Diazirine Photoprobes for the Identification of Vancomycin-Binding Proteins.

Vancomycin's interactions with cellular targets drive its antimicrobial activity and also trigger expression of resistance against the antibiotic. Interaction partners for vancomycin have previously been identified using photoaffinity probes, which have proven to be useful tools for exploring vancomycin's interactome. This work seeks to develop diazirine-based vancomycin photoprobes that display enhanced specificity and bear fewer chemical modifications as compared to previous photoprobes. Using proteins fused to vancomycin's main cell-wall target, d-alanyl-d-alanine, we used mass spectrometry to show that these photoprobes specifically label known vancomycin-binding partners within minutes. In a complementary approach, we developed a Western-blot strategy targeting the vancomycin adduct of the photoprobes, eliminating the need for affinity tags and simplifying the analysis of photolabeling reactions. Together, the probes and identification strategy provide a novel and streamlined pipeline for identifying vancomycin-binding proteins.

Novel Diazirine Photoprobes for the Identification of Vancomycin-Binding Proteins.

Vancomycin's interactions with cellular targets drive its antimicrobial activity, and also trigger expression of resistance against the antibiotic. Interaction partners for vancomycin have previously been identified using photoaffinity probes, which have proven to be useful tools for exploring vancomycin's interactome. This work seeks to develop diazirine-based vancomycin photoprobes that display enhanced specificity and bear fewer chemical modifications, as compared to previous photoprobes. Using proteins fused to vancomycin's main cell-wall target, D-alanyl-D-alanine, we use mass spectrometry to show that these photoprobes specifically label known vancomycin-binding partners within minutes. In a complementary approach, we developed a Western-blot strategy targeting the vancomycin adduct of the photoprobes, eliminating the need for affinity tags and simplifying the analysis of photolabeling reactions. Together, the probes and identification strategy provide a novel and streamlined pipeline for identifying novel vancomycin-binding proteins.

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.