A symmetrical tetramer for S. aureus pyruvate carboxylase in complex with coenzyme A

Abstract

Pyruvate carboxylase (PC) is a conserved metabolic enzyme with important cellular functions. We report crystallographic and cryo-electron microscopy (EM) studies of Staphylococcus aureus PC (SaPC) in complex with acetyl-CoA, an allosteric activator, and mutagenesis, biochemical, and structural studies of the biotin binding site of its carboxyltransferase (CT) domain. The disease-causing A610T mutation abolishes catalytic activity by blocking biotin binding to the CT active site, and Thr908 might play a catalytic role in the CT reaction. The crystal structure of SaPC in complex with CoA reveals a symmetrical tetramer, with one CoA molecule bound to each monomer, and cryo-EM studies confirm the symmetrical nature of the tetramer. These observations are in sharp contrast to the highly asymmetrical tetramer of Rhizobium etli PC in complex with ethyl-CoA. Our structural information suggests that acetyl-CoA promotes a conformation for the dimer of the biotin carboxylase domain of PC that might be catalytically more competent.

Figure 1

Structure of S. aureus pyruvate carboxylase (SaPC). (a). Schematic drawing of the structure of wild-type SaPC tetramer (Xiang and Tong, 2008), viewed from the bottom layer. The domains in monomer 1 are given separate colors: BC in red, CT in green, PT in gold, and BCCP in blue, also indicated in the bar graph at the bottom. The other three monomers are colored in magenta, cyan and yellow. The biotin moiety is shown as a stick model in black. The gray circle highlights the active site of CT domain in monomer 3, with a bound BCCP-biotin from monomer 4. (b). Stereo drawing of the active site of the CT domain in SaPC. The biotin moiety is shown in gray. The side chains of residues selected for mutagenesis are shown as stick models. (c). Conservation of residues in the active site of CT. All the structure figures were produced with PyMOL (DeLano, 2002).

Figure 2

Structures of the A610T and T908A mutants of SaPC. (a). Schematic drawing of the tetramer of the A610T mutant of SaPC, viewed from the top layer and colored as in Fig. 1a. (b). Molecular surface of the A610T mutant showing the steric clash between the Thr610 residue and the bound position of biotin as observed in the wild-type structure. (c). Schematic drawing showing the overlay of the biotin binding site in the A610T mutant (in color) and the wild-type enzyme (in gray). Large conformational differences for the C-terminal segment of CT are visible, associated with the relocation of biotin to the exo site in the mutant structure. The bound position of pyruvate (in black) as observed in the wild-type SaPC structure is also shown. (d). Schematic drawing showing the overlay of the biotin binding site in the T908A mutant (in color) and the wild-type enzyme (in gray).

Figure 3

A symmetrical tetramer for SaPC in complex with CoA. (a). Activation of SaPC catalysis by various acetyl-CoA analogs. A representative titration for each compound is shown. (b). Omit F_o–F_c electron density at 2.9 Å resolution for CoA. The contour level is at 3σ. (c). Schematic drawing of the tetramer of the CoA complex of SaPC, colored as in Fig. 1a. The CoA molecules are shown as space-filling models in black. One binding site for CoA is highlighted with the gray circle. (d). Schematic drawing of the tetramer of the CoA complex of SaPC, viewed from the bottom layer. (e). Overlay of the structures of the four monomers of SaPC in the CoA complex, based on their CT domains. The BC domains show a 6° difference in their relative orientations.

Figure 4

CryoEM studies confirm a symmetrical tetramer for SaPC in complex with acetyl-CoA. The cryoEM density of SaPC in the presence of acetyl-CoA, top view, (a), side view, (b) and bottom view, (c). (d). Fit of the symmetrical tetramer of SaPC in complex with CoA into the cryoEM map. The atomic model is colored in green. (e). Close-up showing the fit between the PT domains of SaPC and the cryoEM map. (f). The asymmetrical tetramer of RePC cannot be completely accommodated into the cryoEM map. (g). Close-up showing the PT domains of RePC lying outside the cryoEM envelope.

Figure 5

Binding mode of CoA in SaPC. (a). SaPC displays positive cooperativity towards acetyl-CoA binding in the presence of aspartate. (b). Detailed interactions between CoA and the BC and PT domains in the complex with SaPC. The CoA molecule is shown in black. (c). Molecular surface of SaPC near the CoA binding site, colored as in panel b.

Figure 6

Conformational changes in SaPC upon CoA binding. (a). Overlay of the structure of the BC domain dimer in the CoA complex (in color) and free enzyme (in gray) of SaPC. The structure of one monomer is overlayed, showing a large difference in the positions of the other monomer. The two-fold axis of the dimer is indicated by the horizontal line in blue. (b). Overlay of the PT domain dimers in the CoA complex (in color) and free enzyme (in gray) of SaPC, as well as the two equivalent PT domains in RePC (in magenta).