title: Engineered variants of InlB with an additional leucine-rich repeat discriminate between physiologically relevant and packing contacts in crystal structures of the InlB:MET complex.
abstract: The physiological relevance of contacts in crystal lattices often remains elusive. This was also the case for the complex between the invasion protein internalin B (InlB) from Listeria monocytogenes and its host cell receptor, the human receptor tyrosine kinase (RTK) MET. InlB is a MET agonist and induces bacterial host cell invasion. Activation of RTKs generally involves ligand-induced dimerization of the receptor ectodomain. The two currently available crystal structures of the InlB:MET complex show the same arrangement of InlB and MET in a 1:1 complex, but different dimeric 2:2 assemblies. Only one of these 2:2 assemblies is predicted to be stable by a computational procedure. This assembly is mainly stabilized by a contact between the Cap domain of InlB from one and the Sema domain of MET from another 1:1 complex. Here, we probe the physiological relevance of this interaction. We generated variants of the leucine-rich repeat (LRR) protein InlB by inserting an additional repeat between the first and the second LRR. This should allow formation of the 1:1 complex but disrupt the potential 2:2 complex involving the Cap-Sema contact due to steric distortions. A crystal structure of one of the engineered proteins showed that it folded properly. Binding affinity to MET was comparable to that of wild-type InlB. The InlB variant induced MET phosphorylation and cell scatter like wild-type InlB. These results suggest that the Cap-Sema interaction is not physiologically relevant and support the previously proposed assembly, in which a 2:2 InlB:MET complex is built around a ligand dimer.
authors.affiliation: Department of Chemistry, Bielefeld University, 33501 Bielefeld, Germany. Hartmut.Niemann@uni-bielefeld.de
authors.forename: Hartmut H
authors.initials: HH
authors.lastname: Niemann
authors.forename: Ermanno
authors.initials: E
authors.lastname: Gherardi
authors.forename: Willem M
authors.initials: WM
authors.lastname: Bleymüller
authors.forename: Dirk W
authors.initials: DW
authors.lastname: Heinz
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003461:Crystallography; D006801:Humans; D007930:Leucine / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D015202:Protein Engineering; D019859:Proto-Oncogene Proteins c-met / Q000737:chemistry* / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; C095768:inlB protein, Listeria monocytogenes; C491743:MET protein, human; D019859:Proto-Oncogene Proteins c-met; D007930:Leucine