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

Area of Science:

• Structural Biology
• Biochemistry
• Microbiology

Background:

• Insulin-cleaving membrane protease (ICMP) from Pseudomonas aeruginosa is crucial for bacterial pathogenesis and iron uptake.
• Canonical M75 peptidases utilize a zinc-dependent HxxE motif for catalytic activity.

Purpose of the Study:

• To determine the high-resolution structure of ICMP.
• To investigate the metal ion coordination and potential catalytic mechanisms of ICMP.

Main Methods:

• High-resolution structure determination of ICMP using single-wavelength anomalous diffraction (SAD).
• Crystallization of selenomethionine-substituted ICMP (Se-Met ICMP) yielding crystals diffracting to 1.9 Å resolution.
• Analysis of the protein structure, including helix bundle arrangement and metal ion coordination sites.

Main Results:

• The ICMP structure reveals an atypical HxxE motif and a unique V-shaped cleft for metal ion coordination.
• Unlike canonical M75 peptidases, no metal ions were found near the HxxE motif.
• A metal ion was identified at the opening of the V-shaped cleft, coordinated by specific ICMP residues (Asp211, Glu316, Cys319, Asp322, Asp397) via hydrogen bonds.

Conclusions:

• The study reveals a novel metal-binding site and potential catalytic mechanism for ICMP, distinct from known M75 peptidases.
• These findings offer new insights into the structural diversity of HxxE-motif-containing peptidases.
• The results lay the groundwork for future research into ICMP's catalytic mechanisms and substrate interactions.