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1.
Figure 6

Figure 6. A conserved catalytic blueprint in hPPIP5K2KD. From: Structural basis for an inositol pyrophosphate kinase surmounting phosphate crowding.

This figure shows a spatially conserved catalytic blueprint in hPPIP5K2KD (PDB code 3T9D), several other inositol phosphate kinases (PDB codes: hITPK1 (ref. 24), 2Q7D; hIP3K32, 1W2C; AtIP5K33, 2XAN), and PKA34 (PDB code 1L3R). The structures were aligned by superimposing the bound nucleotides; the color key denotes the origins of the lysine and aspartic acid side chains and the donor (leaving) and the acceptor (entering) groups.

Huanchen Wang, et al. Nat Chem Biol. 2011 November 27;8(1):111-116.
2.
Figure 5

Figure 5. The relative topologies of nucleotide and substrate in inositol phosphate kinases. From: Structural basis for an inositol pyrophosphate kinase surmounting phosphate crowding.

Comparison of inositol phosphate substrate orientation relative to the nucleotide cofactor by several inositol phosphate kinases (Protein Data Bank codes: hPPIP5K2KD, 3T9D; EhITPK1 (ref. 24), 1Z2P; hIP3K32, 1W2C; AtIP5K33, 2XAN) is shown. We have highlighted the angle between the plane of the nucleotide’s β- and γ-phosphates and the plane of the inositol ring substrate. Numbers indicate phosphorylation positions on the inositol rings.

Huanchen Wang, et al. Nat Chem Biol. 2011 November 27;8(1):111-116.
3.
Figure 2

Figure 2. Comparison of substrate binding pockets for hPPIP5K2KD and ITPK1. From: Structural basis for an inositol pyrophosphate kinase surmounting phosphate crowding.

(a,b) Substrates for hPPIP5K2KD (5-IP7) (a) and EhITPK1 (1,3,4-IP3) (b) are shown in gray with orange and red phosphate groups. Residues that contact the substrate are also shown (gray spheres, carbon; red spheres, oxygen; blue spheres, nitrogen). ATP analogs are depicted as brown stick models. (ce) Electrostatic surface representations of inositol phosphate substrate binding pockets of hPPIP5K2KD (c), EhITPK1 (d) and hITPK1 (e). The overall sequence identity of hPPIP5K2KD with EhITPK1 and hITPK1 is 8% and 11%, respectively.

Huanchen Wang, et al. Nat Chem Biol. 2011 November 27;8(1):111-116.
4.
Figure 1

Figure 1. Overall structure of hPPIP5K2KD, a member of the ATP–grasp fold superfamily. From: Structural basis for an inositol pyrophosphate kinase surmounting phosphate crowding.

(a) Ribbon diagram of hPPIP5K2KD. The αβα domain (residues 42–124 and 330–366) is shown in yellow. The two antiparallel β-sheet clusters of the ATP-grasp domain are colored green (residues 125–148 and 244–329) and blue (residues 149–243). Lighter shading of each color highlights structural elements that are present in hPPIP5K2KD but not in ITPK1 (see later panels). For example, the three-stranded β-sheet that is unique to hPPIP5K2KD is colored cyan. Shown in stick models are 5-IP7 and AMP-PNP; the latter is deeply buried (9% solvent accessible). (b) Electrostatic surface plot colored in blue and red, which respectively indicate positive and negative electrostatic potentials at physiological pH. (c) Ribbon diagram of EhITPK1 shown with bound 1,3,4-IP3 as a gray stick model with orange and red phosphate groups. (d) Ribbon diagram of hITPK1 (crystal structure with an inositol phosphate has not yet been determined).

Huanchen Wang, et al. Nat Chem Biol. 2011 November 27;8(1):111-116.
5.
Figure 3

Figure 3. Structural and mutagenic analysis of residues in hPPIP5K2KD that participate in substrate binding. From: Structural basis for an inositol pyrophosphate kinase surmounting phosphate crowding.

(a) Active site of hPPIP5K2KD. Shown in green are residues that make polar contacts with substrates. 5-IP7 is shown with gray carbon and orange and red phosphate groups, and IP6 is shown in light blue. Carbon atoms on the inositol ring are numbered. The interactions of Lys214 with 5-IP7 are highlighted by dashed yellow lines. Mg4 and Mg5 (sky blue spheres) are two magnesium atoms that are only present in crystal complexes that also contain substrate (Mg5 was present only in the hPPIP5K2KD–5-IP7 complex). (b) Relative catalytic activities of wild-type and single-site mutants of hPPIP5K2KD. First-order rate constants are shown relative to wild-type enzyme. The horizontal broken line emphasizes a 90% decrease in catalytic activity. Rates for wild-type enzyme: 5-IP7 = 346 ± 15 μg−1 min−1, IP6 = 16 ± 1 μg−1 min−1. n = 3, mean ± s.e.m. The data for the various mutants are mean values from two determinations, and s.d. were less than 10% of the mean values.

Huanchen Wang, et al. Nat Chem Biol. 2011 November 27;8(1):111-116.
6.
Figure 4

Figure 4. Snapshots of the reaction mechanism. From: Structural basis for an inositol pyrophosphate kinase surmounting phosphate crowding.

(ac) Ground (a), transition (b) and product (c) states of hPPIP5K2KD. Key catalytic residues are shown as green and blue sticks. Inositol pyrophosphates and the nucleotide cofactors are shown (white, carbon; red, oxygen; orange, phosphorus; blue, nitrogen), as are catalytic magnesium atoms (magenta spheres) and a water molecule (WAT; blue sphere). The 1-carbon atom in the inositol ring is labeled. Bond distances (dotted lines) are denoted in angstroms (Å). The MgF3 transition state analog is depicted in purple (for Mg) and cyan (for F). The simulated-annealing OMIT difference map for MgF3 is contoured at 5.0 σ. (d) Induced-fit motions of hPPIP5K2 upon 5-IP7 binding. The hPPIP5K2KD–AMP-PNP structure (orange) is superimposed with the structure of hPPIP5K2KD–AMP-PNP–5-IP7 (green). Metals in the catalytic center are colored magenta. Movements of side chains are highlighted with black arrows. (e) View highlighting some differences in the interactions of substrates with amino acids in the ground (green) and transition (purple) states. (f) Superimposition of inositol pyrophosphate bound in the ground (green), transition (purple) and product states (brown). Leaving and entering oxygen atoms are colored red. Movements are highlighted by solid arrows (detailed further in text). The dashed line shows the direction of nucleophilic attack. During catalysis, the relative positions of the three oxygen atoms of the active phosphoryl group are preserved, but the phosphorus atom moves about 1.4 Å and its configuration is inverted.

Huanchen Wang, et al. Nat Chem Biol. 2011 November 27;8(1):111-116.

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