Conformational Dynamics of a Mobile Loop in the NAD(H)‐Binding Subunit of Proton‐Translocating Transhydrogenases from Rhodospirillum Rubrum and Escherichia Coli

Abstract

Transhydrogenase catalyses the reversible transfer of reducing equivalents between NAD(H) and NADP(H) to the translocation of protons across a membrane. Uniquely in Rhodospirillum rubrum, the NAD(H)-binding subunit (called Th,) exists as a separate subunit which can be reversibly dissociated from the membrane-located subunits. We have expressed the gene for R. rubrum Th, in Escherichia coli to yield large quantities of protein. Low concentrations of either trypsin or endoproteinase Lys-C lead to cleavage of purified Th, specifically at Lys227-Thr228 and Lys237-Glu238. Observations on the onedimensional 'H-NMR spectra of Th, before and after proteolysis indicate that the segment which straddles the cleavage sites forms a mobile loop protruding from the surface of the protein. Alanine dehydrogenase, which is very similar in sequence to the NAD(H)-binding subunit of transhydrogenase, lacks this segment. Limited proteolytic cleavage has little effect on some of the structural characteristics of Th, (its dimeric nature, its ability to bind to the membrane-located subunits of transhydrogenase, and the short-wavelength fluorescence emission of a unique Trp residue) but does decrease the NADH-binding affinity, and does lower the catalytic activity of the reconstituted complex. The presence of NADH protects against trypsin or Lys-C cleavage, and leads to broadening, and in some cases, shifting, of NMR spectral signals associated with amino acid residues in the surface loop. This indicates that the loop becomes less mobile after nucleotide binding. Observation by NMR during a titration of Th, with NAD' provides evidence of a two-step nucleotide binding reaction. By introducing an appropriate stop codon into the gene coding for the polypeptide of E. coli transhydrogenase cloned into an expression vector, we have prepared the NAD(H)-binding domain equivalent to Th,. The E. coli protein is sensitive to proteolysis by either trypsin or Lys-C in the mobile loop. Judging by the effect of NADH on its NMR spectrum and on the fluorescence of its Trp residues, the protein is capable of binding the nucleotide though it is unable to dock with the membrane-located subunits of transhydrogenase from R. rubrum.

Mapping of Residues in the NADP(H)-binding Site of Proton-translocating Nicotinamide Nucleotide Transhydrogenase fromEscherichia coli

“…The rates of release of NADP ϩ must, at a minimum, be as fast as the turnover of the reverse reaction 4 J. Rydström, X., Hu, and J. B. Jackson, unpublished results.…”

“…Domain II (ϳ␣405 to ␣510 and ␤1 to ϳ␤260) spans the membrane. Domain I (dI) from E. coli (4,5), Rhodospirillum rubrum (rrI) (6), and bovine (7), and domain III (dIII) from E. coli (5), R. rubrum (8,9), and bovine (7,9) have been overexpressed, purified, and partially characterized. So far, domain II has not been expressed as a separate entity.…”

Review and Hypothesis. New insights into the reaction mechanism of transhydrogenase: Swivelling the dIII component may gate the proton channel

“…1), interact with the bound nucleotide. The former, detectable in proton NMR spectra because of its segmental mobility [25], closes down on the surface of the protein following nucleotide binding. Mutations in the mobile loop of Rhodospirillum rubrum dI (Rra 1 M226F, Rra 1 T231C, Rra 1 G234A, Rra 1 Y235F, Rra 1 A236G, Rra 1 K237M -see Fig.…”

Catalytic Properties of Hybrid Complexes of the NAD(H)-Binding and NADP(H)-Binding Domains of the Proton-Translocating Transhydrogenases from Escherichia coli and Rhodospirillum rubrum

“…The dI proteins are isolated as apoprotein dimers, which bind NADH with moderately high affinity and NAD + with lower affinity (15). The binding of NAD(H) to recombinant dI protein is accompanied by closure of a surface mobile loop onto the adenosine part of the nucleotide (10). Single site mutations 1 Abbreviations: dI, dII, and dIII, domain I, domain II, and domain III, respectively, of transhydrogenases in general; rrI and rrIII, domain I and domain III constructs, respectively, of Rhodospirillum rubrum transhydrogenase; ecI and ecIII, domain I and domain III constructs, respectively, of Escherichia coli transhydrogenase; ec , preparation of E. coli transhydrogenase in which at least the portion of the R-subunit extruding from the membrane has been degraded; AcPyAD + , 3-acetylpyridine adenine dinucleotide (oxidized form).…”