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Structural and functional characterization of the PNT and SAM domains : diverse roles in protein-protein interactions Mackereth, Cameron David


The PNT (or Pointed) domain, and highly related SAM (sterile alpha motif) domain, are a conserved region of 70 to 85 amino acids that is found in a diverse range of eukaryotic proteins including the Ets family of transcription factors and components of the yeast pheromone-response pathway. These domains, which can be expressed and purified in isolation, are structurally characterized by a core bundle of four a-helices. Additional helices in specific variants highlight plasticity in the architecture of these domains. The PNT domain structure from the Ets protein Erg, determined by using nuclear magnetic resonance (NMR) spectroscopy, reveals that only the four core helices are present; in contrast, the Ets proteins Ets-1 and GABPα have PNT domains that display an intimately associated N-terminal helix. The generalized role of the PNT domain as a self-association module has been investigated. Biophysical characterization of various PNT domains reveals a diverse range of oligomeric states, with those from Ets members Ets-1, Ets-2, Pnt-P2, GABPα, Erg and Fli-1 being monomeric in solution, whereas Tel and Yan are polymeric. In contrast, Ste4 and Byr2 of the Schizosaccharomyces pombe mating pathway form an unexpected 3:1 complex dependent on the interaction of their respective SAM domains coupled with trimerization of Ste4 via an adjacent coiled-coil region. For the Ets-1 and GABPα PNT domains, an alternative role in providing a mitogen activated protein (MAP) kinase docking site has been investigated. These docking sites increase the specificity of MAP kinase-mediated phosphorylation of phosphoacceptor sites N- and C-terminal to the PNT domains of Ets-1 and GABPα, respectively. The residues flanking the PNT domain in the Ets family transcription factors have additional importance with respect to post-translational modification and protein association. Limited proteolysis and NMR-based relaxation studies confirm that the peptide regions adjacent to the PNT domains of Ets-1, GABPα and Erg are conformationally disordered in solution. In addition, phosphorylation of threonine 38 in Ets-1 or threonine 280 in GABPα does not significantly change the dynamic nature of these regions, nor does this modification perturb the structure of the adjoining PNT domains. For Ets-1, a consensus sumoylation site has also been identified on lysine 15 within its disordered N-terminus. Using chemical shift perturbation mapping, the binding interface on both Ets-1 and the small ubiquitin-like modifier (SUMO)-conjugating enzyme 9 (UBC9) were identified. This latter surface region coincides with that site of binding by previously characterized sumoylation targets. No direct interaction between Ets-1 and SUMO was detected, highlighting the role of UBC9 in mediating the chemical linkage of these two proteins. The biological consequence of this modification is still unclear, although sumoylation of the Ets family member Tel has been reported to differentially target the protein to sub-nuclear assemblies. However, in vitro analyses with Tel indicate that a previously proposed SUMO-acceptor lysine within its PNT domain does not appear to interact with UBC9. Rather, an N-terminal site, corresponding to the conserved sequence in Ets-1, may reflect a more prominent sumoylation site for Tel.

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