Interactions of the NADP(H)-binding domain III of proton-translocating transhydrogenase from escherichia coli with NADP(H) and the NAD(H)-binding domain I studied by NMR and site-directed mutagenesis.
Using the purified NADP(H)-binding domain of proton-translocating Escherichia coli transhydrogenase (ecIII) overexpressed in (15)N- and (2)H-labeled medium, together with the purified NAD(H)-binding domain from E. coli (ecI), the interface between ecIII and ecI, the NADP(H)-binding site and the influence on the interface by NAD(P)(H) was investigated in solution by NMR chemical shift mapping. Mapping of the NADP(H)-binding site showed that the NADP(H) substrate is bound to ecIII in an extended conformation at the C-terminal end of the parallel beta-sheet. The distribution of chemical shift perturbations in the NADP(H)-binding site, and the nature of the interaction between ecI and ecIII, indicated that the nicotinamide moiety of NADP(H) is located near the loop comprising residues P346-G353, in agreement with the recently determined crystal structures of bovine [Prasad, G. S., et al. (1999) Nat. Struct. Biol. 6, 1126-1131] and human heart [White, A. W., et al. (2000) Structure 8, 1-12] transhydrogenases. Further chemical shift perturbation analysis also identified regions comprising residues G389-I406 and G430-V434 at the C-terminal end of ecIII's beta-sheet as part of the ecI-ecIII interface, which were regulated by the redox state of the NAD(P)(H) substrates. To investigate the role of these loop regions in the interaction with domain I, the single cysteine mutants T393C, R425C, G430C, and A432C were generated in ecIII and the transhydrogenase activities of the resulting mutant proteins characterized using the NAD(H)-binding domain I from Rhodospirillum rubrum (rrI). All mutants except R425C showed altered NADP(H) binding and domain interaction properties. In contrast, the R425C mutant showed almost exclusively changes in the NADP(H)-binding properties, without changing the affinity for rrI. Finally, by combining the above conclusions with information obtained by a further characterization of previously constructed mutants, the implications of the findings were considered in a mechanistic context.
Bergkvist
A
A
Department of Biochemistry and Biophysics, Göteborg University, S-413 90 Göteborg, Sweden. anders.bergkvist@bcbp.gu.se
Johansson
C
C
Johansson
T
T
Rydström
J
J
Karlsson
B G
BG
D000595:Amino Acid Sequence; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000737:chemistry* / Q000235:genetics; D009249:NADP / Q000737:chemistry* / Q000235:genetics; D009250:NADP Transhydrogenases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011485:Protein Binding / Q000235:genetics; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011522:Protons; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012247:Rhodospirillum rubrum / Q000201:enzymology / Q000235:genetics; D012996:Solutions; D013379:Substrate Specificity / Q000235:genetics
D011522:Protons; D011994:Recombinant Proteins; D012996:Solutions; D009243:NAD; D009249:NADP; D009250:NADP Transhydrogenases