The receptor tyrosine kinase MET is essential for embryonic development of vertebrates and the healing of skin wounds in adults. In addition MET signaling is subverted during infection by the facultative intracellular pathogen Listeria monocytogenes, which induces its own uptake into non-phagocytic cells upon MET activation. The surface associated protein InlB functions as bacterial MET agonist by specifically binding to the MET extracellular domain. Structurally, InlB does not resemble the endogenous MET ligand hepatocyte growth factor/scatter factor (HGF/SF). However, the cellular response to MET stimulation by InlB is very similar to that elicited by HGF/SF.
We try to understand the mechanism of ligand-induced MET activation at the molecular level. To this end we solved crystal structures of the complex formed by InlB and the MET ectodomain. (Niemann et al. 2007; top figure). The structure revealed that the binding site for InlB does not overlap with that previously described for the β-chain of HGF/SF. MET dimerization is brought about by a small, low-affinity contact formed by the back-sides of two InlB molecules in the 2:2 complex. (Ferraris et al. 2010). As discussed in a recent review, this is reminiscent of MET activation by the HGF/SF splice variant NK1 (Niemann 2013). In cooperation with Prof. Heilemann (Frankfurt) we now use single-molecule fluorescence microscopy to study InlB mediated MET dimerization on cells. (Dietz et al. 2013).
InlB is a multi-domain protein. MET only interacts with the N-terminal internalin domain of InlB, but the other InlB domains contribute to full activation of the MET receptor. We investigate the function of the central domain of InlB called B-repeat. We solved its crystal structure revealing an ubiquitin-like β-grasp fold (central figure) and we showed that the B-repeat is required for InlB to induce cell motility. (Ebbes et al. 2011). The InlB B-repeat probably binds to a host cell receptor other than MET. Currently, we try to identify this receptor.
MET is a target structure of medical importance due to its role in tissue regeneration. MET agonists could serve as protein therapeutics, e.g. to stimulate wound healing. The structure of the 2:2 InlB/MET signaling active complex, allowed the rational design of a potent MET agonist. (Ferraris et al. 2010; Kolditz et al. 2013; bottom figure).
Resembling a macromolecular syringe, type III secretion systems are multi-protein complexes used by various Gram-negative pathogens to inject effector proteins directly into the host cell cytoplasm. Hydrophobic so-called translocator proteins form the necessary pore in the host cell membrane. Within in the bacterial cytosol, these translocators bind to a chaperone, which itself is not exported. In contrast to classic chaperones, T3S chaperones do not bind or hydrolyze nucleotides.
We solved the first crystal structure of a T3S translocator chaperone, the Yersinia enterocolitica protein SycD. ( Büttner et al. 2008). SycD forms a curved structure consisting of three tetratrico peptide repeats (TPRs; top figure). A second structure of SycD in complex with a chaperone-binding peptide derived from the translocator protein YopD shows that the concave face of the curved TPR fold serves as binding site for YopD. ( Schreiner et al. 2012; bottom figure).
In addition to the translocators YopB and YopD, SycD binds to several other T3S proteins enabling it to exert regulatory functions. Currently, we investigate the interaction of SycD with several Y. enterocolitica T3S proteins in vitro. in vitro.
The gastric pathogen H. pylori employs a type IV secretion system (T4SS) to inject the oncogenic effector protein CagA into the stomach epithelium of its human host. The T4SS protein CagL localizes to the T4SS pilus and is essential for injection of CagA into host cells. CagL harbors a RGD motif that probably mediates binding to host cell integrins.
We solved the crystal structure of H. pylori CagL. (Barden et al. 2013). An elongated three-helix bundle forms the structural core of CagL, to which the N-terminal helix is associated only loosely. Structure comparisons suggest that CagL is a H. pylori specific protein. The RGD motif is exposed at the protein surface and thus accessible for interaction with a receptor molecule. In contrast to previously characterized RGD motifs, the RGD motif of CagL is not located in an extended or flexible loop structure but in the middle of a long helix.
We now investigate the molecular basis of the interaction between CagL and host cell receptors.