Biology subjects

Grasty, K. C.

Publications and source records attributed to Grasty, K. C..

3 recordsLinked to original sources

bioRxiv2023

Novel Diazirine Photoprobes for the Identification of Vancomycin-Binding Proteins

Vancomycins 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 vancomycins 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 vancomycins 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. For Table of Contents Only. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/545140v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1d21b8forg.highwire.dtl.DTLVardef@10f4d4borg.highwire.dtl.DTLVardef@19bb1f0org.highwire.dtl.DTLVardef@bf3dd5_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Structure of VanS from Vancomycin-Resistant Enterococci: A Sensor Kinase with Weak ATP Binding

The VanRS two-component system regulates the resistance phenotype of vancomycin-resistant enterococci (VRE). VanS is a sensor histidine kinase that responds to the presence of vancomycin by autophosphorylating and subsequently transferring the phosphoryl group to the response regulator, VanR. The phosphotransfer activates VanR as a transcription factor, which initiates the expression of resistance genes. Structural information about VanS proteins has remained elusive, hindering the molecular-level understanding of their function. Here, we present X-ray crystal structures for the catalytic and ATP-binding (CA) domains of two VanS proteins, derived from VRE types A and C. Both proteins adopt the canonical Bergerat fold that has been observed for CA domains of other prokaryotic histidine kinases. We attempted to determine structures for the nucleotide-bound forms of both proteins; however, despite repeated efforts, these forms could not be crystallized, prompting us to measure the proteins binding affinities for ATP. Unexpectedly, both CA domains displayed low affinities for the nucleotide, with KD values in the low millimolar range. Since these KD values are comparable to intracellular ATP concentrations, this weak substrate binding could reflect a way of regulating expression of the resistance phenotype.

biochemistry↗

bioRxiv2020

Ubiquitin Substrates Dramatically Increase Ataxin3 Deubiquitinating Activity: Allosteric crosstalk connects three distinct sites

Ataxin3 is the founding member of the MJD family of deubiquitinating enzymes, and plays important roles in maintaining protein homeostasis and promoting DNA repair. The enzyme also contains a polyglutamine tract of variable length, and in its expanded form the protein becomes the causative agent of a neurodegenerative disorder known as Machado-Joseph disease. In vitro, ataxin3 displays low catalytic activity, prompting questions about how the enzyme is regulated and what signals might lead to its activation. Recently, it has been demonstrated that ataxin3 activity can be stimulated by either mono-ubiquitination or high concentrations of free ubiquitin. Here, we show that ubiquitin conjugates with cleavable bonds can stimulate ataxin3 activity much more strongly than free ubiquitin, with physiological levels of these conjugates increasing activity up to 60-fold over basal levels. Our data are consistent with a model in which ubiquitin conjugates activate the enzyme allosterically by binding in a site adjacent to the catalytic center, known as Site 1. We further show that two additional ubiquitin-binding sites in the enzyme work in concert to modulate enzyme activation, and we propose a model in which ubiquitin conjugates bridge these two sites to drive the enzyme into a high-activity conformation. SignificanceUbiquitin signaling networks modulate almost all aspects of eukaryotic biology, and their outputs reflect the dynamic balance between ubiquitin attachment and removal. The latter process is catalyzed by deubiquitinating enzymes (DUBs), which must be carefully regulated to ensure that their activities are applied appropriately. Ataxin3 is a DUB that participates in quality-control pathways that support cellular health; however, the regulation of its activity has remained poorly understood. Here, we show that ataxin3 can be dramatically activated by naturally occurring ubiquitin species, and that this activation involves a previously uncharacterized interplay between three distinct sites on the enzyme. Our improved understanding of ataxin3 regulation provides insights into allosteric mechanisms that may prove applicable to other enzymes in the ubiquitin universe.

biochemistry↗