Role of structural and dynamical plasticity in Sin3: the free PAH2 domain is a folded module in mSin3B

The co-repressor Sin3 is the essential scaffold protein of the Sin3/HDAC co-repressor complex, which is recruited to the DNA by a diverse group of transcriptional repressors, targeting genes involved in the regulation of the cell cycle, proliferation and differentiation. Sin3 contains four repeats commonly denoted as paired amphipathic helix (PAH1-4) domains that provide the principal interaction surface for various repressors. Here, we present the first structure of the free state of the PAH2 domain and discuss its implications for interaction with the repressors. The unbound conformation is very similar to the conformation observed when bound to either the Mad1 or HBP1 repressor, suggesting that the PAH2 domain serves as a template that guides proper folding of the unstructured repressor. The free PAH2 domain shows micro- to millisecond conformational exchange between the folded, major state and a partially unfolded, minor state. Upon complex formation, we observe a significant decrease in fast time-scale flexibility of local regions of the protein, correlated with the formation of intermolecular contacts, and an overall decrease in the slow time-scale conformational exchange. On the basis of our data and using a multiple sequence alignment of all PAH domains, we suggest that the PAH1, PAH2 and PAH3 domains form pre-folded binding modules in full-length Sin3 like beads-on-a-string, and act as folding templates for the interaction domains of their targets

Nmr-Studies of the Major Coat Protein of Bacteriophage-M13 - Structural Information of Gviiip in Dodecylphosphocholine Micelles

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Kinetic folding mechanism of PDZ2 from PTP-BL

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Structure, dynamics and binding characteristics of the second PDZ domain of PTP-BL.

Contains fulltext : 187660.pdf (publisher's version ) (Closed access)The PDZ domains of the protein tyrosine phosphatase PTP-BL mediate interactions by binding to specific amino acid sequences in target proteins. The solution structure of the second PDZ domain of PTP-BL, PDZ2, displays a compact fold with six beta strands and two alpha-helices. A unique feature of this domain compared to the canonical PDZ fold is an extended flexible loop at the base of the binding pocket, termed L1, that folds back onto the protein backbone, a feature that is shared by both the murine and human orthologues. The structure of PDZ2 differs significantly from the orthologous human structure. A comparison of structural quality indicators clearly demonstrates that the PDZ2 ensemble is statistically more reasonable than that of the human orthologue. The analysis of (15)N relaxation data for PDZ2 shows a normal pattern, with more rigid secondary structures and more flexible loop structures. Close to the binding pocket, Leu85 and Thr88 display greater mobility when compared to surrounding residues. Peptide binding studies demonstrated a lack of interaction between murine PDZ2 and the C terminus of the murine Fas/CD95 receptor, suggesting that the Fas/CD95 receptor is not an in vivo target for PDZ2. In addition, PDZ2 specifically binds the C termini of both human Fas/CD95 receptor and the RIL protein, despite RIL containing a non-canonical PDZ-interacting sequence of E-x-V. A model of PDZ2 with the RIL peptide reveals that the PDZ2 binding pocket is able to accommodate the bulkier side-chain of glutamic acid while maintaining crucial protein to peptide hydrogen bond interactions

Sequence-specific assignment of the PAH2 domain of Sin3B free and bound to Mad1.

Contains fulltext : 79476.pdf (publisher's version ) (Closed access

The interaction of PTP-BL PDZ domains with RIL: an enigmatic role for the RIL LIM domain.

Contains fulltext : 59363.pdf (publisher's version ) (Closed access)PDZ domains are protein-protein interaction modules that are crucial for the assembly of structural and signaling complexes. PDZ domains specifically bind short carboxyl-terminal peptides and occasionally internal sequences that structurally resemble peptide termini. Previously, using yeast two-hybrid methodology, we studied the interaction of two PDZ domains present in the large submembranous protein tyrosine phosphatase PTP-BL with' the C-terminal half of the LIM domain-containing protein RIL. Deletion of the extreme RIL C-terminus did not eliminate binding, suggesting the presence of a PDZ binding site within the RIL LIM moiety. We have now performed experiments in mammalian cell lysates and found that the RIL C-terminus proper, but not the RIL LIM domain, can interact with PTP-BL, albeit very weakly. However, this interaction with PTP-BL PDZ domains is greatly enhanced when the combined RIL LIM domain and C-terminus is used, pointing to synergistic effects. NMR titration experiments and site-directed mutagenesis indicate that this result is not dependent on specific interactions that require surface exposed residues on the RIL LIM domain, suggesting a stabilizing role in the association with PTP-BL