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

Abstract

Proton pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains an alpha subunit with the NAD(H)-binding domain I and a beta subunit with the NADP(H)-binding domain III. The membrane domain (domain II) harbors the proton channel and is made up of the hydrophobic parts of the alpha and beta subunits. The interface in domain II between the alpha and the beta subunits has previously been investigated by cross-linking loops connecting the four transmembrane helices in the alpha subunit and loops connecting the nine transmembrane helices in the beta subunit. However, to investigate the organization of the nine transmembrane helices in the beta subunit, a split was introduced by creating a stop codon in the loop connecting transmembrane helices 9 and 10 by a single mutagenesis step, utilizing an existing downstream start codon. The resulting enzyme was composed of the wild-type alpha subunit and the two new peptides beta1 and beta2. As compared to other split membrane proteins, the new transhydrogenase was remarkably active and catalyzed activities for the reduction of 3-acetylpyridine-NAD(+) by NADPH, the cyclic reduction of 3-acetylpyridine-NAD(+) by NADH (mediated by bound NADP(H)), and proton pumping, amounting to about 50-107% of the corresponding wild-type activities. These high activities suggest that the alpha subunit was normally folded, followed by a concerted folding of beta1 + beta2. Cross-linking of a betaS105C-betaS237C double cysteine mutant in the functional split cysteine-free background, followed by SDS-PAGE analysis, showed that helices 9, 13, and 14 were in close proximity. This is the first time that cross-linking between helices in the same beta subunit has been demonstrated.

Transmembrane Domain Interactions and Residue Proline 378 Are Essential for Proper Structure, Especially Disulfide Bond Formation, in the Human Vitamin K-Dependent γ-Glutamyl Carboxylase

“…When limited structural information about a protein is available, functional splitting and reassembly of multispanning membrane proteins can be employed to study their structure and function (17)(18)(19)(20)(21)(22). It is a useful approach to study the function of protein domains, the role of individual transmembrane helices, and the helical packing within the membrane.…”

“…Given the limited amount of structural information about the carboxylase, it is difficult to incorporate the information we have about residues important for function into a coherent model of how this multisubstrate enzyme simultaneously catalyzes the carboxylation and epoxidation reactions. When limited structural information about a protein is available, functional splitting and reassembly of multispanning membrane proteins can be employed to study their structure and function – . It is a useful approach to study the function of protein domains, the role of individual transmembrane helices, and the helical packing within the membrane.…”

Characterization of a Unique MutantlysEGene, Originating fromCorynebacterium glutamicum, Encoding a Product That InducesL-Lysine Production inMethylophilus methylotrophus

“…A unique variant of the LysE protein might be needed for L-lysine export in M. methylotrophus and M. glycogenes. Artificial splitting of membrane proteins, which can form a catalytically active heterodimer complex, have been described earlier, [23][24][25] but such a conversion is not feasible in some cases, presumably due to insufficient assembly or improper folding of fragments. 26) Several split proteins were active only in the case of coexpression of the polypeptides in a restricted space in the cell, from a dicistronic operon on a single plasmid for example.…”