Structure of a triclinic ternary complex of horse liver alcohol dehydrogenase at 2.9 A resolution.

Abstract

Abstract The structure of a triclinic complex between liver alcohol dehydrogenase, reduced coenzyme NADH, and the inhibitor dimethylsulfoxide has been determined to 2.9 A resolution using isomorphous replacement methods. The heavy-atom positions were derived by molecular replacement methods using phase angles derived from a model of the orthorhombic apoenzyme structure previously determined to 2.4 A resolution. A model of the present holoenzyme molecule was built on a Vector General 3400 display system using the RING system of programs. This model gave a crystallographic R -value of 37.9%. There are extensive conformational differences between the protein molecules in the two forms. The conformational change involves a rotation of 7.5 ° of the catalytic domains relative to the coenzyme binding domains. A hinge region for this rotation is defined within a hydrophobic core between two helices. The internal structures of the domains are preserved with the exception of a movement of a small loop in the coenzyme binding domain. A cleft between the domains is closed by this coenzyme-induced conformational change, making the active site less accessible from solution and thus more hydrophobic. The two crystallographically independent subunits are very similar and bind both coenzyme and inhibitor in an identical way within the present limits of error. The coenzyme molecule is bound in an extended conformation with the two ends in hydrophobic crevices on opposite sides of the central pleated sheet of the coenzyme binding domain. There are hydrogen bonds to oxygen atoms of the ribose moities from Asp223, Lys228 and His51. The pyrophosphate group is in contact with the side-chains of Arg47 and Arg369. No new residues are brought into the active site compared to the apoenzyme structure. The active site zinc atom is close to the hinge region, where the smallest structural changes occur. Small differences in the co-ordination geometry of the ligands Cys46, His67 and Cysl74 are not excluded and may account for the ordered mechanism. The oxygen atom of the inhibitor dimethylsulfoxide is bound directly to zinc confirming the structural basis for the suggested mechanism of action based on studies of the apoenzyme structure.