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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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Cofactor induced ordering of S-loop. A: Surface drawing showing the packing of CpGAPDH subunits A and D in the apoenzyme. A loop comprising of residues 185–197 is disordered in the apoenzyme structure. CpGAPDH holoenzyme structure in which the above mentioned loop is ordered (shown in cyan), is superposed on the apoenzyme structure. B: Surface representation of the interface between subunits A and D of CpGAPDH. A and D subunits of apo-CpGAPDH and Holo-CpGAPDH superposed. The S-loop, which is disordered in the apoenzyme structure but ordered in the holoenzyme, is shown as surface colored by charge (red: negative, blue: positive, green: neutral). Interactions occur between molecules A & D and B & C.
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Figure 1: Cofactor induced ordering of S-loop. A: Surface drawing showing the packing of CpGAPDH subunits A and D in the apoenzyme. A loop comprising of residues 185–197 is disordered in the apoenzyme structure. CpGAPDH holoenzyme structure in which the above mentioned loop is ordered (shown in cyan), is superposed on the apoenzyme structure. B: Surface representation of the interface between subunits A and D of CpGAPDH. A and D subunits of apo-CpGAPDH and Holo-CpGAPDH superposed. The S-loop, which is disordered in the apoenzyme structure but ordered in the holoenzyme, is shown as surface colored by charge (red: negative, blue: positive, green: neutral). Interactions occur between molecules A & D and B & C.

Mentions: Cofactor binding induces ordering of the S-loop (residues 185–197) in all four subunits of CpGAPDH (Fig. 1A). In the apoenzyme this loop, which forms part of the NAD binding pocket, is not visible in the electron density map apparently due to disorder in the absence of NAD. The loop is well defined in all four subunits in the holoenzyme structure as well as in the ternary complex. Ordering of this loop increases interactions between subunits positioned across from each other in the tetramer (A:D, and B:C subunits) and changes packing environment at the subunit interfaces (Fig. 1B). However, there is no direct contact between NAD and any amino acid residue in this loop. In the holoenzyme one water molecule is involved in bridging the pyrophosphate of the nicotinamide moiety with the peptide nitrogen atom of A184 in subunits A (W425), C (W717) and D (W379). The corresponding oxygen and nitrogen atoms in the B subunit are ~1Å further than in the other three subunits and no water molecule could be located for bridging them. In addition, in each subunit pair (A, D and B, C) one water molecule acts as a bridge between the main chain oxygen atom on D190 of one subunit and the adenosine pyrophosphate group of the NAD molecule belonging to the other subunit. In each subunit, one or both of the carboxyl oxygen atoms of D34 forms hydrogen bonds to the hydroxyl oxygen atoms of adenosine ribose of NAD.


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)

Cofactor induced ordering of S-loop. A: Surface drawing showing the packing of CpGAPDH subunits A and D in the apoenzyme. A loop comprising of residues 185–197 is disordered in the apoenzyme structure. CpGAPDH holoenzyme structure in which the above mentioned loop is ordered (shown in cyan), is superposed on the apoenzyme structure. B: Surface representation of the interface between subunits A and D of CpGAPDH. A and D subunits of apo-CpGAPDH and Holo-CpGAPDH superposed. The S-loop, which is disordered in the apoenzyme structure but ordered in the holoenzyme, is shown as surface colored by charge (red: negative, blue: positive, green: neutral). Interactions occur between molecules A & D and B & C.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Cofactor induced ordering of S-loop. A: Surface drawing showing the packing of CpGAPDH subunits A and D in the apoenzyme. A loop comprising of residues 185–197 is disordered in the apoenzyme structure. CpGAPDH holoenzyme structure in which the above mentioned loop is ordered (shown in cyan), is superposed on the apoenzyme structure. B: Surface representation of the interface between subunits A and D of CpGAPDH. A and D subunits of apo-CpGAPDH and Holo-CpGAPDH superposed. The S-loop, which is disordered in the apoenzyme structure but ordered in the holoenzyme, is shown as surface colored by charge (red: negative, blue: positive, green: neutral). Interactions occur between molecules A & D and B & C.
Mentions: Cofactor binding induces ordering of the S-loop (residues 185–197) in all four subunits of CpGAPDH (Fig. 1A). In the apoenzyme this loop, which forms part of the NAD binding pocket, is not visible in the electron density map apparently due to disorder in the absence of NAD. The loop is well defined in all four subunits in the holoenzyme structure as well as in the ternary complex. Ordering of this loop increases interactions between subunits positioned across from each other in the tetramer (A:D, and B:C subunits) and changes packing environment at the subunit interfaces (Fig. 1B). However, there is no direct contact between NAD and any amino acid residue in this loop. In the holoenzyme one water molecule is involved in bridging the pyrophosphate of the nicotinamide moiety with the peptide nitrogen atom of A184 in subunits A (W425), C (W717) and D (W379). The corresponding oxygen and nitrogen atoms in the B subunit are ~1Å further than in the other three subunits and no water molecule could be located for bridging them. In addition, in each subunit pair (A, D and B, C) one water molecule acts as a bridge between the main chain oxygen atom on D190 of one subunit and the adenosine pyrophosphate group of the NAD molecule belonging to the other subunit. In each subunit, one or both of the carboxyl oxygen atoms of D34 forms hydrogen bonds to the hydroxyl oxygen atoms of adenosine ribose of NAD.

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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