Thermal unfolding used as a probe to characterize the intra- and intersubunit stabilizing interactions in phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus.
Tetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus can be described as a dimer of dimers with three nonequivalent interfaces. To investigate the contribution of intra- and intersubunit interactions to GAPDH thermostability, 10 residues located either at the cofactor domain (amino acids 1-148 and 313-333) or at the catalytic domain (amino acids 149-312) were mutated and the thermal unfolding of the mutants was studied by differential scanning calorimetry in the absence and presence of saturating concentrations of NAD. Disruptions of intrasubunit interactions lead to a drastic decrease in thermostability of the N313T, Y283V, and W310F mutants. Moreover, for the N313T mutant, a weakening of cooperative interactions between the catalytic and the cofactor domains and an inefficient binding of NAD are observed. This is likely the consequences of modification or loss of the hydrogen bonding network associating N313 and residues 236-238 and N313 and the nicotinamide carboxyamide of NAD, respectively. For the residues Y283 and W310, which are involved in stacking hydrophobic interactions, mutating both positions does not affect the efficiency of NAD binding. This shows that the factors involved in the thermostability of the tetrameric apo GAPDH are then different from those induced by NAD binding. Disruption of intersubunit hydrogen bonds between the catalytic domain and the NAD-binding domain of a neighboring subunit also leads to a significant destabilization of the apo tetrameric form as observed for the D282G mutant. Moreover, no efficient binding of NAD is observed. Both results are likely the consequence of a loss of hydrogen bonds across the P-axis and the Q-axis between D282 and R197 and between D282 and R52, respectively. Similar results, i.e., a destabilizing effect and inefficient NAD binding, are observed with the T34Q/T39S/L43Q mutant in which steric hindrance is introduced at the S-loop of the R-axis-related subunit via mutations at the adenosine subsite. The dimeric form of the D282G mutant exhibits a single partial heat absorption peak, whereas the Y46G/R52G mutant which exists only as a dimer shows two peaks. Taking into account the recent small-angle X-ray scattering studies which suggested that the dimeric form of the D282G mutant and of the dimeric Y46G/R52G mutant are of the O-R and O-P types, respectively (Vachette, unpublished results), we propose that the presence of one or two peaks in thermal unfolding of dimers is a signature of the dimer type.
Roitel
Olivier
O
Maturation des ARN et Enzymologie Moléculaire, UMR 7567 CNRS-UHP, Université Henri Poincaré Nancy I, BP 239, 54506 Vandoeuvre-lès-Nancy Cedex, France.
Ivinova
Olga
O
Muronetz
Vladimir
V
Nagradova
Natalia
N
Branlant
Guy
G
D001051:Apoenzymes / Q000737:chemistry / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D019281:Dimerization; D004795:Enzyme Stability / Q000235:genetics; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000737:chemistry* / Q000235:genetics; D020035:Holoenzymes / Q000737:chemistry / Q000235:genetics; D006358:Hot Temperature; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D011489:Protein Denaturation / Q000235:genetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry
D001051:Apoenzymes; D020035:Holoenzymes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)