Structural tightening and interdomain communication in the catalytic cycle of phosphoglycerate kinase.
Changes in amide-NH chemical shift and hydrogen exchange rates as phosphoglycerate kinase progresses through its catalytic cycle have been measured to assess whether they correlate with changes in hydrogen bonding within the protein. Four representative states were compared: the free enzyme, a product complex containing 3-phosphoglyceric acid (3PG), a substrate complex containing ADP and a transition-state analogue (TSA) complex containing a 3PG-AlF(4)(-)-ADP moiety. There are an overall increases in amide protection from hydrogen exchange when the protein binds the substrate and product ligands and an additional increase when the TSA complex is formed. This is consistent with stabilisation of the protein structure by ligand binding. However, there is no correlation between the chemical shift changes and the protection factor changes, indicating that the protection factor changes are not associated with an overall shortening of hydrogen bonds in the protected ground state, but rather can be ascribed to the properties of the high-energy, exchange-competent state. Therefore, an overall structural tightening mechanism is not supported by the data. Instead, we observed that some cooperativity is exhibited in the N-domain, such that within this domain the changes induced upon forming the TSA complex are an intensification of those induced by binding 3PG. Furthermore, chemical shift changes induced by 3PG binding extend through the interdomain region to the C-domain beta-sheet, highlighting a network of hydrogen bonds between the domains that suggests interdomain communication. Interdomain communication is also indicated by amide protection in one domain being significantly altered by binding of substrate to the other, even where no associated change in the structure of the substrate-free domain is indicated by chemical shifts. Hence, the communication between domains is also manifested in the accessibility of higher-energy, exchange-competent states. Overall, the data that are consistent with structural tightening relate to defined regions and are close to the 3PG binding site and in the hinge regions of 3-phosphoglycerate kinase.
Marston
James P
JP
Department of Molecular Biology and Biotechnology, Firth Court, The University of Sheffield, Western Bank, Sheffield, UK.
Cliff
Matthew J
MJ
Reed
Michelle A C
MA
Blackburn
G Michael
GM
Hounslow
Andrea M
AM
Craven
C Jeremy
CJ
Waltho
Jonathan P
JP
D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D005988:Glyceric Acids / Q000737:chemistry / Q000378:metabolism; D008954:Models, Biological; D008958:Models, Molecular; D010735:Phosphoglycerate Kinase / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary / Q000502:physiology
D005988:Glyceric Acids; D000244:Adenosine Diphosphate; C005156:3-phosphoglycerate; D010735:Phosphoglycerate Kinase