Mutation of a key residue in the type II secretion system ATPase uncouples ATP hydrolysis from protein translocation.
Membrane-associated ATPase constitutes an essential element common to all secretion machineries in Gram-negative bacteria. How ATP hydrolysis by these ATPases is coupled to secretion process remains unclear. Here we identified R286 as a key residue in the type II secretion system (T2SS) ATPase XpsE of Xanthomonas campestris that plays a pivotal role in coupling ATP hydrolysis to protein translocation. Mutation of R286 to alanine made XpsE hydrolyse ATP at a rate five times that of the wild-type XpsE. Yet the mutant XpsE(R286A) is non-functional in protein secretion via T2SS. Detailed analyses indicated that the mutant XpsE(R286A) lost the ability co-ordinating the N- and C-domain of XpsE. Without significantly influencing XpsE binding affinity with ATP or its oligomerization, R286A mutation however, caused XpsE lose the ability to associate with the cytoplasmic membrane via XpsL(N). As a consequence, ATP hydrolysis by XpsE was uncoupled from protein secretion. Because R286 is highly conserved among members of the secretion NTPase superfamily, we speculate that its equivalent in other homologues may also play a critical energy coupling role for T2SS, type IV pilus assembly and type IV secretion system.
Shiue
Sheng-Jie
SJ
Institute of Biochemistry, National Chung Hsing University, Taichung, Taiwan.
Chien
I-Ling
IL
Chan
Nei-Li
NL
Leu
Wei-Ming
WM
Hu
Nien-Tai
NT
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000409:Alanine / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport; D016959:Xanthomonas campestris / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; C071567:general secretion pathway protein, Xanthomonas campestris; D000255:Adenosine Triphosphate; D001120:Arginine; D000251:Adenosine Triphosphatases; D000409:Alanine