Structural analysis of inositol monophosphatase complexes with substrates

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Human inositol monophosphatase (HINP) is a crucial enzyme involved in cellular signaling pathways.
• Understanding HINP's active site is vital for developing targeted therapeutics.

Purpose of the Study:

• To elucidate the structural basis of HINP inhibition by Gd3+ and substrate binding.
• To determine the precise interactions within the HINP active site during catalysis.

Main Methods:

• X-ray crystallography was employed to determine the structures of ternary complexes.
• High-resolution (2.2-2.3 Å) structural data provided detailed atomic information.
• Mutagenesis experiments were used to validate key residue interactions.

Main Results:

• Substrate (D- or L-myo-inositol 1-phosphate) and Gd3+ bind identically in the phosphatase dimer active site.
• Gd3+ occupancy was 35%, suggesting partial replacement by Li+ from crystallization solvent.
• Active site orientations of D- and L-inositol 1-phosphate differ by ~60° rotation around the phosphate ester bond.
• Key residues (Asp 93, Ala 196, Glu 213, Asp 220) form identical hydrogen bonds with both substrates.
• Structural data suggest a metal-bound water molecule as the active site nucleophile, activated by Glu 70 and Thr 95.
• Aspartate residues (90, 93, 220) are implicated in promoting ester oxygen expulsion.

Conclusions:

• The study provides high-resolution structural insights into HINP's mechanism of action.
• Identified interactions highlight the roles of specific residues and metal ions in catalysis.
• Findings contribute to a deeper understanding of inositol phosphate metabolism and phosphatase function.