The VanS sensor histidine kinase from type-B VRE recognizes vancomycin directly.

V ancomycin-resistant e nterococci (VRE) are among the most common causes of nosocomial infections, which can be challenging to treat. VRE have acquired a suite of resistance genes that function together to confer resistance to vancomycin. Expression of the resistance phenotype is controlled by the VanRS two-component system. This system senses the presence of the antibiotic, and responds by initiating transcription of resistance genes. VanS is a transmembrane sensor histidine kinase, and plays a fundamental role in antibiotic resistance by detecting vancomycin and then transducing this signal to VanR. Despite the critical role played by VanS, fundamental questions remain about its function, and in particular about how it senses vancomycin. Here, we focus on purified VanRS systems from the two most clinically prevalent forms of VRE, types A and B. We show that in a native-like membrane environment, the enzymatic activities of type-A VanS are insensitive to vancomycin, suggesting that the protein functions by an indirect mechanism that detects a downstream consequence of antibiotic activity. In contrast, the autokinase activity of type-B VanS is strongly stimulated by vancomycin. We additionally demonstrate that this effect is mediated by a direct physical interaction between the antibiotic and the type-B VanS protein, and localize the interacting region to the protein's periplasmic domain. This represents the first time that a direct sensing mechanism has been confirmed for any VanS protein. Significance Statement: When v ancomycin-resistant e nterococci (VRE) sense the presence of vancomycin, they remodel their cell walls to block antibiotic binding. This resistance phenotype is controlled by the VanS protein, a sensor histidine kinase that senses the antibiotic and signals for transcription of resistance genes. However, the mechanism by which VanS detects the antibiotic has remained unclear. Here, we show that VanS proteins from the two most common types of VRE use very different sensing mechanisms. Vancomycin does not alter the signaling activity of VanS from type-A VRE, suggesting an indirect sensing mechanism; in contrast, VanS from type-B VRE is activated by direct binding of the antibiotic. Such mechanistic insights will likely prove useful in circumventing vancomycin resistance.

Structures of full-length VanR from Streptomyces coelicolor in both the inactive and activated states.

Vancomycin has historically been used as a last-resort treatment for serious bacterial infections. However, vancomycin resistance has become widespread in certain pathogens, presenting a serious threat to public health. Resistance to vancomycin is conferred by a suite of resistance genes, the expression of which is controlled by the VanR-VanS two-component system. VanR is the response regulator in this system; in the presence of vancomycin, VanR accepts a phosphoryl group from VanS, thereby activating VanR as a transcription factor and inducing expression of the resistance genes. This paper presents the X-ray crystal structures of full-length VanR from Streptomyces coelicolor in both the inactive and activated states at resolutions of 2.3 and 2.0 Å, respectively. Comparison of the two structures illustrates that phosphorylation of VanR is accompanied by a disorder-to-order transition of helix 4, which lies within the receiver domain of the protein. This transition generates an interface that promotes dimerization of the receiver domain; dimerization in solution was verified using analytical ultracentrifugation. The inactive conformation of the protein does not appear intrinsically unable to bind DNA; rather, it is proposed that in the activated form DNA binding is enhanced by an avidity effect contributed by the receiver-domain dimerization.

Voltage-Dependent Profile Structures of a Kv-Channel via Time-Resolved Neutron Interferometry.

Available experimental techniques cannot determine high-resolution three-dimensional structures of membrane proteins under a transmembrane voltage. Hence, the mechanism by which voltage-gated cation channels couple conformational changes within the four voltage sensor domains, in response to either depolarizing or polarizing transmembrane voltages, to opening or closing of the pore domain's ion channel remains unresolved. Single-membrane specimens, composed of a phospholipid bilayer containing a vectorially oriented voltage-gated K+ channel protein at high in-plane density tethered to the surface of an inorganic multilayer substrate, were developed to allow the application of transmembrane voltages in an electrochemical cell. Time-resolved neutron reflectivity experiments, enhanced by interferometry enabled by the multilayer substrate, were employed to provide directly the low-resolution profile structures of the membrane containing the vectorially oriented voltage-gated K+ channel for the activated, open and deactivated, closed states of the channel under depolarizing and hyperpolarizing transmembrane voltages applied cyclically. The profile structures of these single membranes were dominated by the voltage-gated K+ channel protein because of the high in-plane density. Importantly, the use of neutrons allowed the determination of the voltage-dependent changes in both the profile structure of the membrane and the distribution of water within the profile structure. These two key experimental results were then compared to those predicted by three computational modeling approaches for the activated, open and deactivated, closed states of three different voltage-gated K+ channels in hydrated phospholipid bilayer membrane environments. Of the three modeling approaches investigated, only one state-of-the-art molecular dynamics simulation that directly predicted the response of a voltage-gated K+ channel within a phospholipid bilayer membrane to applied transmembrane voltages by utilizing very long trajectories was found to be in agreement with the two key experimental results provided by the time-resolved neutron interferometry experiments.

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.