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

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

Structural and biochemical characterization of Chlamydia trachomatis DsbA reveals a cysteine-rich and weakly oxidising oxidoreductase

Copyright © 2016 Christensen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The Gram negative bacteria Chlamydia trachomatis is an obligate intracellular human pathogen that can cause pelvic inflammatory disease, infertility and blinding trachoma. C. trachomatis encodes a homolog of the dithiol oxidoreductase DsbA. Bacterial DsbA proteins introduce disulfide bonds to folding proteins providing structural bracing for secreted virulence factors, consequently these proteins are potential targets for antimicrobial drugs. Despite sharing functional and structural characteristics, the DsbA enzymes studied to date vary widely in their redox character. In this study we show that the truncated soluble form of the predicted membrane anchored protein C. trachomatis DsbA (CtDsbA) has oxidase activity and redox properties broadly similar to other characterized DsbA proteins. However CtDsbA is distinguished from other DsbAs by having six cysteines, including a second disulfide bond, and an unusual dipeptide sequence in its catalytic motif (Cys-Ser-Ala-Cys). We report the 2.7 Å crystal structure of CtDsbA revealing a typical DsbA fold, which is most similar to that of DsbA-II type proteins. Consistent with this, the catalytic surface of CtDsbA is negatively charged and lacks the hydrophobic groove found in EcDsbA and DsbAs from other enterobacteriaceae. Biochemical characterization of CtDsbA reveals it to be weakly oxidizing compared to other DsbAs and with only a mildly destabilizing active site disulfide bond. Analysis of the crystal structure suggests that this redox character is consistent with a lack of contributing factors to stabilize the active site nucleophilic thiolate relative to more oxidizing DsbA proteins

Protein–ligand interactions investigated by thermal shift assays (TSA) and dual polarization interferometry (DPI)

Over the last decades, a wide range of biophysical techniques investigating protein-ligand interactions have become indispensable tools to complement high-resolution crystal structure determinations. Current approaches in solution range from high-throughput-capable methods such as thermal shift assays (TSA) to highly accurate techniques including microscale thermophoresis (MST) and isothermal titration calorimetry (ITC) that can provide a full thermodynamic description of binding events. Surface-based methods such as surface plasmon resonance (SPR) and dual polarization interferometry (DPI) allow real-time measurements and can provide kinetic parameters as well as binding constants. DPI provides additional spatial information about the binding event. Here, an account is presented of new developments and recent applications of TSA and DPI connected to crystallography

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

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

Crystal structure of CtDsbA.

<p>A. The crystal structure of CtDsbA contains a thioredoxin domain (light green) and a helical domain (dark green.) Loops on the catalytic surface that constitute the active site of CtDsbA and determine redox activity are colored orange and labeled. The active site catalytic disulfide is highlighted with sulfurs shown as yellow spheres. The non-catalytic disulfide (between Cys84 and Cys145) and the single thiol (Cys70) in L1 are shown in stick representation. The most N-terminal region of CtDsbA is unstructured. Crystal packing interactions with the second monomer in the asymmetric unit and a symmetry related molecule (shown in white) stabilize this region of the protein such that is well resolved in the electron density map. B. Close view of the four loops (L1, <i>cis</i>Pro L2, L3 and the Cys-Ser-Ala-Cys motif) which constitute the active site surface of CtDsbA. C. In the crystal structure the active site cysteines are oxidized. Analysis of bond distances indicates that the Cys 38 thiolate could be stabilized by favorable bond interactions with Thr 172 (3.4 Å between Thr 172 OH and Cys 38 SG in the oxidized structure) of the neighboring cisPro L2 consistent with an oxidizing protein character. The Cys 41 sulfur atom is 3.5 Å from the Thr 172 hydroxyl in the oxidized structure. 2Fo-Fc and Fo-Fc electron density maps for the active site and cisPro Loop 2 were generated from calculated phases using phenix.maps and are shown contoured at 1.0 σ and 3.0 σ respectively. The maps are shown within a 1 Å radius of each atom of each loop.</p

Surface properties of CtDsbA.

<p>Surface representation for CtDsbA of the catalytic (left) and non-catalytic (right) faces. The active site residues Cys-Ser-Ala-Cys are colored yellow and the nucleophilic cysteine sulfur highlighted in orange. Pockets formed on the posterior face of the protein between H1 and H3 (pocket 1) and the N-terminal unstructured region and H6 (pocket 2) are labeled. B. Electrostatic surface representation of CtDsbA. Views are oriented as above. Electrostatic surface potential is contoured between -5 (red) and +5 (blue) kT/e. The nucleophilic cysteine is annotated with an S.</p

Redox potential determination for CtDsbA-SSS by electrophoretic motility shift.

<p><b>A</b> SDS-PAGE gel of oxidized CtDsbA (3 μM) incubated for 24 h with increasing concentration of DTT (0 μM -12 mM). <b>B</b> The fraction of thiolate as a function of reduced DTT versus oxidized DTT is plotted. Fitting of the data revealed a Keq of 3.8 ± 0.8 x 10⁻⁴ M equivalent to a redox potential of -229 mV. Mean and SD calculated from 4 biological replicates are plotted.</p

X-ray data measurement and refinement statistics for CtDsbA.

<p>X-ray data measurement and refinement statistics for CtDsbA.</p