MgtB is a P-type ATPase of Salmonella typhimurium that mediates the influx of Mg2+ against its electrochemical gradient, has minimal homology to known prokaryotic P-type ATPases and is most homologous to mammalian Ca2+-ATPases thus making it an excellent model of sarcoplasmic reticular Ca2+-ATPases. Because the molecular genetic techniques available in prokaryotes are easier, faster and in many cases more advanced than in eukaryotic systems, important experimental approaches are available for study of the structure of membrane proteins that are not feasible in eukaryotes. We propose to examine the membrane domain of MgtB using an iterative process combining cysteine scanning mutagenesis and crosslinking with molecular modeling to provide an enhanced structure of the membrane domain. The end result with be a molecularly detailed picture of the spatial orientation of each transmembrane segment to every other segment. Succinctly, we will determine for each transmembrane segment """"""""which face faces which face."""""""" The following specific questions will be asked: 1) What is the structure of the membrane domain of a P-type ATPase? For a given transmembrane segment of MgtB, we will determine which other transmembrane segments are immediately adjacent to allow construction of a medium resolution model of the membrane domain of a P-type ATPase. A """"""""library"""""""" of mutant MgtB isoforms will be created by mutagenizing selected residues within membrane domains to cysteine; double mutants will be then constructed with cysteine substitutions in two different membrane domains. Subsequent oxidation will form disulfide bonds and crosslink only those cysteines with appropriately close spatial orientations within the membrane. This approach is only feasible in a prokaryotic cell. 2) Which membrane domains shift position during the reaction cycle of a P-type ATPase? The evolving membrane domain model will allow prediction of specific amino acid residues within neighboring helices whose relative positions may shift, via rotation and/or translation, during the phosphorylation-dephosphorylation cycle of a P-type ATPase. The predictions will be tested by determining the efficiency of cysteine crosslinking between selected pairs of residues as a function of the enzyme's phosphorylation state. 3) What changes occur in metal binding sites during the reaction cycle of a P-type ATPase? MgtB will be purified using a 6xHis tag. In collaboration with Dr. J.K. Blasie, purified, detergent-solubilized enzyme will be covalently tethered via an extracytoplasmic cysteine to the sulfhydryl endgroup surface of an organic self-assembled monolayer chemisorbed onto the surface of a Ge/Si multilayer substrate. This will provide vectorially oriented enzyme to determine the profile structure of MgtB using nonresonance x-ray diffraction, thereby more firmly establishing its membrane domain structure. Within this profile structure, using resonance x-ray diffraction, the distribution of bound Ni2+, as surrogate for Mg2+ on the enzyme's high-affinity sites will then be determined.
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