2.1 A crystal structure of the Mycobacterium tuberculosis serine hydrolase, Hip1, in its anhydro-form (Anhydrohip1)

The 2.6 angstrom crystal structure of the apo form of Hip1 (hydrolase important for pathogenesis) has been previously reported. However, very little is known about the active site architecture of this M. tuberculosis (Mtb), serine hydrolase drug target. To begin mapping the active site of Hip1, we cocrystallized Hip1 with the irreversible serine protease inhibitor, 4-(2-aminoethyl)-benzenesulfonylfluoride (AEBSF). We chose AEBSF for cocrystallization with Hip1 since the similar inhibitor, phenylmethylsulfonyl fluoride (PMSF), interestingly exhibited no activity against Hip1. We obtained crystals that diffracted to 2.1 angstrom but to our bewilderment, we did not observe any electron density for the inhibitor in the omit map for the Hip1-AEBSF complex. Rather, in the active site, dehydroalanine (dAla) was found to occupy the expected position of the catalytic Ser228, thus yielding anhydrohip1. Here we present a comparative analysis of the crystal structures of anhydrohip1 and Hip1 and provide a mechanism for the conversion of the enzyme to the anhydro-form through reaction with AEBSF. With the aid of molecular docking, we propose an explanation for the differential inhibition of Hip1 by AEBSF and PMSF. We also present a preliminary definition of the S1 and S2 pockets of the protease's active site and propose a mechanism for a ligand-induced conformational change within the S2 pocket. Finally, we expand upon the previous demarcation of the putative lipid binding pocket in the a-domain of the enzyme. We believe that this detailed analysis of the structures of anhydrohip1 and Hip1 provides valuable information useful for the structure-based drug design of novel Hip1-directed Mtb therapeutics. (c) 2022 Elsevier Inc. All rights reserved.

Automated summary

This study presents the crystal structure of the apo form of the hydrolase Hip1, revealing insights into the active site architecture of this drug target. By co-crystallizing Hip1 with the irreversible serine protease inhibitor AEBSF, the researchers discovered a mechanism for the conversion of the enzyme to an anhydro form. They also provide a comparative analysis of the structures of anhydrohip1 and Hip1, propose a mechanism for ligand-induced conformational changes, and expand upon the putative lipid binding pocket in the enzyme. These findings are valuable for the structure-based drug design of novel therapeutics targeting Hip1 in M. tuberculosis.