodulin adopts drastically different conformations with different affinities for Ca2+ at its C-lobe. Such target protein induced conformational changes make calmodulin a dynamic Ca2+ sensor, capable of responding to different Ca2+ concentrations in cellular Ca2+ signaling

Structural Basis for Calmodulin as a Dynamic Calcium Sensor

Calmodulin is a prototypical and versatile Ca2+ sensor with EF-hands as its high-affinity Ca2+ binding domains. Calmodulin is present in all eukaryotic cells, mediating Ca2+-dependent signaling. Upon binding Ca2+, calmodulin changes its conformation to form complexes with a diverse array of target proteins. Despite a wealth of knowledge on calmodulin, little is known on how target proteins regulate calmodulin’s ability to bind Ca2+. Here, we take advantage of two splice variants of SK2 channels, which are activated by Ca2+-bound calmodulin, but show different sensitivity to Ca2+ for their activation. Protein crystal structures and other experiments show that depending on which SK2 splice variant it binds to calmodulin adopts drastically different conformations with different affinities for Ca2+ at its C-lobe. Such target protein induced conformational changes make calmodulin a dynamic Ca2+ sensor, capable of responding to different Ca2+ concentrations in cellular Ca2+ signaling

Recognition of Anesthetic Barbiturates by a Protein Binding Site: A High Resolution Structural Analysis

Barbiturates potentiate GABA actions at the GABAA receptor and act as central nervous system depressants that can induce effects ranging from sedation to general anesthesia. No structural information has been available about how barbiturates are recognized by their protein targets. For this reason, we tested whether these drugs were able to bind specifically to horse spleen apoferritin, a model protein that has previously been shown to bind many anesthetic agents with affinities that are closely correlated with anesthetic potency. Thiopental, pentobarbital, and phenobarbital were all found to bind to apoferritin with affinities ranging from 10–500 µM, approximately matching the concentrations required to produce anesthetic and GABAergic responses. X-ray crystal structures were determined for the complexes of apoferritin with thiopental and pentobarbital at resolutions of 1.9 and 2.0 Å, respectively. These structures reveal that the barbiturates bind to a cavity in the apoferritin shell that also binds haloalkanes, halogenated ethers, and propofol. Unlike these other general anesthetics, however, which rely entirely upon van der Waals interactions and the hydrophobic effect for recognition, the barbiturates are recognized in the apoferritin site using a mixture of both polar and nonpolar interactions. These results suggest that any protein binding site that is able to recognize and respond to the chemically and structurally diverse set of compounds used as general anesthetics is likely to include a versatile mixture of both polar and hydrophobic elements

Vancomycin does not affect the enzymatic activities of purified VanSA.

VanS is a membrane-bound sensor histidine kinase responsible for sensing vancomycin and activating transcription of vancomycin-resistance genes. In the presence of vancomycin, VanS phosphorylates the transcription factor VanR, converting it to its transcriptionally active form. In the absence of vancomycin, VanS dephosphorylates VanR, thereby maintaining it in a transcriptionally inactive state. To date, the mechanistic details of how vancomycin modulates VanS activity have remained elusive. We have therefore studied these details in an in vitro system, using the full-length VanS and VanR proteins responsible for type-A vancomycin resistance in enterococci. Both detergent- and amphipol-solubilized VanSA display all the enzymatic activities expected for a sensor histidine kinase, with amphipol reconstitution providing a marked boost in overall activity relative to detergent solubilization. A putative constitutively activated VanSA mutant (T168K) was constructed and purified, and was found to exhibit the expected reduction in phosphatase activity, providing confidence that detergent-solubilized VanSA behaves in a physiologically relevant manner. In both detergent and amphipol solutions, VanSA's enzymatic activities were found to be insensitive to vancomycin, even at levels many times higher than the antibiotic's minimum inhibitory concentration. This result argues against direct activation of VanSA via formation of a binary antibiotic-kinase complex, suggesting instead that either additional factors are required to form a functional signaling complex, or that activation does not require direct interaction with the antibiotic