Characterization of the calcium-binding sites of Listeria monocytogenes InlB.
The Listeria monocytogenes protein InlB promotes invasion of mammalian cells through activation of the receptor tyrosine kinase Met. The InlB N-cap, a approximately 40 residue part of the domain that binds Met, was previously observed to bind two calcium ions in a novel and unusually exposed manner. Because subsequent work raised questions about the existence of these calcium-binding sites, we assayed calcium binding in solution to the InlB N-cap. We show that calcium ions are bound with dissociation constants in the low micromolar range at the two identified sites, and that the sites interact with one another. We demonstrate that the calcium ions are not required for structure, and also find that they have no appreciable effect on Met activation or intracellular invasion. Therefore, our results indicate that the sites are fortuitous in InlB, but also suggest that the simple architecture of the sites may be adaptable for protein engineering purposes.
Marino
Michael
M
Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0375, USA.
Banerjee
Manidipa
M
Copp
Jeremy
J
Dramsi
Shaynoor
S
Chapman
Tara
T
van der Geer
Peter
P
Cossart
Pascale
P
Ghosh
Partho
P
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008089:Listeria monocytogenes; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D020449:Repetitive Sequences, Amino Acid
D001426:Bacterial Proteins; D002135:Calcium-Binding Proteins; D008565:Membrane Proteins; C095768:inlB protein, Listeria monocytogenes; D002118:Calcium