Probing the functional tolerance of the b subunit of Escherichia coli ATP synthase for sequence manipulation through a chimera approach.
A dimer of 156-residue b subunits forms the peripheral stator stalk of eubacterial ATP synthase. Dimerization is mediated by a sequence with an unusual 11-residue (hendecad) repeat pattern, implying a right-handed coiled coil structure. We investigated the potential for producing functional chimeras in the b subunit of Escherichia coli ATP synthase by replacing parts of its sequence with corresponding regions of the b subunits from other eubacteria, sequences from other polypeptides having similar hendecad patterns, and sequences forming left-handed coiled coils. Replacement of positions 55-110 with corresponding sequences from Bacillus subtilis and Thermotoga maritima b subunits resulted in fully functional chimeras, judged by support of growth on nonfermentable carbon sources. Extension of the T. maritima sequence N-terminally to position 37 or C-terminally to position 124 resulted in slower but significant growth, indicating retention of some capacity for oxidative phosphorylation. Portions of the dimerization domain between 55 and 95 could be functionally replaced by segments from two other proteins having a hendecad pattern, the distantly related E subunit of the Chlamydia pneumoniae V-type ATPase and the unrelated Ag84 protein of Mycobacterium tuberculosis. Extension of such sequences to position 110 resulted in loss of function. None of the chimeras that incorporated the leucine zipper of yeast GCN4, or other left-handed coiled coils, supported oxidative phosphorylation, but substantial ATP-dependent proton pumping was observed in membrane vesicles prepared from cells expressing such chimeras. Characterization of chimeric soluble b polypeptides in vitro showed their retention of a predominantly helical structure. The T. maritima b subunit chimera melted cooperatively with a midpoint more than 20 degrees C higher than the normal E. coli sequence. The GCN4 construct melted at a similarly high temperature, but with much reduced cooperativity, suggesting a degree of structural disruption. These studies provide insight into the structural and sequential requirements for stator stalk function.
Bi
Yumin
Y
Department of Biochemistry, University of Western Ontario, London, Ontario, Canada N6A 5C1.
Watts
Joel C
JC
Bamford
Pamela Krauss
PK
Briere
Lee-Ann K
LK
Dunn
Stanley D
SD
D025181:ATP Synthetase Complexes / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002462:Cell Membrane / Q000201:enzymology; D004251:DNA Transposable Elements; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism
D004251:DNA Transposable Elements; D029968:Escherichia coli Proteins; D021122:Protein Subunits; D025181:ATP Synthetase Complexes