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An unexpected phosphate binding site in glyceraldehyde 3-phosphate dehydrogenase: crystal structures of apo, holo and ternary complex of Cryptosporidium parvum enzyme.

Cook WJ, Senkovich O, Chattopadhyay D - BMC Struct. Biol. (2009)

Bottom Line: The structures of the C. parvum GAPDH ternary complex and other GAPDH complexes demonstrate the plasticity of the substrate binding site.We propose that the active site of GAPDH can accommodate the substrate in multiple conformations at multiple locations during the initial encounter.However, the C-3 phosphate group clearly prefers the 'new Pi' site for initial binding in the active site.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294, USA. wjcook@uab.edu

ABSTRACT

Background: The structure, function and reaction mechanism of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) have been extensively studied. Based on these studies, three anion binding sites have been identified, one 'Ps' site (for binding the C-3 phosphate of the substrate) and two sites, 'Pi' and 'new Pi', for inorganic phosphate. According to the original flip-flop model, the substrate phosphate group switches from the 'Pi' to the 'Ps' site during the multistep reaction. In light of the discovery of the 'new Pi' site, a modified flip-flop mechanism, in which the C-3 phosphate of the substrate binds to the 'new Pi' site and flips to the 'Ps' site before the hydride transfer, was proposed. An alternative model based on a number of structures of B. stearothermophilus GAPDH ternary complexes (non-covalent and thioacyl intermediate) proposes that in the ternary Michaelis complex the C-3 phosphate binds to the 'Ps' site and flips from the 'Ps' to the 'new Pi' site during or after the redox step.

Results: We determined the crystal structure of Cryptosporidium parvum GAPDH in the apo and holo (enzyme + NAD) state and the structure of the ternary enzyme-cofactor-substrate complex using an active site mutant enzyme. The C. parvum GAPDH complex was prepared by pre-incubating the enzyme with substrate and cofactor, thereby allowing free movement of the protein structure and substrate molecules during their initial encounter. Sulfate and phosphate ions were excluded from purification and crystallization steps. The quality of the electron density map at 2A resolution allowed unambiguous positioning of the substrate. In three subunits of the homotetramer the C-3 phosphate group of the non-covalently bound substrate is in the 'new Pi' site. A concomitant movement of the phosphate binding loop is observed in these three subunits. In the fourth subunit the C-3 phosphate occupies an unexpected site not seen before and the phosphate binding loop remains in the substrate-free conformation. Orientation of the substrate with respect to the active site histidine and serine (in the mutant enzyme) also varies in different subunits.

Conclusion: The structures of the C. parvum GAPDH ternary complex and other GAPDH complexes demonstrate the plasticity of the substrate binding site. We propose that the active site of GAPDH can accommodate the substrate in multiple conformations at multiple locations during the initial encounter. However, the C-3 phosphate group clearly prefers the 'new Pi' site for initial binding in the active site.

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Comparison of the D-G3H binding sites in CpGAPDH, A and D subunit. A: Subunit A of CpGAPDH. Stick models are shown in white and red for subunit A; residues that bind to the C-3 phosphate and the active site residues (S153 and H180) are shown as sphere. The C3-phosphate occupies the 'new Pi site' in A, B and C subunits. B: Orientation of D-G3H molecule in subunit D of CpGAPDH (white and red stick) is different. There is no interaction between the phosphate oxygens and the phosphate binding loop.
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Figure 4: Comparison of the D-G3H binding sites in CpGAPDH, A and D subunit. A: Subunit A of CpGAPDH. Stick models are shown in white and red for subunit A; residues that bind to the C-3 phosphate and the active site residues (S153 and H180) are shown as sphere. The C3-phosphate occupies the 'new Pi site' in A, B and C subunits. B: Orientation of D-G3H molecule in subunit D of CpGAPDH (white and red stick) is different. There is no interaction between the phosphate oxygens and the phosphate binding loop.

Mentions: Two distinct positions for the substrate have been described in different GAPDH structures (Table 2). In CpGAPDH subunits A, B and C the substrate binds with the C-3 phosphate in the classical 'new Pi site' as defined by Kim et al [17]. The phosphate oxygen atoms form hydrogen bonds with residues G215 (N), T154 (OG1), S152 (OG), A216 (N) and T214 (OG1) (Fig. 4A and Table 3). In subunit D, the phosphate moiety of D-G3H occupies a site that does not correspond to any previously reported phosphate binding site (Fig. 4B). The phosphorus atom in this subunit is ~2.6–2.8Å from the corresponding position in the other three subunits. The phosphate oxygen atoms form hydrogen bonds with residues S153 (OG), H180 (NE2), T154 (OG1) (Table 3). In subunits A, B and C the hydroxyl oxygen atom of S152 is involved in hydrogen bonding with the C3 phosphate oxygen of the substrate. As in the holoenzyme this hydroxyl oxygen also forms hydrogen bonds with the side chain hydroxyl group of T155. In subunit D, the S152 hydroxyl group retains the hydrogen bond with T155, but the phosphate is oriented away from the S152 hydroxyl oxygen.


An unexpected phosphate binding site in glyceraldehyde 3-phosphate dehydrogenase: crystal structures of apo, holo and ternary complex of Cryptosporidium parvum enzyme.

Cook WJ, Senkovich O, Chattopadhyay D - BMC Struct. Biol. (2009)

Comparison of the D-G3H binding sites in CpGAPDH, A and D subunit. A: Subunit A of CpGAPDH. Stick models are shown in white and red for subunit A; residues that bind to the C-3 phosphate and the active site residues (S153 and H180) are shown as sphere. The C3-phosphate occupies the 'new Pi site' in A, B and C subunits. B: Orientation of D-G3H molecule in subunit D of CpGAPDH (white and red stick) is different. There is no interaction between the phosphate oxygens and the phosphate binding loop.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Comparison of the D-G3H binding sites in CpGAPDH, A and D subunit. A: Subunit A of CpGAPDH. Stick models are shown in white and red for subunit A; residues that bind to the C-3 phosphate and the active site residues (S153 and H180) are shown as sphere. The C3-phosphate occupies the 'new Pi site' in A, B and C subunits. B: Orientation of D-G3H molecule in subunit D of CpGAPDH (white and red stick) is different. There is no interaction between the phosphate oxygens and the phosphate binding loop.
Mentions: Two distinct positions for the substrate have been described in different GAPDH structures (Table 2). In CpGAPDH subunits A, B and C the substrate binds with the C-3 phosphate in the classical 'new Pi site' as defined by Kim et al [17]. The phosphate oxygen atoms form hydrogen bonds with residues G215 (N), T154 (OG1), S152 (OG), A216 (N) and T214 (OG1) (Fig. 4A and Table 3). In subunit D, the phosphate moiety of D-G3H occupies a site that does not correspond to any previously reported phosphate binding site (Fig. 4B). The phosphorus atom in this subunit is ~2.6–2.8Å from the corresponding position in the other three subunits. The phosphate oxygen atoms form hydrogen bonds with residues S153 (OG), H180 (NE2), T154 (OG1) (Table 3). In subunits A, B and C the hydroxyl oxygen atom of S152 is involved in hydrogen bonding with the C3 phosphate oxygen of the substrate. As in the holoenzyme this hydroxyl oxygen also forms hydrogen bonds with the side chain hydroxyl group of T155. In subunit D, the S152 hydroxyl group retains the hydrogen bond with T155, but the phosphate is oriented away from the S152 hydroxyl oxygen.

Bottom Line: The structures of the C. parvum GAPDH ternary complex and other GAPDH complexes demonstrate the plasticity of the substrate binding site.We propose that the active site of GAPDH can accommodate the substrate in multiple conformations at multiple locations during the initial encounter.However, the C-3 phosphate group clearly prefers the 'new Pi' site for initial binding in the active site.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294, USA. wjcook@uab.edu

ABSTRACT

Background: The structure, function and reaction mechanism of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) have been extensively studied. Based on these studies, three anion binding sites have been identified, one 'Ps' site (for binding the C-3 phosphate of the substrate) and two sites, 'Pi' and 'new Pi', for inorganic phosphate. According to the original flip-flop model, the substrate phosphate group switches from the 'Pi' to the 'Ps' site during the multistep reaction. In light of the discovery of the 'new Pi' site, a modified flip-flop mechanism, in which the C-3 phosphate of the substrate binds to the 'new Pi' site and flips to the 'Ps' site before the hydride transfer, was proposed. An alternative model based on a number of structures of B. stearothermophilus GAPDH ternary complexes (non-covalent and thioacyl intermediate) proposes that in the ternary Michaelis complex the C-3 phosphate binds to the 'Ps' site and flips from the 'Ps' to the 'new Pi' site during or after the redox step.

Results: We determined the crystal structure of Cryptosporidium parvum GAPDH in the apo and holo (enzyme + NAD) state and the structure of the ternary enzyme-cofactor-substrate complex using an active site mutant enzyme. The C. parvum GAPDH complex was prepared by pre-incubating the enzyme with substrate and cofactor, thereby allowing free movement of the protein structure and substrate molecules during their initial encounter. Sulfate and phosphate ions were excluded from purification and crystallization steps. The quality of the electron density map at 2A resolution allowed unambiguous positioning of the substrate. In three subunits of the homotetramer the C-3 phosphate group of the non-covalently bound substrate is in the 'new Pi' site. A concomitant movement of the phosphate binding loop is observed in these three subunits. In the fourth subunit the C-3 phosphate occupies an unexpected site not seen before and the phosphate binding loop remains in the substrate-free conformation. Orientation of the substrate with respect to the active site histidine and serine (in the mutant enzyme) also varies in different subunits.

Conclusion: The structures of the C. parvum GAPDH ternary complex and other GAPDH complexes demonstrate the plasticity of the substrate binding site. We propose that the active site of GAPDH can accommodate the substrate in multiple conformations at multiple locations during the initial encounter. However, the C-3 phosphate group clearly prefers the 'new Pi' site for initial binding in the active site.

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