PDBsum entry 2x0s

Transferase

PDB id: 2x0s

Contents

Protein chain

899 a.a.

Waters ×36

References listed in PDB file

Key reference

Title

The 3.0 a resolution crystal structure of glycosomal pyruvate phosphate dikinase from trypanosoma brucei.

Authors

L.W.Cosenza, F.Bringaud, T.Baltz, F.M.Vellieux.

Ref.

J Mol Biol, 2002, 318, 1417-1432. [DOI no: 10.1016/S0022-2836(02)00113-4]

PubMed id

12083528

Abstract

The crystal structure of the glycosomal enzyme pyruvate phosphate dikinase from the African protozoan parasite Trypanosoma brucei has been solved to 3.0 A resolution by molecular replacement. The search model was the 2.3 A resolution structure of the Clostridium symbiosum enzyme. Due to different relative orientations of the domains and sub-domains in the two structures, molecular replacement could be achieved only by positioning these elements (four bodies altogether) sequentially in the asymmetric unit of the P2(1)2(1)2 crystal, which contains one pyruvate phosphate dikinase (PPDK) subunit. The refined model, comprising 898 residues and 188 solvent molecules per subunit, has a crystallographic residual index Rf = 0.245 (cross-validation residual index Rfree = 0.291) and displays satisfactory stereochemistry. Eight regions, comprising a total of 69 amino acid residues at the surface of the molecule, are disordered in this crystal form. The PPDK subunits are arranged around the crystallographic 2-fold axis as a dimer, analogous to that observed in the C. symbiosum enzyme. Comparison of the two structures was carried out by superposition of the models. Although the fold of each domain or sub-domain is similar, the relative orientations of these constitutive elements are different in the two structures. The trypanosome enzyme is more "bent" than the bacterial enzyme, with bending increasing from the center of the molecule (close to the molecular 2-fold axis) towards the periphery where the N-terminal domain is located. As a consequence of this increased bending and of the differences in relative positions of subdomains, the nucleotide-binding cleft in the amino-terminal domain is wider in T. brucei PPDK: the N-terminal fragment of the amino-terminal domain is distant from the catalytic, phospho-transfer competent histidine 482 (ca 10 A away). Our observations suggest that the requirements of domain motion during enzyme catalysis might include widening of the nucleotide-binding cleft to allow access and departure of the AMP or ATP ligand.

Figure 6.
Figure 6. A GRASP representation of the electrostatic potential at the surface of the glycosomal PPDK molecule. This Figure shows the PPDK dimer viewed from opposite sides (related by a 180° rotation). The separation between the two monomers is indicated by a thick line. The entire surface of the molecule is covered by basic residues, giving a blue surface. The only area where acidic residues are in excess (red area) is located in the inter-domain depression where the central domain is situated.

Figure 7.
Figure 7. Superimposed subunits of T. brucei and C. symbiosum PPDK. The superposition operation was carried out using only the C-terminal domains. The C^a tracings of the superimposed subunits are shown, with T. brucei PPDK in black and bacterial PPDK in gray. For easier understanding, the equivalent domains and sub-domains in the two subunits are also represented by vectors between corresponding residues in the two structures (shown as thick lines).

The above figures are reprinted by permission from Elsevier: J Mol Biol (2002, 318, 1417-1432) copyright 2002.

Secondary reference #1

Title

Crystallization and preliminary crystallographic investigation of glycosomal pyruvate phosphate dikinase from trypanosoma brucei.

Authors

L.W.Cosenza, F.Bringaud, T.Baltz, F.M.Vellieux.

Ref.

Acta Crystallogr D Biol Crystallogr, 2000, 56, 1688-1690.

PubMed id

11092947

Secondary reference #2

Title

Functional and molecular characterization of a glycosomal ppi-Dependent enzyme in trypanosomatids: pyruvate, Phosphate dikinase.

Authors

F.Bringaud, D.Baltz, T.Baltz.

Ref.

Proc Natl Acad Sci U S A, 1998, 95, 7963-7968. [DOI no: 10.1073/pnas.95.14.7963]

PubMed id

9653123

Figure 1.
Fig. 1. Map of pTSA-3'proc vector designed for gene expression in procyclic forms of T. brucei (kindly provided by D. Salmon and E. Pays). The coding sequences (PPDK: pyruvate, phosphate dikinase; HYG: hygromycine resistance; -TUB and -TUB: - and -tubulin) are indicated by hatched boxes. The black boxes flanking the PPDK gene contain the procyclin promoter indicated by an arrow (5' proc) or the polyadenylation signal present in the 3' noncoding region of the procyclin (3' proc), and the splice leaders (SL) are shown. The white boxes represent the tubulin intergenic region used to target the insertion of the recombinant pTSA-3'proc (B) into the tubulin gene cluster (A), and the pUC18 vector is shown as a hatched thin box. Abbreviations: B, BssHII; Xb, XbaI and Xh, XhoI.

Figure 3.
Fig. 3. Immunofluorescence analysis of T. brucei procyclic forms (EATRO 1125). Untransfected cells were stained with anti-aldolase (A) or anti-PPDK H121 (B) mAbs; anti-poly(His) was used to stain cells expressing histidine-tagged PPDK, which contains the AKL glycosomal targeting motif (PPDK34) (C) or histidine-tagged PPDK depleted of AKL glycosomal targeting motif (PPDK35) (D).