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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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Possible rotation or movement of the substrate in the active site. 2Fo-Fc electron density map computed after the final cycle of refinement contoured at 1σ level (in blue) and Fo-Fc electron density map (3.0σ level) shown in pink around the substrate molecule in C subunit.
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Figure 6: Possible rotation or movement of the substrate in the active site. 2Fo-Fc electron density map computed after the final cycle of refinement contoured at 1σ level (in blue) and Fo-Fc electron density map (3.0σ level) shown in pink around the substrate molecule in C subunit.

Mentions: The orientation of the O2 hydroxyl group and O1 carbonyl oxygen of D-G3H with respect to the active site residues (S153 and H180) varies in different subunits of CpGAPDH tetramer. In subunit A, the O1 oxygen is within hydrogen bonding distance of NE2 of H180 as well as the hydroxyl oxygen atom of S153 (in the wild type enzyme a cysteine residue is present in its place). The O2 oxygen atom forms a hydrogen bond with a water molecule. In subunits B and C, the D-G3H is rotated approximately 180 about the C2-C3 bond (with respect to the substrate in the A subunit). In both B and C subunits, the C2 hydroxyl oxygen atom is within hydrogen bonding distance of H180 NE2 and S153 OG. In these subunits the O1 oxygen of the substrate forms a hydrogen bond with a water molecule. An Fo-Fc electron density map calculated after refinement, showed unexplained density (connecting the position of the C3 atom to a neighboring water molecule) near the D-G3H molecule in the C subunit (Fig. 6) suggesting some movement or conformational flexibility of this molecule. Attempts to fit an alternate conformation of D-G3H in this density were not successful. In molecule D, however, the substrate is rotated 90° so that one of the phosphate oxygen atoms forms a hydrogen bond with the H180 NE2 and S153 OG. There is also a hydrogen bond between the T154 side chain and a phosphate oxygen. In this subunit, the orientation of the substrate places the O1 oxygen away from the active site residues; the O2 oxygen forms a contact with side chain nitrogen atom (NH2) of R237. The position of the substrate in this subunit is clearly not suitable for catalysis and may represent an initial (Pre-Michaelis) complex. Comparison of the holoenzyme subunits with those in the ternary complex reveals that upon substrate binding the side chain of R237 residue in each subunit moves towards the substrate.


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)

Possible rotation or movement of the substrate in the active site. 2Fo-Fc electron density map computed after the final cycle of refinement contoured at 1σ level (in blue) and Fo-Fc electron density map (3.0σ level) shown in pink around the substrate molecule in C subunit.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2662861&req=5

Figure 6: Possible rotation or movement of the substrate in the active site. 2Fo-Fc electron density map computed after the final cycle of refinement contoured at 1σ level (in blue) and Fo-Fc electron density map (3.0σ level) shown in pink around the substrate molecule in C subunit.
Mentions: The orientation of the O2 hydroxyl group and O1 carbonyl oxygen of D-G3H with respect to the active site residues (S153 and H180) varies in different subunits of CpGAPDH tetramer. In subunit A, the O1 oxygen is within hydrogen bonding distance of NE2 of H180 as well as the hydroxyl oxygen atom of S153 (in the wild type enzyme a cysteine residue is present in its place). The O2 oxygen atom forms a hydrogen bond with a water molecule. In subunits B and C, the D-G3H is rotated approximately 180 about the C2-C3 bond (with respect to the substrate in the A subunit). In both B and C subunits, the C2 hydroxyl oxygen atom is within hydrogen bonding distance of H180 NE2 and S153 OG. In these subunits the O1 oxygen of the substrate forms a hydrogen bond with a water molecule. An Fo-Fc electron density map calculated after refinement, showed unexplained density (connecting the position of the C3 atom to a neighboring water molecule) near the D-G3H molecule in the C subunit (Fig. 6) suggesting some movement or conformational flexibility of this molecule. Attempts to fit an alternate conformation of D-G3H in this density were not successful. In molecule D, however, the substrate is rotated 90° so that one of the phosphate oxygen atoms forms a hydrogen bond with the H180 NE2 and S153 OG. There is also a hydrogen bond between the T154 side chain and a phosphate oxygen. In this subunit, the orientation of the substrate places the O1 oxygen away from the active site residues; the O2 oxygen forms a contact with side chain nitrogen atom (NH2) of R237. The position of the substrate in this subunit is clearly not suitable for catalysis and may represent an initial (Pre-Michaelis) complex. Comparison of the holoenzyme subunits with those in the ternary complex reveals that upon substrate binding the side chain of R237 residue in each subunit moves towards the substrate.

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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