Inhibition of preprotein translocation and reversion of the membrane inserted state of SecA by a carboxyl terminus binding mAb.
SecA is the peripheral subunit of the preprotein translocase of Escherichia coli. SecA consists of two independently folding domains, i.e., the N-domain bearing the high-affinity nucleotide binding site (NBS-I) and the C-domain that harbors the low-affinity NBS-II. ATP induces SecA insertion into the membrane during preprotein translocation. Domain-specific monoclonal antibodies (mAbs) were developed to analyze the functions of the SecA domains in preprotein translocation. The antigen binding sites of the obtained mAbs were confined to five epitopes. One of the mAbs, i.e., mAb 300-1K5, recognizes an epitope in the C-domain in a region that has been implicated in membrane insertion. This mAb, either as IgG or as Fab, completely inhibits in vitro proOmpA translocation and SecA translocation ATPase activity. It prevents SecA membrane insertion and, more strikingly, reverses membrane insertion and promotes the release of SecA from the membrane. Surface plasmon resonance measurements demonstrate that the mAb recognizes the ADP- and the AMP-PNP-bound state of SecA either free in solution or bound at the membrane at the SecYEG protein. It is concluded that the mAb actively reverses a conformation essential for membrane insertion of SecA. The other mAbs directed to various epitopes in the N-domain were found to be without effect, although all bind the native SecA. These results demonstrate that the C-domain plays an important role in the SecA membrane insertion, providing further evidence that this process is needed for preprotein translocation.
den Blaauwen
T
T
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands.
de Wit
J G
JG
Gosker
H
H
van der Does
C
C
Breukink
E J
EJ
de Leij
L
L
Driessen
A J
AJ
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors* / Q000276:immunology / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000378:metabolism / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000276:immunology / Q000378:metabolism; D001666:Binding Sites, Antibody; D001692:Biological Transport / Q000276:immunology; D015374:Biosensing Techniques; D018604:Epitope Mapping; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D008565:Membrane Proteins / Q000037:antagonists & inhibitors* / Q000276:immunology / Q000378:metabolism; D026901:Membrane Transport Proteins; D011487:Protein Conformation; D011498:Protein Precursors / Q000037:antagonists & inhibitors* / Q000276:immunology / Q000378:metabolism; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D012995:Solubility
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011498:Protein Precursors; D000069816:SEC Translocation Channels; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins