The Study Of Crystal Structure Of Myroilysin From Myroides Sp. CSLB8

Myroilysin is a bacterial source M12 family metalloprotease,which has elastinolytic activity and plays an important role in swelling collagen.An about 23 kDa protease was purified from Myroides sp.cslb8 in our lab,it has 99%sequence identity with the reported myroilysin.In order to elucidate the catalytic mechanism of myroilysin,its crystal structure was studied.The myroilysin protein was first purified from the supernatant of CSLB8.The crystal of myroilysin was harvested,but it was not diffracted with X-ray.Though the crystallization condition of the purified myroilysin was optimized,there was no improvment for the diffraction of the crystals.Then heterologous expression for the gene of mature peptide of myroilysin was then attempted in E.coli,but no product was got.Whereas the recombinant pro-myroilysin could be successfully expressed in the E.coli,purified,methylated.The purified pro-myroilysin was used for crystallization,and the crystals of pro-myroilysin were obtained at the condition of 0.1 M Bis-Tris pH 6.5,40%PEG4000 and 0.1 M HEPES-NaOH pH7.5,1.4 M sodium citrate.Since myroilysin is a zinc-dependent metalloprotease in family M12,a zinc ion is supposed to be presenting in pro-myroilysin.To solve the phase problem,we collected a dataset at the peak wavelength of zinc(1.2816 A).The structure was determined with a single wavelength anomalous dispersion method(SAD)and refined to 1.89 A.Another 1.6 A dataset was collected from condition(0.1 M Bis-Tris pH6.5,40%PEG4000),and the structure was determined using molecular replacement method by using the 1.89 A model as template.The crystal of pro-myroilysin from 0.1 M Bis-Tris pH 6.5,40%PEG4000 belongs to space group P12i 1,and there is only a pro-myroilysin molecule in the asymmetric unit cell.The three-dimensional structure shows that the whole pro-myroilysin molecule is composed of the pro-peptide,N-terminal domain and C-terminal domain.The N-terminal domain and the C-terminal domain form a V-shaped cleft.In the long and deep active site cleft,the catalytic zinc ion at the bottom is respectively coordinated in a triangular pyramidal geometry by three NE2 atoms of His 142,His 146 and His 152 belonging to the signature motif HEXXHXXGXXH,and by the SG atom of side chain of cysteine residue(Cys28)of the pro-peptide.The side chains of His142,Glu143 and His146 from the central helix a6 are projected into the deep active site cleft,and then the helix a6 extends up to Gly 149,where it turns sharply to bring the third coordinator(His 152)of zinc ion to the active site.In pro-myroilysin,a cysteine residue(Cys28)in the pro-peptide coordinates the zinc ion,and forms a hydrogen bond with catalytic residue Glu143.The presence of Cys28 expels the catalytic water molecule away from the activity site and results in the inhibition of activity.