Mapping the oligomeric interface of diacylglycerol kinase by engineered thiol cross-linking: Homologous sites in the transmembrane domain

This work represents the first stage of thiol-based cross-linking studies t o map the oligomeric interface of the homotrimeric membrane protein diacylg lycerol kinase (DAGK). A total of 53 single-cysteine mutants spanning DAGK' s three transmembrane segments and the first part of a cytoplasmic domain w ere purified and subjected to catalytic oxidation in mixed micelles. Four m utants (A52C, I53C, A74C, and I75C) were observed to undergo intratrimer di sulfide bond formation between homologous sites on adjacent subunits, To es tablish whether the homologous sites are proximal in the ground-state confo rmation of DAGK or whether the disulfide bonds formed as a result of motion s that brought normally distal sites into transient proximity, additional c ross-linking experiments were carried out in three different milieus of var ying fluidity [mixed micelles, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocho line (POPC) vesicles, and Escherichia coli membranes]. Cross-linking experi ments included disulfide bond formation under three different catalytic con ditions [Cu(II)-phenanthroline oxidation, I-2 oxidation. and thionitrobenzo ate-based thiol exchange] and reactions with a set of bifunctional thiol-re active chemical cross-linkers presenting two different reactive chemistries and several spacer lengths. On the basis of these studies, residues 53 and 75 are judged to be in stable proximity within the DAGK homotrimer, while position 52 appears to be more distal and forms disulfide bonds only as a r esult of protein motions. Results for position 74 were ambiguous, In lipid vesicles and mixed micelles DAGK appears to execute motions that are not pr esent in native membranes, with mobility also being higher for DAGK in mixe d micelles than in POPC vesicles.