Cofilin plays a key role in the choreography of actin dynamics via its ability to sever actin filaments and increase the rate of monomer dissociation from pointed ends. The exact manner by which phosphoinositides bind to cofilin and inhibit its interaction with actin has proven difficult to ascertain. We determined the structure of chick cofilin and used NMR chemical shift mapping and structure-directed mutagenesis to unambiguously locate its recognition site for phosphoinositides (PIs). This structurally unique recognition site requires both the acyl chain and head group of the PI for a productive interaction, and it is not inhibited by phosphorylation of cofilin. We propose that the interaction of cofilin with membrane-bound PIs abrogates its binding to both actin and actin-interacting protein 1, and facilitates spatiotemporal regulation of cofilin activity.

Ticks transmit multiple pathogens to different hosts without compromising their health. Their ability to evade microbial infections is largely a result of their effective innate immune response including various antimicrobial peptides. Therefore, a deep understanding of how ticks (and other arthropod vectors) control microbial loads could lead to the design of broad-spectrum antimicrobial agents. In this paper we study the role of the aminoterminal copper and nickel (ATCUN)-binding sequence in the peptide ixosin, isolated from the salivary glands of the hard tick Ixodes sinensis. Our results indicate that the ATCUN motif is not essential to the potency of ixosin, but is indispensable to its oxidative mechanism of action. Specifically, the ATCUN motif promotes dioxygen-and copper-dependent lipid (per)oxidation of bacterial membranes in a temporal fashion coinciding with the onset of bacterial death. Microscopy and studies on model membranes indicate that the oxidized phospholipids are utilized as potential targets of ixosin B (another tick salivary gland peptide) involving its delocalization to the bacterial membrane, thus resulting in a synergistic effect. Our proposed mechanism of action highlights the centrality of the ATCUN motif to ixosin's mechanism of action and demonstrates a novel way in which (tick) antimicrobial peptides (AMPs) utilize metal ions in its activity. This study suggests that ticks employ a variety of effectors to generate an amplified immune response, possibly justifying its vector competence.

Adaptor protein Shc plays a key role in mitogen-activated protein kinase (MAPK) signaling pathway, which can be mediated through a number of different receptors including integrins. By specifically recognizing the tyrosine-phosphorylated integrin ␤ 3 , Shc has been shown to trigger integrin outside-in signaling, although the structural basis of this interaction remains nebulous. Here we present the detailed structural analysis of Shc phosphotyrosine-binding (PTB) domain in complex with the bi-phosphorylated ␤ 3 integrin cytoplasmic tail (CT). We show that this complex is primarily defined by the phosphorylation state of the integrin C-terminal Tyr 759 , which fits neatly into the classical PTB pocket of Shc. In addition, we have identified a novel binding interface which concurrently accommodates phosphorylated Tyr 747 of the highly conserved NPXY motif of ␤ 3 . The structure represents the first snapshot of an integrin cytoplasmic tail bound to a target for mediating the outside-in signaling. Detailed comparison with the known Shc PTB structure bound to a target TrkA peptide revealed some significant differences, which shed new light upon the PTB domain specificity.Integrins, a major class of non-covalent heterodimeric, glycoprotein cell surface receptors, are among the most studied and best characterized cell adhesion molecules. Integrins mediate a plethora of cell-cell, cell-extracellular matrix (ECM), 2 and cell-pathogen interactions and hence are responsible for controlling a wide array of biological processes including homeostasis, cell migration, differentiation, adhesion, immune response etc. The unique bidirectional flow of information through integrins involves inside-out signals, which allow them to interact with extracellular ligands (such as fibrinogen, von Willebrand factor, fibronectin) and ligand-dependent outside-in signals which adjust the cellular response to cell-cell adhesion (1). Although our understanding of the molecular details of inside-out integrin signaling (2, 3) has grown by leaps and bounds over the past decade, the early intracellular events following the integrin-mediated ECM engagement, outside-in signal transduction, still require further clarification. With respect to the outside-in signaling, the important unanswered questions center on selective recognition of proximal effectors by integrin cytoplasmic domains at different stages of cell spreading. Phosphorylation of the integrin tails is considered to be one of the spatiotemporal mechanisms for imparting such selectivity and, indeed, phosphorylation switches are thought to be a common principle of integrin regulation. Platelet integrin ␤ 3 cytoplasmic tail (CT) is laden with various phosphorylation sites, including two tyrosines, one serine, and multiple threonines. However, only tyrosine phosphorylation is found to be specific for the outside-in signaling (4 -8) and Shc (in particular its p52 isoform) was identified as a primary signaling partner for the tyrosine-phosphorylated ␤ 3 CT (8).Adaptor protein Shc (Src homology ...

Onconase (rONC), otherwise known as ranpirnase or P-30 protein, which was initially purified from extracts of Rana pipiens oocytes and early embryos, exhibits anticancer activity both in vitro and in vivo and is in phase III clinical trials for tumor therapy. We have determined the solution NMR structure of a recombinant onconase with Met ؊1 , Gln 1 , and Leu 23 residues (M؊1, Q1, M23L)rONC. The 20 best solution structures had a backbone root mean square deviation of 0.41 ؎ 0.09 Å with respect to the average structure. The energy-minimized average NMR structure had a backbone root mean square deviation of 0.72 Å from the x-ray crystallographic structure of native onconase; however, the orientation of the N-terminal residue in the two structures was very different. Comparison of the 15 N HSQC spectrum of (M؊1, Q1, M23L)rONC with that of a mutant E1S-rONC, which is identical to the nONC except with the N-terminal pyroglutamyl residue replaced by Ser, showed that N-terminal and residue 23 mutations induced structural changes in regions beyond the mutation sites. Model-free analysis of the backbone amide 15 N-T 1 , 15 N-T 2 , and 15 N-1 H NOE relaxation data for (M؊1, Q1, M23L)rONC and E1S-rONC revealed that the E1S-rONC molecule showed very little flexibility, whereas (M؊1, Q1, M23L)rONC exhibited substantial flexibility, which may account for the previously observed reduced stability and increased protease susceptibility. The ␣ 1 helix and ␤-sheets of (M؊1, Q1, M23L)rONC displayed bending motions. These data provided strong evidence for the presence of an N-terminal hydrogen bond network in E1S-rONC, but not in (M؊1, Q1, M23L)rONC.

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