COMPARISON OF THE STRUCTURES OF WILD-TYPE AND A N313T MUTANT OF ESCHERICHIA-COLI GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASES - IMPLICATION FOR NAD BINDING AND COOPERATIVITY

The crystal structure of wild-type and N313T mutant glyceraldehyde 3-p hosphate dehydrogenases from Escherichia coli was determined in the pr esence of NAD at 1.8 Angstrom and 2.17 Angstrom, respectively. The str ucture of the monomer and of the tetramer are similar to those observe d for other GAPDHs. An exhaustive analysis of the hydrophobic clusters and the hydrogen bond networks explain the high degree of sequence co nservation in GAPDHs. The structural effect of the N313T mutation is a change in the (phi,psi) angles of nearby residues Asn236 and Val237, while the structure around the mutated residue remains unchanged. A de tailed comparison of the wild-type and N313T mutant E. coli GAPDH with the apo and hole forms of Bacillus stenrothermophilus GAPDH is carrie d out in relation to the apo --> hole transition. An unbiased set of a bout 60 residues, whose C-alpha atoms remain in the same relative posi tion in the different forms of the tetramer, is defined as the tetrame r ''core'' which acts as a fixed scaffold around which structural rear rangements occur during the apo --> holo transition. This core essenti ally includes beta-strands from the beta-sheets forming the O-P and Q- R interfaces, in particular strand beta 1 which bears catalytic residu e His176. During the apo --> holo transition, dimer O-P rotates around the molecular P-axis by about +1 degrees and dimer O-R by about -1 de grees. Further rotations of the NAD binding domain relative to the cat alytic domain are discussed in relation to the molecular symmetry. The possible effect on NAD binding cooperativity of mutations around the tetramer core is exemplified by residue 252. The presence of a conserv ed hydrophilic patch embedded in the hydrophobic O-P interface is high lighted. A mechanism for substrate binding, different from those curre ntly proposed, is described where the hydroxyl group of the substrate C-(2) atom is hydrogen bonded to Cys149N. (C) 1996 Academic Press Limi ted