Protein Complex Production from the Drug Discovery Standpoint.

Small molecule drug discovery critically depends on the availability of meaningful in vitro assays to guide medicinal chemistry programs that are aimed at optimizing drug potency and selectivity. As it becomes increasingly evident, most disease relevant drug targets do not act as a single protein. In the body, they are instead generally found in complex with protein cofactors that are highly relevant for their correct function and regulation.

From autoinhibition to inhibition in trans: the Raf-1 regulatory domain inhibits Rok-alpha kinase activity.

The activity of Raf-1 and Rok-alpha kinases is regulated by intramolecular binding of the regulatory region to the kinase domain. Autoinhibition is relieved upon binding to the small guanosine triphosphatases Ras and Rho. Downstream of Ras, Raf-1 promotes migration and tumorigenesis by antagonizing Rok-alpha, but the underlying mechanism is unknown.

Structural basis for SH3 domain-mediated high-affinity binding between Mona/Gads and SLP-76.

SH3 domains are protein recognition modules within many adaptors and enzymes. With more than 500 SH3 domains in the human genome, binding selectivity is a key issue in understanding the molecular basis of SH3 domain interactions. The Grb2-like adaptor protein Mona/Gads associates stably with the T-cell receptor signal transducer SLP-76.

Determinants of the in vivo folding of the prion protein. A bipartite function of helix 1 in folding and aggregation.

Misfolding of the mammalian prion protein (PrP) is implicated in the pathogenesis of prion diseases. We analyzed wild type PrP in comparison with different PrP mutants and identified determinants of the in vivo folding pathway of PrP. The complete N terminus of PrP including the putative transmembrane domain and the first beta-strand could be deleted without interfering with PrP maturation.

Dual-specific Src and Abl kinase inhibitors, PP1 and CGP76030, inhibit growth and survival of cells expressing imatinib mesylate-resistant Bcr-Abl kinases.

The leukemogenic tyrosine kinase Bcr-Abl contains a highly conserved inhibitor-binding pocket (IBP), which serves as a binding site for imatinib mesylate. Mutations at the IBP may lead to resistance of the Abl kinase against imatinib mesylate. To examine the mechanisms of imatinib mesylate binding and resistance in more detail, we created several point mutations at amino acid positions 315 and 380 of Abl, blocking the access to the IBP and rendering Bcr-Abl imatinib mesylate-resistant.

The protein import motor of mitochondria: a targeted molecular ratchet driving unfolding and translocation.

Unfolding and import of preproteins into mitochondria are facilitated by a molecular motor in which heat shock protein 70 (Hsp70) in the matrix plays an essential role. Here we present two different experimental approaches to analyze mechanisms underlying this function of Hsp70. First, preproteins containing stretches of glutamic acid (polyE) or glycine (polyG) repeats in front of folded domains were imported into mitochondria.

Prediction of novel Bag-1 homologs based on structure/function analysis identifies Snl1p as an Hsp70 co-chaperone in Saccharomyces cerevisiae.

Polypeptide binding by the chaperone Hsp70 is regulated by its ATPase activity, which is itself regulated by co-chaperones including the Bag domain nucleotide exchange factors. Here, we tested the functional contribution of residues in the Bag domain of Bag-1M that contact Hsp70. Two point mutations, E212A and E219A, partially reduced co-chaperone activity, whereas the point mutation R237A completely abolished activity in vitro.

Ligand discrimination by TPR domains. Relevance and selectivity of EEVD-recognition in Hsp70 x Hop x Hsp90 complexes.

Protein-protein interaction modules containing so-called tetratricopeptide repeats (TPRs) mediate the assembly of Hsp70/Hsp90 multi-chaperone complexes. The TPR1 and TPR2A domains of the Hsp70/Hsp90 adapter protein p60/Hop specifically bind to short peptides corresponding to the C-terminal tails of Hsp70 and Hsp90, respectively, both of which contain the highly conserved sequence motif EEVD-COOH. Here, we quantitatively assessed the contribution of TPR-mediated peptide recognition to Hsp70.