Biochemical, Structural and Molecular Dynamics Analyses of the Potential Virulence Factor RipA from Yersinia pestis

Abstract

Human diseases are attributed in part to the ability of pathogens to evade the eukaryotic immune systems. A subset of these pathogens has developed mechanisms to survive in human macrophages. Yersinia pestis, the causative agent of the bubonic plague, is a predominately extracellular pathogen with the ability to survive and replicate intracellularly. A previous study has shown that a novel rip (required for intracellular proliferation) operon (ripA, ripB and ripC) is essential for replication and survival of Y. pestis in postactivated macrophages, by playing a role in lowering macrophage-produced nitric oxide (NO) levels. A bioinformatics analysis indicates that the rip operon is conserved among a distally related subset of macrophage-residing pathogens, including Burkholderia and Salmonella species, and suggests that this previously uncharacterized pathway is also required for intracellular survival of these pathogens. The focus of this study is ripA, which encodes for a protein highly homologous to 4-hydroxybutyrate-CoA transferase; however, biochemical analysis suggests that RipA functions as a butyryl-CoA transferase. The 1.9 Å X-ray crystal structure reveals that RipA belongs to the class of Family I CoA transferases and exhibits a unique tetrameric state. Molecular dynamics simulations are consistent with RipA tetramer formation and suggest a possible gating mechanism for CoA binding mediated by Val227. Together, our structural characterization and molecular dynamic simulations offer insights into acyl-CoA specificity within the active site binding pocket, and support biochemical results that RipA is a butyryl-CoA transferase. We hypothesize that the end product of the rip operon is butyrate, a known anti-inflammatory, which has been shown to lower NO levels in macrophages. Thus, the results of this molecular study of Y. pestis RipA provide a structural platform for rational inhibitor design, which may lead to a greater understanding of the role of RipA in this unique virulence pathway.

Structural snapshots along the reaction pathway ofYersinia pestisRipA, a putative butyryl-CoA transferase

“…The previously solved apo RipA structure contains two monomers in the asymmetric unit, where each monomer has a different G(V/I)G loop conformation ( Fig. 1b), suggesting open and closed configurations; this loop flexibility was also observed in apo RipA molecular-dynamics (MD) simulations (Torres et al, 2011). Furthermore, the conformational flexibility of the G(V/I)G loop has been proposed to be important for the protection of reaction intermediates from hydrolysis in its closed state (Macieira et al, 2012;Torres et al, 2011) configurations.…”

“…1b), suggesting open and closed configurations; this loop flexibility was also observed in apo RipA molecular-dynamics (MD) simulations (Torres et al, 2011). Furthermore, the conformational flexibility of the G(V/I)G loop has been proposed to be important for the protection of reaction intermediates from hydrolysis in its closed state (Macieira et al, 2012;Torres et al, 2011) configurations. We also observe conformational diversity of a conserved phenylalanine (Phe85) which gates access to the acyl-group binding pocket, and of the G(V/I)G loop, which facilitates access to the catalytic active site.…”

“…The RipA mutants were overexpressed as described previously for WT RipA (Torres et al, 2011). In brief, the constructs in a pET28 plasmid were grown in E. coli BL21(DE3) Gold cells (Stratagene) at 310 K in LB medium containing 30 mg ml À1 kanamycin.…”

“…Within the pgm locus, studies demonstrate that the ripA gene is necessary for Y. pestis replication in postactivated macrophages and for a decrease in host NO levels. Recently, RipA was characterized as a putative butyryl-CoA transferase (butyryl-CoAT; Torres et al, 2011), thus alluding to a role for Y. pestis butyrate production in observed host NO reduction, as butyrate is a known anti-inflammatory that has been shown to reduce NO levels in interferon -activated macrophages (Park et al, 2007;Stempelj et al, 2007).…”

Characterizing the virulence factor YapE in Yersinia pestis.

“…It would be assumed that if the pgm locus is present the bacteria would have an advantage to survive intracellularly where the removal should result in the reduction of bacterial numbers. One reason I did not see the reduction in bacterial numbers with the ∆pgm strain is that the rip operon is important for Y. pestis replication in activated macrophages [146,147]. When performing the gentamicin protection assay, the macrophages were not stimulated prior to infection.…”