Structural Analysis of the Essential Resuscitation Promoting Factor YeaZ Suggests a Mechanism of Nucleotide Regulation through Dimer Reorganization

Abstract

BackgroundThe yeaZ gene product forms part of the conserved network YjeE/YeaZ/YgjD essential for the survival of many Gram-negative eubacteria. Among other as yet unidentified roles, YeaZ functions as a resuscitation promoting factor required for survival and resuscitation of cells in a viable but non-culturable (VBNC) state.Methodology/Principal FindingsIn order to investigate in detail the structure/function relationship of this family of proteins we have performed X-ray crystallographic studies of Vibrio parahaemolyticus YeaZ. The YeaZ structure showed that it has a classic actin-like nucleotide-binding fold. Comparisons of this crystal structure to that of available homologues from E. coli, T. maritima and S. typhimurium revealed two distinctly different modes of dimer formation. In one form, prevalent in the absence of nucleotide, the putative nucleotide-binding site is incomplete, lacking a binding pocket for a nucleotide base. In the second form, residues from the second subunit complete the nucleotide-binding site. This suggests that the two dimer architectures observed in the crystal structures correspond to a free and a nucleotide-bound form of YeaZ. A multiple sequence alignment of YeaZ proteins from different bacteria allowed us to identify a large conserved hydrophobic patch on the protein surface that becomes exposed upon nucleotide-driven dimer re-arrangement. We hypothesize that the transition between two dimer architectures represents the transition between the ‘on’ and ‘off’ states of YeaZ. The effect of this transition is to alternately expose and bury a docking site for the partner protein YgjD.Conclusions/SignificanceThis paper provides the first structural insight into the putative mechanism of nucleotide regulation of YeaZ through dimer reorganization. Our analysis suggests that nucleotide binding to YeaZ may act as a regulator or switch that changes YeaZ shape, allowing it to switch partners between YjeE and YgjD.

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

“…In contrast, St YeaZ has lobes A and B configured in different relative positions than in most homologs, 28 which together with short connecting loops might contribute to the inability to bind nucleotides. 28,30 The relative domain positions of YeaZ do not change on formation of the complex with YgjD (Fig. 6).…”

“…By contrast, this C-terminal region is disordered in the StYeaZ structure 28 and proteolytically removed in VpYeaZ. 30 The third set of contacts involves a region near the C-terminus of YgjD. However, as it is about ten residues from the C-terminus, the latter thus does not extend over the surface of the partner protein in the manner observed for the C-terminus of YeaZ binding to YgjD.…”

“…The configuration of the YgjD-YeaZ heterodimer mimics one of the two putative YeaZ homodimers suggested from the crystal packing of earlier YeaZ structures. [27][28][29][30] Although this so-called A 2 /form 1 dimer has a smaller surface contact area than does the AB/ form 2 dimer, it is the most commonly observed in the previously reported YeaZ crystal structures.…”

“…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.…”

“…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. 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.…”