Crystal Structure of a Hidden Protein, YcaC, a Putative Cysteine Hydrolase from Pseudomonas aeruginosa, with and without an Acrylamide Adduct

Abstract

As part of the ongoing effort to functionally and structurally characterize virulence factors in the opportunistic pathogen Pseudomonas aeruginosa, we determined the crystal structure of YcaC co-purified with the target protein at resolutions of 2.34 and 2.56 Å without a priori knowledge of the protein identity or experimental phases. The three-dimensional structure of YcaC adopts a well-known cysteine hydrolase fold with the putative active site residues conserved. The active site cysteine is covalently bound to propionamide in one crystal form, whereas the second form contains an S-mercaptocysteine. The precise biological function of YcaC is unknown; however, related prokaryotic proteins have functions in antibacterial resistance, siderophore production and NADH biosynthesis. Here, we show that YcaC is exceptionally well conserved across both bacterial and fungal species despite being non-ubiquitous. This suggests that whilst YcaC may not be part of an integral pathway, the function could confer a significant evolutionary advantage to microbial life.

Keywords: X-ray crystallography; YcaC; isochorismate family; micro-crystals; molecular replacement; protein octamer.

Figure 1

(a) Ribbon representation of P. aeruginosa YcaC octamer in crystal form I with chains alternately colored purple and silver, chloride as green spheres and sulfate as sticks with orange sulfur and red oxygens; (b) Close-up of the sulfate and chloride binding sites in chain A shown in purple with chain B depicted in silver. Stacked Arg 7 and 174 with the sulfate are shown in ball-and-stick representation. The chloride is shown with coordinating residues Gly86, Asn87 and Asn93 and the lid-forming Arg177 from chain B.

Figure 2

Ribbon representation of the least-squares superposition of chain A and B of Pa YcaC shown in purple and silver, the isochorismatase PhzD from P. aeruginosa (1NF9) [11] in lime and Mtb PncA (3PL1-a nicotinamidase) [12] in teal. N-terminal residues are colored blue, and C-terminal residues are colored red. (i) PhzD and PncA are both missing the YcaC C-terminal helices that form the active site with the neighboring chain; (ii) the large loop would prevent tetramerization if present in YcaC; (iii) as would the additional N-terminal helix present in PhzD. N- and C-termini are highlighted in blue and red, respectively.

Figure 3

Sequence alignment of YcaC homologues with secondary structural elements annotated based on the Pa YcaC crystal structure presented here. Conserved residues are highlighted in red (highly conserved) and white on red background (absolutely conserved).