Structural basis for phosphoserine-proline recognition by group IV WW domains.

Pin1 contains an N-terminal WW domain and a C-terminal peptidyl-prolyl cis-trans isomerase (PPIase) domain connected by a flexible linker. To address the energetic and structural basis for WW domain recognition of phosphoserine (P.Ser)/phosphothreonine (P. Thr)- proline containing proteins, we report the energetic and structural analysis of a Pin1-phosphopeptide complex. The X-ray crystal structure of Pin1 bound to a doubly phosphorylated peptide (Tyr-P.Ser-Pro-Thr-P.Ser-Pro-Ser) representing a heptad repeat of the RNA polymerase II large subunit's C-terminal domain (CTD), reveals the residues involved in the recognition of a single P.Ser side chain, the rings of two prolines, and the backbone of the CTD peptide. The side chains of neighboring Arg and Ser residues along with a backbone amide contribute to recognition of P.Ser. The lack of widespread conservation of the Arg and Ser residues responsible for P.Ser recognition in the WW domain family suggests that only a subset of WW domains can bind P.Ser-Pro in a similar fashion to that of Pin1.

Structure of the human anti-apoptotic protein survivin reveals a dimeric arrangement.

Survivin is a 16.5 kDa protein that is expressed during the G2/M phase of the cell cycle and is hypothesized to inhibit a default apoptotic cascade initiated in mitosis. This inhibitory function is coupled to survivin's localization to the mitotic spindle. To begin to address the structural basis of survivin's function, we report the X-ray crystal structure of a recombinant form of full length survivin to 2.58 A resolution. Survivin consists of two defined domains including an N-terminal Zn2+-binding BIR domain linked to a 65 A amphipathic C-terminal alpha-helix. The crystal structure reveals an extensive dimerization interface along a hydrophobic surface on the BIR domain of each survivin monomer. A basic patch acting as a sulfate/phosphate-binding module, an acidic cluster projecting off the BIR domain, and a solvent-accessible hydrophobic surface residing on the C-terminal amphipathic helix, are suggestive of functional protein-protein interaction surfaces.

The C-terminal domain of the Cdc2 inhibitory kinase Myt1 interacts with Cdc2 complexes and is required for inhibition of G(2)/M progression.

Activation of Cdc2, is the universal event controlling the onset of mitosis. In higher eukaryotes, Cdc2 activity is in part regulated by inhibitory phosphorylation of Thr14 and Tyr15, catalyzed by Wee1 and Myt1, which prevents catastrophic premature entry into mitosis. In this study we defined the function of Myt1 by overexpression studies in both S. pombe and a human osteosarcoma cell line. Similar to Wee1, overexpression of human Myt1 prevented entry into mitosis in both cell types; however, Myt1 catalytic activity was not essential for the cell cycle delay observed with human cells. Myt1 expression was restricted to proliferating cells. Furthermore, we detected no major decline in Myt1 protein abundance prior to the entry into mitosis, which coincides with the loss of Myt1 activity. We localized mitotic phosphoepitopes, recognized by the monoclonal antibody MPM-2, to the C-terminal domain of Myt1. The mitotic peptidyl-prolyl isomerase, Pin1, was able to associate with this domain in a phosphorylation-dependent manner. Truncation of the C-terminal domain of Myt1 prevented its ability to induce G(2)/M phase arrest in overexpression studies in human cells and dramatically reduced its ability to phosphorylate Cdc2 in vitro. We demonstrate that the C-terminal domain of Myt1 was required for recruitment of Cdc2, and we infer that this domain lies in the cytoplasm because it can interact with and is phosphorylated by Cdc2. In conclusion, we propose that Myt1 can negatively regulate Cdc2/cyclin B1 and inhibit G(2)/M progression by two means, both of which require the C-terminal domain; first, Myt1 can bind and sequester Cdc2/cyclin B1 in the cytoplasm preventing entry into the nucleus, and, second, it can phosphorylate associated Cdc2/cyclin B1 at Thr14 and Tyr15 thus inhibiting its catalytic activity.