The high affinity ATP binding site modulates the SecA-precursor interaction.
SecA is the central component of the protein-translocation machinery of Escherichia coli. It is able to interact with the precursor protein, the chaperone SecB, the integral membrane protein complex SecYEG, acidic phospholipids and its own mRNA. We studied the interaction between prePhoE and SecA by using a site-specific photocrosslinking strategy. We found that SecA is able to interact with both the signal sequence and the mature domain of prePhoE. Furthermore, this interaction was dependent on the type of nucleotide bound. SecA in the ADP-bound conformation was unable to crosslink with the precursor, whereas the ATP-bound conformation was active in precursor crosslinking. The SecA-precursor interaction was maintained in the presence of E. coli phospholipids but was loosened by the presence of phosphatidylglycerol bilayers. Examining SecA ATP binding site mutants demonstrated that ATP hydrolysis at the N-terminal high affinity binding site is responsible for the changed interaction with the preprotein.
van Voorst
F
F
CBLE, Institute of Biomembranes, Department of Biochemistry of Membranes, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands. f.vanvoorst@chem.uu.nl
Vereyken
I J
IJ
de Kruijff
B
B
D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001386:Azides / Q000494:pharmacology; D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism; D004164:Diphosphonates / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006868:Hydrolysis / Q000187:drug effects; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D026901:Membrane Transport Proteins; D009154:Mutation / Q000235:genetics; D010743:Phospholipids / Q000378:metabolism; D018272:Porins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000502:physiology; D021381:Protein Transport / Q000187:drug effects; D000069816:SEC Translocation Channels; D000081416:SecA Proteins
D001386:Azides; D001426:Bacterial Proteins; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D004164:Diphosphonates; D029968:Escherichia coli Proteins; D008051:Lipid Bilayers; D008081:Liposomes; D026901:Membrane Transport Proteins; D010743:Phospholipids; D018272:Porins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D000069816:SEC Translocation Channels; C057842:SecB protein, Bacteria; D000266:Adenylyl Imidodiphosphate; C031876:azacycloheptane-2,2-diphosphonate; D000244:Adenosine Diphosphate; C082922:PhoE protein, E coli; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins