Structural characterization of Salmonella typhimurium YeaZ, an M22 O‐sialoglycoprotein endopeptidase homolog

Abstract

The Salmonella typhimurium "yeaZ" gene (StyeaZ) encodes an essential protein of unknown function (StYeaZ), which has previously been annotated as a putative homolog of the Pasteurella haemolytica M22 O-sialoglycoprotein endopeptidase Gcp. YeaZ has also recently been reported as the first example of an RPF from a gram-negative bacterial species. To further characterize the properties of StYeaZ and the widely occurring MK-M22 family, we describe the purification, biochemical analysis, crystallization, and structure determination of StYeaZ. The crystal structure of StYeaZ reveals a classic two-lobed actin-like fold with structural features consistent with nucleotide binding. However, microcalorimetry experiments indicated that StYeaZ neither binds polyphosphates nor a wide range of nucleotides. Additionally, biochemical assays show that YeaZ is not an active O-sialoglycoprotein endopeptidase, consistent with the lack of the critical zinc binding motif. We present a detailed comparison of YeaZ with available structural homologs, the first reported structural analysis of an MK-M22 family member. The analysis indicates that StYeaZ has an unusual orientation of the A and B lobes which may require substantial relative movement or interaction with a partner protein in order to bind ligands. Comparison of the fold of YeaZ with that of a known RPF domain from a gram-positive species shows significant structural differences and therefore potentially distinctive RPF mechanisms for these two bacterial classes.

Crystal structure of the dimer of two essential Salmonella typhimurium proteins, YgjD & YeaZ and calorimetric evidence for the formation of a ternary YgjD–YeaZ–YjeE complex

“…25 An ATP-ase activity for HiYjeE was observed, although another report revealed tight binding of ADP but only slow hydrolysis of ATP once contaminating ATPases were removed. 26 Crystal structures for YeaZ from Escherichia coli (Ec), 27 Salmonella typhimurium (St), 28 Thermotoga maritime (Tm), 29 and Vibrio parahaemolyticus(Vp) 30 have been determined. YeaZ has a 2-lobed structure which belongs to the ASKHA superfamily.…”

“…In order for YeaZ to bind nucleotides it seemed that relative domain movements and/or binding to a partner protein might be necessary. 28 It has been proposed that an alternative dimer interface AB/form 2, observed in the TmYeaZ crystal structure, if utilised in the YgjD-YeaZ complex, might allow nucleotide binding to extend from YeaZ across the heterodimer boundary. 29,30 It was further suggested that the different conformational states of these two dimeric complexes could form the basis for a switching mechanism between partner proteins.…”

“…29,30 It was further suggested that the different conformational states of these two dimeric complexes could form the basis for a switching mechanism between partner proteins. StYeaZ has been reported to act as a resuscitation promoting factor (RPF) in Salmonellae, 31 Our studies are aimed at the characterization of the structure and biochemical properties of essential proteins of unknown function in Salmonella typhimurium that might represent novel drug targets, including YeaZ, 28 YegS, 33 and YcbL. 34 New drugs are urgently required as the continual rise of high level resistance to current anti-bacterials presents increasing threats to human and animal health.…”

“…7]. Similarly, StYeaZ, whether present in the heterodimeric binary complex with St YgjD or as a single protein 28 adopts an overall similar conformation but with one notable exception. Although the C-terminal $13 amino acids are disordered in the crystal structure of StYeaZ, 28 11 of these residues become fully ordered in the complex with YgjD by formation of an extended arm that has many contacts with YgjD, which are described in more detail below.…”

Conserved Network of Proteins Essential for Bacterial Viability

“…The YeaZ proteins form a family assigned as COG1214 (http://www.ncbi.nlm.nih.gov/Structure /cdd/cddsrv.cgi?uidϭCOG1214) which is annotated as "Inactive homolog of metal-dependent proteases, putative molecular chaperone." A recent crystal structure of S. enterica serovar Typhimurium YeaZ shows that the protein is part of the HSP70 actin-like-fold (HALF) protein family and suggests the likelihood that upon domain rearrangement, the protein would bind a nucleotide (51). Purification of an affinity-tagged version of YeaZ produced at native levels identified YgjD as a copurifying partner (9), suggesting that these two homologous proteins form a complex in vivo.…”