Functional insights into human HMG-CoA lyase from structures of Acyl-CoA-containing ternary complexes

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.

SCHEME 1.

HMG-CoA lyase reaction scheme.

FIGURE 1.

Sequence alignment of HMGCLs from Homo sapiens (human), Rattus norvegicus (rat), Brucella melitensis, Pseudomonas aeruginosa, Bacillus subtilis, and Bacillus licheniformis. The dollar sign indicates the start of the mature protein at Thr²⁸. Identical residues in all six organisms are highlighted in gray. Secondary structural elements of human HMGCL are shown as cylinders (12 α-helices) and arrows (nine β-strands). Catalytic residues and residues that coordinate Mg²⁺ ion are marked highlighted in black. Residues interacting with the CoA ligand are indicated (#). The underlined helical CoA-binding region displays some mobility upon ligand binding. The underlined glycine-rich loop delineates the signature sequence for the HMGCL family of enzymes. Residues after position 290 are largely helical, and this part of the polypeptide is external to the TIM barrel.

FIGURE 2.

Ribbon diagrams of the human HMGCL structure. A, an overlay of four monomer structures is shown. Light gray, WT lyase; blue, WT lyase complexed with the inhibitor HG-CoA; green, R41M; pink, R41M-HMG-CoA. Mg²⁺ is shown as a blue ball; the inhibitor HG-CoA is shown with atomic coloring; and the substrate HMG-CoA is displayed in gray. Hydrogen bonds with protein or bonds to Mg²⁺ are indicated by dotted lines. Significant differences between the overlaid monomers are marked in red. The glycine-rich loop containing Leu²⁶³–Asn²⁷⁵ shows significant movement when the R41M-HMG-CoA complex is compared with the other three monomers. Ala¹²⁹–Cys¹⁴¹ also shows mobility when CoA ligands are bound in the L-shaped conformation. B, the dimer structure of mutant R41M complexed with the substrate HMG-CoA and Mg²⁺. The tan lower monomer shows bound HMG-CoA with atomic coloring. The cyan upper monomer displays the apo-R41M structure, and a modeled magenta HMG-CoA is shown only to indicate the active site. The barrel of the cyan monomer is rotated by ∼90° relative to the tan lower monomer. The magenta C terminus of the lower monomer is interacting with the green Leu²⁶³–Asn²⁷⁵ loop of the cyan upper monomer. The dark cyan C terminus of the upper monomer is no longer interacting with the red Leu²⁶³–Asn²⁷⁵ loop of the bottom monomer due to the loop movement upon ligand binding. Lys⁴⁸ is located far from the active site and does not interact with the bound CoA ligand.

FIGURE 3.

Active site of human HMGCL. A, overlay of monomer structures of WT lyase with (blue) and without (gray) the bound inhibitor HG-CoA. Atom coloring is used for all ligands, and bond interactions are indicated by dotted lines. B, monomer overlay of mutant R41M in green and R41M with the substrate HMG-CoA in pink. Red indicates the mobility of the Cys²⁶⁶-containing loop residues Leu²⁶³–Asn²⁷⁵. This loop moves closer into the active site when the substrate is bound. C–E, electron density maps showing bound ligands: HGA (a hydrolysis product of the inhibitor HG-CoA; C), the inhibitor HG-CoA (D), and the substrate HMG-CoA (E) fitted into densities from |F_o| − |F_c| maps at the 2.5σ level. The adenine ring of HMG-CoA (gray) is disordered.

FIGURE 4.

Interactions between the HMGCL polypeptide and bound acyl-CoA ligands. A, schematic drawing of interactions between WT human lyase and HG-CoA. Hydrogen and coordination bonds are indicated by dashed lines. Thicker lines reflect the interaction of the HG-CoA analog with ordered water (A) or the interactions of HMG-CoA and ordered water with Cys²⁶⁶ (B). The curved line at Trp⁸¹ indicates a hydrophobic interaction between the indole ring and bound inhibitor. B, interactions between HMGCL R41M and HMG-CoA. The disordered adenine of HMG-CoA is depicted as thinner lines. Most interactions are similar when WT-HG-CoA and R41M-HMG-CoA are compared. Some minor differences in the binding region of the pyrophosphoryl adenosine moiety of CoA are observed due to the disordered adenine ring in R41M-HMG-CoA. However, the most significant changes are seen where arginine is replaced with methionine.

FIGURE 5.

Proposed reaction mechanism for HMGCL. This schematic drawing shows functionally important active-site side chains, Mg²⁺, an ordered water molecule, and the HMGCL substrate and products. The left side of the diagram indicates the six Mg²⁺ coordination ligands, as found in the R41M-Mg²⁺-HMG-CoA ternary complex. In the absence of the HMG-CoA or HG-CoA ternary complex ligands, the substrate C5 carboxyl and C3 hydroxyl oxygen ligands are replaced with water and Asn²⁷⁵ oxygens (not shown) (8).