Projects
Proline-rich sequences
Proline-rich sequences are amongst the most frequent motifs in eukaryotic cells and play a pivotal role in many signaling pathway. They mediate the assembly of molecu-lar complexes by interacting with versatile recognition domains contained in various intracellular proteins. The currently identified signaling domains that recognize proline-rich sequences are the SH3 domain, the WW domain, the EVH1 domain, pro-filin and the GYF domain. The human T cell adhesion molecule CD2 contains proline- rich stretches in its cytoplasmatic tail. These motifs (e.g. SHRPPPPGHRV) interact with the GYF domain of the CD2 binding protein 2 (CD2BP2). In collaboration with the Protein Engineering Group (Dr. C. Freund), the structure of the GYF- SHRPPPPGHRV complex (1L2Z) could be resolved by NMR. Based on the result that Fyn-knockout mice are severely impaired in regard to CD2 stimulated signaling it has been suggested that the Fyn-SH3 domain interacts with the CD2 cytoplasmic domain. Modeling the peptide conformation on the basis of the crystal structures of 1fyn and 1abl and using the NMR chemical shift mapping data we docked the CD2 ligand SHRPPPPGHRV to the Fyn-SH3 domain. It was found that the prolines 5 and 6 of the peptide adopt the second proline binding pocket of the Fyn- SH3 domain. The complex is stabilized by an salt bridge between arginine 3 of the ligand and D100 of the Fyn- SH3 domain allowing arginine 3 to insert deeply into the Fyn- SH3 binding pocket. This supports the critical rule of the RT- loop residues in Fyn- SH3- CD2 interactions. In contrast, the CD2- ligand is unable to bind to the lck-SH3 domain, which is also involved in T cell signaling. We found that the SH3 typical orientation of the prolines yields a sterical clash of R3 with the lck-SH3- binding domain.
In cooperation with L. Balls Group we were interested to develop compounds recognized by the EVH1 domain and replacing the prolines of the SFEFPPPPTEDEL motif with peptoidic building blocks. On the basis of the observed chemical shift perturba-tions the interaction of the peptomer replacing Proline5 with the EVH1 domain was modeled. We found that depending from the size of the linker the phenyl ring of the peptomer can adopt the binding pocket of Phe4. As a consequence, a new peptomer replacing Phe4 to Ala was synthesized. This compound without the highly conserved FP- motif was able to bind to the EVH1 domain. New ligands of this type may open a way for the selective modulation of the EVH1 domain interaction with their binding partners.
BAG-domains are protein interaction domains involved in apoptosis signaling. Since most of them are proven to interact with HSP70 this project was aimed to explore differences in binding of SODD-BAG and a previously solved BAG-domain from hBag1 to this protein. The structure of the SODD BAG-domain was determined by solution NMR methods. Although it resembles the overall fold of hBag1 there are differences in length and charge distribution, defining a new subfamily of BAG-domains. Nevertheless we could build a homology model of the SODD-BAG / HSP70 complex based the hBag1 / HSP70 X-ray structure to show that these differences do not affect the HSP70 binding abilities of SODD-BAG. Furthermore we were able to suggest additional electrostatic interactions between SODD / HSP70 compared to the hBag1-complex. Interestingly the residues involved in these interactions are conserved throughout all short SODD BAG-domains in addition to those of the consensus binding motif. We therefore expect subfamily-specific differences of BAG-domains to HSP70.
