An interfacial mechanism and a class of inhibitors inferred from two crystal structures of the Mycobacterium tuberculosis 30 kDa major secretory protein (antigen 85B), a mycolyl transferase

The Mycobacterium tuberculosis 30 kDa major secretory protein (antigen 85B) is the most abundant protein exported by M. tuberculosis, as well as a pot ent immunoprotective antigen and a leading drug target. A mycolyl transfera se of 285 residues, it is closely related to two other mycolyl transferases , each of molecular mass 32 kDa: antigen 85A and antigen 85C. All three cat alyze transfer of the fatty acid mycolate from one trehalose monomycolate t o another, resulting in trehalose dimycolate and free trehalose, thus helpi ng to build the bacterial cell wall. We have determined two crystal structu res of M. tuberculosis antigen 85B (ag85B), initially by molecular replacem ent using antigen 85C as a probe. The apo ag85B model is refined against 1. 8 Angstrom data, to an R-factor of 0.196 (R-free is 0.276), and includes al l residues except the N-terminal Phe. The active site immobilizes a molecul e of the cryoprotectant 2-methyl-2,4-pentanediol. Crystal growth with addit ion of trehalose resulted in a second ag85B crystal structure (1.9 Angstrom resolution; R-factor is 0.195; R-free is 0.285). Trehalose binds in two si tes at opposite ends of the active-site cleft. In our proposed mechanism mo del, the trehalose at the active site Ser126 represents the trehalose liber ated by temporary esterification of Ser126, while the other trehalose repre sents the incoming trehalose monomycolate just prior to swinging over to th e first trehalose site to displace the mycolate from its serine ester. Our proposed interfacial mechanism minimizes aqueous exposure of the apolar myc olates. Based on the trehalose-bound structure, we suggest a new class of a ntituberculous drugs, made by connecting two trehalose molecules by an amph ipathic linker. (C) 2001 Academic Press.