Crystallization, X-ray diffraction analysis and structure of ICMP from Pseudomonasaeruginosa.

Insulin-cleaving membrane protease (ICMP), an outmember protein of Pseudomonas aeruginosa (P. aeruginosa), plays a critical role in the pathogenesis of the bacterium. ICMP has been reported to be involved in the process of iron uptake. In this study, we report the high-resolution structure of ICMP determined by single-wavelength anomalous diffraction (SAD), which shows an atypical HxxE motif that differs from the canonical zinc dependent M75 peptidases and a "V-shaped" cleft that is observed to coordinate the metal ion for the first time. Crystals from the selenomethionine-substituted ICMP(Se-Met ICMP) diffract to 1.9 Å resolution and belong to space group P21, with unit-cell parameters a = 87.93, b = 78.14, c = 9.92 Å, α = 90°, ß = 113.5°, γ = 90°. ICMP consists of two up-and-down helix bundles, which are arranged into an inverted "V" shape. Unexpectedly, no electron densities of metal ions are observed around the ICMP HxxE motif, which is shown to be involved in metal coordination in zinc-dependent M75 peptidases. In contrast, we find a metal ion at the opening cleft of the V-shaped structure of ICMP, where the ICMP residues Asp211, Glu316, Cys319, Asp322, and Asp397 are observed to coordinate the metal via hydrogen-bond interactions. Such observations might imply new potential substrate-binding and catalytic sites. The current work therefore provides novel insights into the diversity of the HxxE-motif-containing peptidase and paves the way for future studies aiming to delineate the mechanism of ICMP catalysis.