PML and several other proteins localizing in PML-nuclear bodies (PML-NB) contain phosphoSIMs (SUMO-interacting motifs), and phosphorylation of this motif plays a key role in their interaction with SUMO family proteins. We examined the role that phosphorylation plays in the binding of the phosphoSIMs of PML and Daxx to SUMO1 at the atomic level. The crystal structures of SUMO1 bound to unphosphorylated and tetraphosphorylated PML-SIM peptides indicate that three phosphoserines directly contact specific positively charged residues of SUMO1. Surprisingly, the crystal structure of SUMO1 bound to a diphosphorylated Daxx-SIM peptide indicate that the hydrophobic residues of the phosphoSIM bind in a manner similar to that seen with PML, but important differences are observed when comparing the phosphorylated residues. Together, the results provide an atomic level description of how specific acetylation patterns within different SUMO family proteins can work together with phosphorylation of phosphoSIM's regions of target proteins to regulate binding specificity.

The T cell antigen receptor encounters foreign antigen during the immune response. Receptor engagement leads to activation of specific protein tyrosine kinases, which then phosphorylate multiple enzymes and adapter proteins. One such enzyme, phospholipase-Cγ1, is responsible for cleavage of a plasma membrane lipid substrate, a phosphoinositide, into two second messengers, diacylglycerol, which activates several enzymes including protein kinase C, and an inositol phosphate, which induces intracellular calcium elevation. In T cells, phospholipase-Cγ1 is recruited to the plasma membrane as part of a four-protein complex containing three adapter molecules. We have used recombinant proteins and synthetic phosphopeptides to reconstitute this quaternary complex in vitro. Extending biophysical tools to study concurrent interactions of the four protein components, we demonstrated the formation and determined the composition of the quaternary complex using multisignal analytical ultracentrifugation, and we characterized the thermodynamic driving forces of assembly by isothermal calorimetry. We demonstrate that the four proteins reversibly associate in a circular arrangement of binding interfaces, each protein interacting with two others. Three interactions are of high affinity, and the fourth is of low affinity, with the assembly of the quaternary complex exhibiting significant enthalpy-entropy compensation as in an entropic switch. Formation of this protein complex enables subsequent recruitment of additional molecules needed to activate phospholipase-Cγ1. Understanding the formation of this complex is fundamental to full characterization of a central pathway in T cell activation. Such knowledge is critical to developing ways in which this pathway can be selectively inhibited.phospholipase C | protein complex | signal transduction | T cell antigen receptor | tyrosine phosphorylation T hree decades of investigation have resulted in a description of T cell antigen receptor (TCR) engagement and the biochemical events that follow. The binding of the TCR ligand, an antigenic peptide presented by a protein encoded by the major histocompatibility complex, leads to the activation of associated and recruited protein tyrosine kinases (PTKs) (1). Critical substrates of these PTKs include the adapter molecules, linker for activation of T cells (LAT), and Src homology 2 (SH2) domaincontaining leukocyte protein of 76kDa (SLP-76) (2, 3). The phosphorylation of tyrosine residues on these adapters initiates binding of many other adapters and enzymes to generate molecular complexes of great heterogeneity (4). In this manner enzymes such as phospholipase C-γ1 (PLC-γ1) and son of sevenless homolog 1 (SOS1) are recruited to the plasma membrane where they encounter their substrates. The products that are a consequence of these enzyme activations-diacylglycerol, elevated intracellular calcium, and activated Ras-have complex intracellular effects during the process of T cell activation (5, 6).Sites of protein-protein interactions between these v...

Binding affinity and specificity are crucial factors that influence nanostructure control by biomineralization peptides. In this paper, we analysed the role that the oligomeric state of a silver biomineralization peptide plays in regulating the morphology of silver nanostructure formation. Oligomerization was achieved by conjugating the silver specific TBP biomineralization peptide to the p53 tetramerization domain peptide (p53Tet). Interestingly, the TBP–p53Tet tetrameric peptide acted as a growth catalyst, controlling silver crystal growth, which resulted in the formation of hexagonal silver nanoplates without consuming the peptide. The TBP–p53Tet peptide caps the surface of the silver crystals, which enhances crystal growth on specific faces and thereby regulates silver nanostructure formation in a catalytic fashion. The present findings not only provide an efficient strategy for controlling silver nanostructure formation by biomineralization peptides, but they also demonstrate that in this case the oligomeric peptides play a unique catalytic role.

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