Functional Insights into Human HMG-CoA Lyase from Structures of Acyl-CoA-containing Ternary Complexes

Zhuji Fu,Jennifer A Runquist,Christa Montgomery,Henry M Miziorko,Jung-Ja P Kim

doi:10.1074/jbc.m110.139931

Abstract

HMG-CoA lyase (HMGCL) is crucial to ketogenesis, and inherited human mutations are potentially lethal. Detailed understanding of the HMGCL reaction mechanism and the molecular basis for correlating human mutations with enzyme deficiency have been limited by the lack of structural information for enzyme liganded to an acyl-CoA substrate or inhibitor. Crystal structures of ternary complexes of WT HMGCL with the competitive inhibitor 3-hydroxyglutaryl-CoA and of the catalytically deficient HMGCL R41M mutant with substrate HMG-CoA have been determined to 2.4 and 2.2 A, respectively. Comparison of these beta/alpha-barrel structures with those of unliganded HMGCL and R41M reveals substantial differences for Mg(2+) coordination and positioning of the flexible loop containing the conserved HMGCL "signature" sequence. In the R41M-Mg(2+)-substrate ternary complex, loop residue Cys(266) (implicated in active-site function by mechanistic and mutagenesis observations) is more closely juxtaposed to the catalytic site than in the case of unliganded enzyme or the WT enzyme-Mg(2+)-3-hydroxyglutaryl-CoA inhibitor complex. In both ternary complexes, the S-stereoisomer of substrate or inhibitor is specifically bound, in accord with the observed Mg(2+) liganding of both C3 hydroxyl and C5 carboxyl oxygens. In addition to His(233) and His(235) imidazoles, other Mg(2+) ligands are the Asp(42) carboxyl oxygen and an ordered water molecule. This water, positioned between Asp(42) and the C3 hydroxyl of bound substrate/inhibitor, may function as a proton shuttle. The observed interaction of Arg(41) with the acyl-CoA C1 carbonyl oxygen explains the effects of Arg(41) mutation on reaction product enolization and explains why human Arg(41) mutations cause drastic enzyme deficiency.

Highlights

A variety of human mutations, including many point mutations in protein-coding exons of the gene, have been documented [6]
To more fully address questions regarding the conformation of bound substrate, activator cation liganding, details concerning reaction chemistry and specificity, as well as the molecular basis for certain inherited HMG-CoA lyase (HMGCL) deficiencies, new structural information on enzyme bound to an intact acyl-CoA molecule is required
Complexes of the WT enzyme with the competitive inhibitor 3-hydroxyglutaryl-CoA and of catalytically deficient R41M enzyme with the authentic substrate HMG-CoA have been supplemented with the activator cation Mg2ϩ, and crystallization of the desired ternary complexes has been accomplished

Summary

EXPERIMENTAL PROCEDURES

Expression and Isolation of HMGCL Proteins—The WT and R41M mutant HMGCL proteins were expressed using plasmid pTrc-HL-1 and purified using the protocol developed for the WT enzyme [9]. X-ray data for the WT lyase crystal soaked with the inhibitor HG-CoA (hereafter referred to as WT/HG-CoA) and for the R41M mutant were collected at Ϫ180 °C to resolutions of 2.4 and 2.2 Å, respectively, using an in-house R-AXIS IVϩϩ detector coupled to a Rigaku Micromax 007 x-ray generator operating at 40 kV and 20 mA. The crystal structure of WT/HG-CoA was determined and refined using the WT human lyase structure (Protein Data Bank code 2CW6) [8] as the starting model. The R41M mutant crystal structure was determined by molecular replacement with MOLREP within the CCP4 program suite [13] using the monomer structure of WT lyase (Protein Data Bank code 2CW6) as the search model.

RESULTS

No metal ions

DISCUSSION