Characterization of the translocation-competent complex between the Helicobacter pylori oncogenic protein CagA and the accessory protein CagF.
CagA is a virulence factor that Helicobacter pylori inject into gastric epithelial cells through a type IV secretion system where it can cause gastric adenocarcinoma. Translocation is dependent on the presence of secretion signals found in both the N- and C-terminal domains of CagA and an interaction with the accessory protein CagF. However, the molecular basis of this essential protein-protein interaction is not fully understood. Herein we report, using isothermal titration calorimetry, that CagA forms a 1:1 complex with a monomer of CagF with nM affinity. Peptide arrays and isothermal titration calorimetry both show that CagF binds to all five domains of CagA, each with μM affinity. More specifically, a coiled coil domain and a C-terminal helix within CagF contacts domains II-III and domain IV of CagA, respectively. In vivo complementation assays of H. pylori with a double mutant, L36A/I39A, in the coiled coil region of CagF showed a severe weakening of the CagA-CagF interaction to such an extent that it was nearly undetectable. However, it had no apparent effect on CagA translocation. Deletion of the C-terminal helix of CagF also weakened the interaction with CagA but likewise had no effect on translocation. These results indicate that the CagA-CagF interface is distributed broadly across the molecular surfaces of these two proteins to provide maximal protection of the highly labile effector protein CagA.
From the Institute of Human Virology and.
Daniel A
DA
Bonsor
Evelyn
E
Weiss
Anat
A
Iosub-Amir
Tali H
TH
Reingewertz
Tiffany W
TW
Chen
Rainer
R
Haas
Assaf
A
Friedler
Wolfgang
W
Fischer
Eric J
EJ
Sundberg
D000230:Adenocarcinoma / Q000378:metabolism / Q000382:microbiology; D019943:Amino Acid Substitution; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D058947:Bacterial Secretion Systems / Q000502:physiology; D016480:Helicobacter pylori / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D015513:Oncogene Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013274:Stomach Neoplasms / Q000378:metabolism / Q000382:microbiology
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D058947:Bacterial Secretion Systems; D046912:Multiprotein Complexes; D015513:Oncogene Proteins; C080866:cagA protein, Helicobacter pylori; C507457:cagF protein, Helicobacter pylori
CagA; CagF; Cancer; Helicobacter pylori; Protein Secretion; Protein Translocation; Protein-Protein Interactions; Type IV Secretion System