Structural Analysis of Galactofuranose-Binding Lectins and Biosynthetic Enzymes.

摘要

Galactofuranose (Galf) is a carbohydrate residue essential for pathogenic organisms, such as Mycobacterium tuberculosis, that is not found in mammalian systems. Therefore, tools that recognize Gal f specifically would be useful for diagnostic purposes and the development of targeted therapeutics. In addition, the biosynthetic enzyme UDP-galactopyranose mutase (UGM) essential for Galf incorporation is an attractive antibiotic target.;To examine how Galf is recognized, the structures of microbe-binding intelectins were investigated. Because no structure of any protein in the intelectin family was available, a crystal structure of Xenopus embryonic epidermal lectin (XEEL) was determined using experimental phasing. The XEEL structure enabled solution of the structure of human intelectin-1 (hIntL-1) as well as structures of XEEL and hIntL-1 bound to their carbohydrate ligands. These structures revealed intelectins to be structurally distinct from other lectins. They also showed that intelectins use an unusual ligand recognition mechanism, a protein-bound calcium ion is chelated by the exocyclic vicinal diol on the ligands. These structures expanded our appreciation of lectin diversity, and could aid in rational engineering of intelectins for microbe detection and elimination.;In addition to Galf detection, inhibition of the essential Galf biosynthesis enzyme UGM was investigated. Triazolothiadiazine inhibitors discovered through virtual screening showed low micromolar inhibitory constants and were effective against virulent strains of Mycobacterium tuberculosis. A structure of a triazolothiadiazine inhibitor bound to Corynebacterium diphtheriae UGM (CdUGM) was determined. The structure not only shows the first small molecule, non-substrate inhibitor bound to UGM, but also suggests the role of UGM conformation in inhibitor design. Additional CdUGM and Mycobacterium smegmatis UGM (MsUGM) structures were also determined to examine structural dynamics and rationalize inhibitor affinity across UGM homologs. The inhibitor-bound CdUGM structure, and various forms of CdUGMs and MsUGMs, should enable development of next generation inhibitors that could target a wide range of UGMs.