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Diagnostic chemical shift markers for loop conformation and substrate and cofactor binding in dihydrofolate reductase complexes
Author(s) -
Osborne Michael J.,
Venkitakrishnan Rani P.,
Dyson H. Jane,
Wright Peter E.
Publication year - 2003
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.03219603
Subject(s) - dihydrofolate reductase , ternary complex , cofactor , chemistry , stereochemistry , pterin , active site , chemical shift , binding site , heteronuclear molecule , substrate (aquarium) , ribose , crystallography , nuclear magnetic resonance spectroscopy , enzyme , biochemistry , biology , ecology
Heteronuclear NMR methods have been used to probe the conformation of four complexes of Escherichia coli dihydrofolate reductase (DHFR) in solution. 1 H N , 15 N, and 13 C α resonance assignments have been made for the ternary complex with folate and oxidized NADP + cofactor and the ternary complex with folate and a reduced cofactor analog, 5,6‐dihydroNADPH. The backbone chemical shifts have been compared with those of the binary complex of DHFR with the substrate analog folate and the binary complex with NADPH (the holoenzyme). Analysis of 1 H N and 15 N chemical shifts has led to the identification of marker resonances that report on the active site conformation of the enzyme. Other backbone amide resonances report on the presence of ligands in the pterin binding pocket and in the adenosine and nicotinamide–ribose binding sites of the NADPH cofactor. The chemical shift data indicate that the enzyme populates two dominant structural states in solution, with the active site loops in either the closed or occluded conformations defined by X‐ray crystallography; there is no evidence that the open conformation observed in some X‐ray structures of E. coli DHFR are populated in solution.