Structure of the ternary complex of phosphomevalonate kinase: the enzyme and its family

Abstract

The galacto-, homoserine-, mevalonate-, phosphomevalonate-kinase (GHMP) superfamily encompases a wide-range of protein function. Three members of the family (mevalonate kinase, phosphomevalonate kinase, and diphosphomevalonate decarboxylase) comprise the mevalonate pathway found in S. pneumoniae and other organisms. We have determined the 1.9 A crystal structure of phosphomevalonate kinase (PMK) from S. pneumoniae in complex with phosphomevalonate and AMPPNP.Mg(2+). Comparison of the apo and ternary PMK structures suggests that ligand binding reverses the side-chain orientations of two antiparallel lysines residues (100 and 101) with the result that Lys101 is switched into a position in which its ammonium ion is in direct contact with the beta,gamma-bridging atom of the nucleotide, where it is expected to stabilize both the ground and transition states of the reaction. Analysis of all available GHMP kinase ternary complex structures reveals that while their C(alpha)-scaffolds are highly conserved, their substrates bind in one of two conformations, which appear to be either reactive or nonreactive. The active site of PMK seems spacious enough to accommodate interconversion of the reactive and nonreactive conformers. A substantial fraction of the PMK active site is occupied by ordered water, which clusters near the charged regions of the substrate. Notably, a water pentamer that interacts extensively with the reactive groups of both substrates was discovered at the active site.

Figure 1. The Mevalonate Pathway

The acronyms: MK, mevalonate kinase (ATP: (R)-mevalonate phosphotransferase, 2.7.1.36), PMK, phosphomevalonate kinase (ATP: (R)-phosphomevalonate kinase, 2.7.4.2), and DPM-DC, diphosphomevalonate decarboxylase (ATP:(R)-5-diphosphomevalonate carboxy-lyase, 4.1.1.33).

Figure 2. Structure and sequence alignments of Sp PMK

(A) Superposition of the apo and ternary complex structures reveals the changes that occur upon binding of ligands. Regions insensitive to ligand are gray, and the four responsive elements (L1, L2 and L3, αH) are colored in blue, red, cyan, and green, respectively — the more intense colors are associated with the ternary complex. The active site contains Pmev (magenta), AMPPNP (purple), and Mg²⁺ (turquoise). The structures of the three conserved sequence motifs originally used to define the GHMP superfamily are highlighted in light brown (M1), pale yellow (M2), and dark brown (M3). Yellow dots indicate where the missing/disordered segment of Loop 1 attaches to the apoenzyme. (B) Structure-based sequence alignment of ternary/apo Sp PMK and representative members of the GHMP kinase superfamily. From top to bottom, the structures include the ternary (2pg9) and apo (1k47) forms of PMK from Streptococcus pneumoniae, ATP-bound mevalonate kinase from Rattus norvegicus (1kvk), the ternary complexes of n-acetygalactose kinase (2a2d) and galactose kinase (1wuu) from Homo sapiens, and homoserine-bound homoserine kinase from Methanococcus jannaschii (1h72). Invariant residues are purple and conserved residues (>60% similarity) are green. The nomenclature for secondary structural elements of ternary PMK, which are colored according to the scheme in (A), is identical to that assigned to apo-PMK (53). Boxes surround motifs M1-3. Panels A and B were produced using Pymol (54) and Alscript (55), respectively.

Figure 3. Conformational Changes of SpPMK

(A) Stereoview of the omit F_o-F_c electron density (3.0 sigma level) surrounding ligands at the active site. (B) Stereo representation of the active site of SpPMK in complex with Pmev, AMPPNP and Mg²⁺. The dotted line segments depict the octahedral coordination of the Mg²⁺ ion as well as the interactions between the pentamer of water molecules that comprise a portion of the PMK binding site.

Figure 4. The Active Site of PMK

Ribbon-and-stick stereo superposition of the PMK active site corresponding to apoenzyme (gray) and the complex with ligands (orange). Figures were produced using PyMol (54).

Figure 5. Type I and II ligand conformations in GHMP kinase ternary structures

(A) The Type I conformation. The positioning of the γ- and Pmev-phosphoryl groups in the PMK ternary complex (shown on the left and right-hand sides of the panel, respectively) suggests that they are non-reactive. Similiar conformations are found in three of the seven GHMP-kinase ternary complex structures. (B) The Type II conformation. Positioning of the reactive moieties in the ternary complex of Gal-NAc kinase — a GHMP kinase. The Gal-NAc nucleophillic oxygen and the γ-phosphoryl group of AMPPNP appear to be positioned well to accomplish in-line displacement. This orientation is also found in three of the seven GHMP kinase ternary complex structures. (C) Alignment of the Type I and II M²⁺·nucleotides. The adenine bases of the Type I and II nucleotides were aligned. The PMK nucleotide color coding: carbon (yellow), phosphorus (orange), divalent cation (red); the Gal-NAc kinase nucleotide color coding: carbon and phosphorus (grey), divalent cation (green). (D) Comparison of the Type I and II active-sites. Resides (grey) within 3 Å of the PPP_i·Mg²⁺ moiety (green) of Glc-NAc kinase complex (Type II) are linked to the moiety by blank dotted lines. Analogous residues in the PMK complex (Type I) are shown in orange. Dashed red lines connect the divalent cation to the α, β and γ-phosphoryl groups to which it is coordinated. (E) Positioning the Type II nucleotide·M²⁺ in the Type I active site. The Type I and II active-sites were superposed using C_α-alignments of conserved regions of PMK and Gal-NAc kinase. The GalNac nucleotide (Type II) is shown in grey and its associated cation (Mn²⁺) is red. The transition from Type I to II requires the cation to migrate across the active-site cavity. Blue spheres represent ordered water. Figures were produed using PyMol (54) and the surface, shown in E, was created with Hollow (56).

Figure 6. Ordered water at the active site of PMK

(A) Ordered water molecules in the active site of the PMK ternary complex. Water oxygen atoms are either orange or olive. The olive atoms form a pentamer of water. Ligands and their coordinated Mg²⁺ ion are slate gray. (B) The ternary-complex water pentamer. The olive pentamer oxygens (Panel A) are replaced with water molecules. The proton geometry was adjusted to conform to that predicted for the pentamer and to reflect the likely hydrogen bonding between the radial protons of the pentamer and the charged substrate moieties (carboxyl and phosphoryl groups of Pmev, and the β- and γ-phosphoryl groups of AMPPNP). (C) Pentamer in the absence in the absence of ligand. The C_α-backbone of each of the six subunits of the apo-structure asymmetric unit was aligned with that of the ternary complex, and the ordered water in the vicinity of the ternary-complex pentamer was compared. Oxygen in the ternary complex pentamer is orange; oxygen in the apo structure is color coded according to subunit (A-F) [green (A), grey (B), purple (C), yellow (D), beige (E), white (F)].