The Complex Crystal Structural Analysis Of Metalloproteinase Ttha1264, Ttha1265from Thermus Thermophilus Hb8

Thermus thermophilus is a thermophilic eubacterium isolated from hot springs, TTHA1264and TTHA1265is the metalloproteinase encoded by two adjacent genes on the chromosome of T. thermophilus HB8, this metalloprotease belongs to M16B subfamily. Previous studies showed that most members of M16B subfamily are heterodimers, so we speculate that TTHA1264and TTHA1265may present as a heterodimer in T. thermophilus. The crystal structure of TTHA1264has been reported, it presented as homodimer in the crystal, it has a metal binding site for Zn2+, but there is no Zn2+binding in the site.To coexpress TTHA1264and TTHA1265, the fragment including TTHA1264and TTHA1265genes was amplified by PCR using T. thermophilus HB8genomic DNA as template, and cloned into the expression vector pET28a. Heterologous expression was carried out in E. coli, and the expression products were purified and the result showed that only TTHA1265was expressed. To exclude the failure of co-expression caused by the difference of ribosome binding site between E. coli and T. Thermophilus, we introduced an E. coli favorable ribosome binding site between these two genes by overlapping PCR.Expression and purification showed that only TTHA1265was expressed and purified. To obtain the complex, TTHA1264and TTHA1265were separately cloned into expression vector pET28a and expressed in E. coli. TTHA1264and TTHA1265were purified by IMAC and gel filtration, after purification, TTHA1264and TTHA1265were mixed and the complex was then separated with gel filtration. The complex was concentrated and crystallized using traditional vapor diffusion method. Rod crystals were obtained for TTHA1264-1265complex. The crystals diffracted to3.0-2.5A. A full dataset was collected to2.6A. Structure determination was failed with molecular replacement using TTHA1264and other solved homologs structures due to the low identity of TTHA1265with its homologs. To solve the phase problem, the selenomethinine derivatized TTHA1265crystals were prepared, full dataset was collected to2.0A. TTHA1265structure was solved by using Se-SAD method. The asymmetric unit contains3TTHA1265molecules. Similar to TTHA1264, TTHA1265also forms a homodimer in the crystal.The complex structure was solved with molecular replacement by using the structure of TTHA1264and TTHA1265as templates. TTHA1264and TTHA1265formed a heterodimer in the crystal. Each TTHA1264contains1Zn ion in the active site.