Crystallographic And Functional Studies On ChuZ, A Heme Oxygenase From Campylobacter Jejuni

The gram-negative, micro-aerophilic bacterium Campylobacter jejuni (C. jejuni), is detected in the gut of a wide range of food-supply animals and avian species. It is a major cause of acute gastroenteritis in industrialized countries. C. jejuni infection is also implicated in the development of serious immune-mediated neurological conditions known as Guillain-Barréand Miller-Fischer syndromes. Iron acquisition is essential for survival, persistence, and pathogenicity of the bacterial because free iron is maintained at very low levels by the hosts, partly in order to restrict microbe growth. As a result of iron shortage, C. jejuni tend to use heme as the sole iron source. With the continual emergence of resistant strains, new methods to prevent and control the infection of C. jejuni are urgently needed. The heme oxygenase ChuZ is part of the heme utilization mechanism of C. jejuni, and may serve as a promising candidate for development of novel anti-bacterial drugs, thus we have embarked on structure determination and function analysis of ChuZ from C. jejuni.Methods1. Cloning, expression and purification of ChuZ.The chuZ gene was amplified from the genome of C. jejuni NCTC11168 and cloned into an expression vector derived from the pET22b plasmid and placed between NdeI and XhoI restriction sites, The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells and the protein was expressed under the induction of IPTG and then purified by affinity, gel-filtration and ion exchange chromatography.2. Crystallization of ChuZ-hemin complexHemin solution was slowly added to purified ChuZ, The solution was incubated at overnight and the extra hemin was removed by gel filtration chromatography. The crystals of ChuZ-hemin complex were obtained by the microbatch technique using Hampton 72 well crystallization plates (HR3-086) and several Hampton crystallization kits such as Index, SaltRx, PEG/Ion Screen, Crystal Screen, PEG/Tacsimate, PEGRx were screened. 3. Data collection and structure determinationAll diffraction data sets were collected at Beam line 17U of Shanghai Synchrotron Radiation Facility (Shanghai, China) using a MAR225 CCD detector . Structure of ChuZ was determined by molecular replacement method.4.Enzymes activity assay of wild ChuZ and its mutantsBased on the structural analysis, key residues that involed in substrate binding were mutated and the enzymatic activities of ChuZ and its mutants were assayed by spectroscopic methods5.Analysis of the surface heme binding sitesChuZ-hemin and mutants binding stoichiometry was assayed by using spectroscopic method and ITC to test binding constants of ChuZ and its mutants.6. Crystallization of mutants H9A/H14A, the mutants was purified and crystallized the same way as wild-type ChuZ.Results:1. The recombinant plasmid pET22b-chuZ was constructed successfully and confirmed by sequencing. The pET22b-chuZ plasmid was transformed into Escherichia coli BL21 (DE3) competent cells and highly expressed under the induction of IPTG. The resulting protein was purified by affinity, gel-filtration and ion exchange chromatography; the purity of the protein was up to 95%.2. During the initial crystallization condition screening experiments, needle-like crystals were observed after 2 days and grew to dimensions of about 0.1×0.1×0.7 mm3 in a few days. As the result of optimization, crystals suitable for high resolution diffraction data collection were obtained via microbatch method in 0.1M MES, 24% PEG400 (v/v), and 0.1M imidazole pH 6.5.3. The crystal diffracted to a resolution of 2.4 ? and belonged to space group C2221, with unit cell parameters a = 106.474, b = 106.698, and c = 52.464 ?,α=β=γ=90°. The crystal structure was solved by the molecular replacement method with the program PHASER, using the structure of HugZ (PDB accession code 3GAS) as the search model. There was one ChuZ-hemin complex in the asymmetric unit.4. Several mutants including R166A, H245Q/R166A, H245Q/H9A/H14A, R166A/H9A/H14A, H9A/H14A mutants were successfully constructed, enzymatic activity assay indicate that R166A/H9A/H14A will result in complete loss of enzyme activity.5. UV absorbance measurements and ITC assay showed that: these results correlated very well with the observation that 1 heme molecule is bound on the surface of the ChuZ dimer in addition to 1 heme per ChuZ monomer inside the binding pocket. The results also suggest that mutation of His9 and His14 removes the surface heme-binding site and resulted in a 1:1 ChuZ-hemin binding stoichiometry.6.A data set diffracted to a resolution of 3? was collected with crystal growed by mutant H9A/H14A, no electronic density was observed in the surface hemin binding site, indicting the mutant lost the capacity to bind hemin.Conculsion:Based on structural and biochemical studies on ChuZ and several functionally relevant mutants, this work demonstrates that ChuZ is a new member of the split-barrel HO family. Although the structure of ChuZ and its interactions with heme are highly conserved with respect to those of H. pylori HugZ, a novel heme-binding site on the surface of ChuZ, which was not observed in HugZ, points to the possibility that in addition to its role as an HO, ChuZ may participate in heme accumulation. These studies greatly improve our understanding of this new bacterial HO family.