Cloning of the (Thr6)-phyllokinin precursor from Phyllomedusa sauvagei skin confirms a non-consensus tyrosine 0-sulfation motif.

Nine bradykinin-related peptides were identified in Phyllomedusa sauvagei skin secretion using QTOF MS/MS fragmentation sequencing. The major peptides were (Thr6)-bradykinin, (Hyp3, Thr6)-bradykinin, (Thr6)-phyllokinin and (Hyp3, Thr6)-phyllokinin. The phyllokinins occurred in both sulfated and non-sulfated forms. All (Thr6)-substituted bradykinins/phyllokinins could be generated from a common precursor by differential post-translational processing and modification. The open-reading frame of the cloned precursor cDNA consisted of 62 amino acid residues with a single bradykinin/phyllokinin coding sequence located at the C-terminus. Structural features included a Glu-Arg processing site at the N-terminus of the bradykinin/phyllokinin domain and the absence of an acidic amino acid residue adjacent to the C-terminal Tyr residue in the phyllokinins. However, the neutral amino acid residue (Ile) at position -1 and the basic amino acid residue (Arg) at position -2 from the Tyr residue, constitute a sulfation motif previously identified only in a protochordean.

IQ motif selectivity in human IQGAP1: binding of myosin essential light chain and S100B.

IQGAPs are cytoskeletal scaffolding proteins which link signalling pathways to the reorganisation of actin and microtubules. Human IQGAP1 has four IQ motifs each of which binds to calmodulin. The same region has been implicated in binding to two calmodulin-like proteins, the myosin essential light chain Mlc1sa and the calcium and zinc ion binding protein S100B. Using synthetic peptides corresponding to the four IQ motifs of human IQGAP1, we showed by native gel electrophoresis that only the first IQ motif interacts with Mlc1sa. This IQ motif, and also the fourth, interacts with the budding yeast myosin essential light chain Mlc1p. The first and second IQ motifs interact with S100B in the presence of calcium ions. This clearly establishes that S100B can interact with its targets through IQ motifs in addition to interacting via previously reported sequences. These results are discussed in terms of the function of IQGAP1 and IQ motif recognition.

The cyclic “silver-diphos” motif [Ag2(-diphosphine)2]2+ as a synthon for building up larger structure structures

The dinuclear, cyclic structural motif [Ag-2(diphosphine)(2)](2+), here termed the

DNA-dependent Protein Kinase-mediated Phosphorylation of Protein Kinase B Requires a Specific Recognition Sequence in the C-terminal Hydrophobic Motif

DNA-dependent protein kinase (DNA-PK) has been implicated in a variety of nuclear processes including DNA double strand break repair, V(D)J recombination, and transcription. A recent study showed that DNA-PK is responsible for Ser-473 phosphorylation in the hydrophobic motif of protein kinase B (PKB/Akt) in genotoxic-stressed cells, suggesting a novel role for DNA-PK in cell signaling. Here, we report that DNA-PK activity toward PKB peptides is impaired in DNA-PK knock-out mouse embryonic fibroblast cells when compared with wild type. In addition, human glioblastoma cells expressing a mutant form of DNA-PK (M059J) displayed a lower DNA-PK activity when compared with glioblastoma cells expressing wild-type DNA- PK (M059K) when PKB peptide substrates were tested. DNA- PK preferentially phosphorylated PKB on Ser-473 when compared with its known in vitro substrate, p53. A consensus hydrophobic amino acid surrounding the Ser-473 phospho-acceptor site in PKB containing amino acids Phe at position +1 and +4 and Tyr at position -1 are critical for DNA- PK activity. Thus, these data define the specificity of DNA- PK action as a Ser-473 kinase for PKB in DNA repair signaling.

Bis-guanylhydrazone diimidazo[1,2-a:1,2-c]pyrimidine as a novel and specific G-quadruplex binding motif

A bis-guanylhydrazone derivative of diimidazo[1,2-a:1,2-c]pyrimidine has unexpectedly been found to be a potent stabiliser of several quadruplex DNAs, whereas there is no significant interaction with duplex DNA. Molecular modeling suggests that the guanylhydrazone groups play an active role in quadruplex binding.

Structural basis for the platelet-collagen interaction - The smallest motif within collagen that recognizes and activates platelet glycoprotein VI contains two glycine-proline-hydroxyproline triplets

Collagen-related peptide is a selective agonist for the platelet collagen receptor Glycoprotein VI. The triple helical peptide contains ten GPO triplets/strand (single letter amino acid nomenclature, where O is hydroxyproline) and so over-represents GPO compared with native collagen sequence. To investigate the ability of Glycoprotein VI to recognize GPO triplets in a setting more representative of the collagens, we synthesized a set of triple helical peptides containing fewer GPO triplets, varying their number and spacing within an inert (GPP)(n) backbone. The adhesion of recombinant human Glycoprotein VI ectodomain, like that of human platelets, to these peptides increased with their GPO content, and platelet adhesion was abolished by the specific anti-Glycoprotein VI-blocking antibody, 10B12. Platelet aggregation and protein tyrosine phosphorylation were induced only by cross-linked peptides and only those that contained two or more GPO triplets. Such peptides were less potent than cross-linked collagen-related peptide. Our data suggest that both the sequences GPOGPO and GPO center dot center dot center dot center dot center dot center dot center dot center dot center dot GPO represent functional Glycoprotein VI recognition motifs within collagen. Furthermore, we propose that the (GPO)(4) motif can support simultaneous binding of two glycoprotein VI molecules, in either a parallel or anti-parallel stacking arrangement, which could play an important role in activation of signaling.

IQ-motif selectivity in human IQGAP2 and IQGAP3: binding of calmodulin and myosin essential light chain

The IQGAP [IQ-motif-containing GAP (GTPase-activating protein)] family members are eukaryotic proteins that act at the interface between cellular signalling and the cytoskeleton. As such they collect numerous inputs from a variety of signalling pathways. A key binding partner is the calcium-sensing protein CaM (calmodulin). This protein binds mainly through a series of IQ-motifs which are located towards the middle of the primary sequence of the IQGAPs. In some IQGAPs, these motifs also provide binding sites for CaM-like proteins such as myosin essential light chain and S100B. Using synthetic peptides and native gel electrophoresis, the binding properties of the IQ-motifs from human IQGAP2 and IQGAP3 have been mapped. The second and third IQ-motifs in IQGAP2 and all four of the IQ-motifs of IQGAP3 interacted with CaM in the presence of calcium ions. However, there were differences in the type of interaction: while some IQ-motifs were able to form complexes with CaM which were stable under the conditions of the experiment, others formed more transient interactions. The first IQ-motifs from IQGAP2 and IQGAP3 formed transient interactions with CaM in the absence of calcium and the first motif from IQGAP3 formed a transient interaction with the myosin essential light chain MIc1sa. None of these IQ-motifs interacted with S100B. Molecular modelling suggested that all of the IQ-motifs, except the first one from IQGAP2 formed alpha-helices in solution. These results extend our knowledge of the selectivity of IQ-motifs for CaM and related proteins.