Immunochemical localization of a region of chaperonin-60 important for productive interaction with chaperonin-10.
An IgG1 monoclonal antibody (mAb 54G8) which binds to both Bordetella pertussis chaperonin-60 (cpn60) and Escherichia coli cpn60 (GroEL) was produced. mAb 54G8 as well as Fab fragments prepared from this antibody were found to abolish the ability of chaperonin-10 (cpn10, GroES) to inhibit the ATPase activity of both B. pertussis cpn60 and E. coli cpn60. Electron microscopy was used to localize the binding site of the monoclonal antibody on the B. pertussis cpn60 molecule. In the absence of the antibody, the B. pertussis molecule exhibited the tetradecameric structure typical of cpn60. Both end views (showing 7-fold symmetry of the face of the molecule) and side views were evident. When mAb 54G8 was bound, B. pertussis cpn60 molecules appeared to be cross-linked so that they formed long chains. Only side views of the molecules were seen in these long chains. When B. pertussis cpn60 complexed with Fab fragments of mAb 54G8 was examined, chains were no longer observed. Instead, side views of B. pertussis cpn60 were often seen with Fab fragments extending from the ends of the molecule. These data indicate that mAb 54G8 appears to bind at or near the end of the B. pertussis cpn60 molecule and that binding of mAb 54G8 at this location affects the ability of cpn10 to productively interact with cpn60, most likely either by sterically blocking the binding of cpn10, by affecting the conformation of cpn60 in such a way that it no longer binds cpn10, or by inhibiting proper transduction of the effects of cpn10 binding.
Burns
D L
DL
Division of Bacterial Products, Food and Drug Administration, Bethesda, Maryland 20892.
Kessel
M
M
Arciniega
J L
JL
Karpas
A
A
Gould-Kostka
J
J
D000251:Adenosine Triphosphatases / Q000032:analysis / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D001886:Bordetella pertussis / Q000378:metabolism*; D018835:Chaperonin 10; D018834:Chaperonin 60; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D007140:Immunoglobulin Fab Fragments; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D016253:Microscopy, Immunoelectron
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D018835:Chaperonin 10; D018834:Chaperonin 60; D006360:Heat-Shock Proteins; D007140:Immunoglobulin Fab Fragments; D000251:Adenosine Triphosphatases