Interactions between the soluble domain I of nicotinamide nucleotide transhydrogenase from Rhodospirillum rubrum and transhydrogenase from Escherichia coli

Abstract

Nicotinamide nucleotide transhydrogenase from Escherichia coli is composed of two subunits, the a and the b subunits, each of which contains a hydrophilic domain, domain I and III, respectively, as well as several transmembrane helices, collectively denoted domain II. The interactions between domain I from Rhodospirillum rubrum (rrI) and the intact or the protease-treated enzyme from E. coli was investigated using the separately expressed and purified domain I from R. rubrum, and His-tagged intact and trypsin-treated E. coli transhydrogenase.Despite harsh treatments with, e.g. detergents and denaturing agents, the a and b subunits remained tightly associated. A monoclonal antibody directed towards the a subunit was strongly inhibitory, an effect that was relieved by added rrI. In addition, rrI also reactivated the trypsin-digested E. coli enzyme in which domain I had been partly removed. This suggests that the hydrophilic domains I and III are not in permanent contact but are mobile during catalysis while being anchored to domain II.Replacement of domain I of intact, as well as trypsin-digested, E. coli transhydrogenase with rrI resulted in a markedly different pH dependence of the cyclic reduction of 3-acetyl-pyridine-NAD 1 by NADH in the presence of NADP(H), suggesting that the protonation of one or more protonable groups in domain I is controlling this reaction. The reverse reaction and proton pumping showed a less pronounced change in pH dependence, demonstrating the regulatory role of domain II in these reactions.Keywords: transhydrogenase; NADP, NAD; membrane protein; proton pump. (Fig. 1). The active forms of the enzymes are dimers (220 kDa), either as a homodimer in the case of the mitochondrial H 1 -TH, as (a,b) 2 in the case of the Escherichia coli H 1 -TH or as (a1,a2,b) 2 in the case of Rhodospirillum rubrum H 1 -TH (1). As shown in Fig. 1, the a subunit (54 kDa) of E. coli H 1 -TH comprises domain I (40 kDa) and four transmembraneous helices, which form one component of dII. The b subunit (49 kDa) is composed of domain III (20 kDa) and nine helices spanning the membrane which constitute the second part of dII. The a1 subunit of R. rubrum corresponds only to domain I and is therefore purified as a soluble homodimer of 81 kDa [3]. The structural properties of domain I (dI) and domain III (dIII), from the three sources mentioned previously, have been characterized by expressing them separately. The determination of their structures has been realized by structural predictions [4±6], NMR [6±9] and X-ray crystallography [10,11]. From these studies, it was found that dI was made of two similar domains (dIa and dIb) each of which comprise a parallel b sheet surrounded by mainly b helices. The structure of domain III also exhibited a classical dinucleotide-binding fold [10,11].

Roles of Individual Amino Acids in Helix 14 of the Membrane Domain of Proton-Translocating Transhydrogenase from Escherichia coli As Deduced from Cysteine Mutagenesis

“…Thus, cysteine mutants of the conserved glycines generally showed low or very low activities, especially of the cyclic reaction, without major changes in the K m NADPH . Separately expressed rrI has earlier been shown to be able to replace both functional and degraded dI in E. coli (30). Addition of rrI to the G245C, G249C, and G252C mutants increased the cyclic activities by 70%, 100%, and 55%, respectively, as compared to 30% for cfTH (not shown).…”

Characterization of hampin/MSL1 as a node in the nuclear interactome

“…His 6 -tagged 51HIN was used for immunization of rabbits and mouse, whereas His 6 -tagged NOP17 was used only for immunization of mouse. Monoclonal and polyclonal antibodies to D1HTH domain (2G10) and monoclonal antibodies 2D8 against first cytoplasmic loop and 3C12 against calmodulin-binding domain of hPMCA4b were described earlier [14] (N.B.P., unpublished data). Mouse 3T3 fibroblasts and the myeloma cells were grown in DMEM containing 10% fetal bovine serum.…”

Functional Split and Crosslinking of the Membrane Domain of the β Subunit of Proton-Translocating Transhydrogenase from Escherichia coli

“…This activity was estimated by the reduction of 400 µM AcPyAD + by 200 µM NADH in the presence or absence of 400 µM NADP + in a medium containing 20 mM Hepes, Mes, Tris-HCl, and Ches, 50 mM NaCl, 2 mM MgCl 2 , and 0.01% Brij (pH 6.0). In addition, the cyclic activity was also assayed in the absence and in the presence of 5 µg of rrI as described (23).…”

“…Trypsin Digestion. When trypsin is added to TH, in the absence of NADP(H), dI is degraded, but the rest of the enzyme remains intact ( , ). Digestion was carried out at 10 °C with trypsin at a mass ratio of 1:30 of trypsin/enzyme.…”

“…As shown in Table 1, the activities of the reverse reaction catalyzed by wtTH(S) and cfTH(S) were 58 and 61%, respectively, of wtTH, whereas the corresponding cyclic activities were 49 and 55%, respectively, in the absence of added rrI and 77 and 70% in the presence of added rrI. Inactivation of the cyclic activities by trypsin due to the cleavage of dI was essentially restored by adding rrI, which has been shown to replace the function of dI in E. coli TH (23). All cyclic activities in the absence of NADP(H) were nil, suggesting that no NADP(H) was bound to dIII (6).…”