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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Despite advances in resolution accompanying the development of high-field superconducting magnets, biomolecular applications of NMR require multiple dimensions in order to resolve individual resonances, and the achievable resolution is typically limited by practical constraints on measuring time. In addition to the need for measuring long evolution times to obtain high resolution, the need to distinguish the sign of the frequency constrains the ability to shorten measuring times. Sign discrimination is typically accomplished by sampling the signal with two different receiver phases or by selecting a reference frequency outside the range of frequencies spanned by the signal and then sampling at a higher rate. In the parametrically sampled (indirect) time dimensions of multidimensional NMR experiments, either method imposes an additional factor of 2 sampling burden for each dimension. We demonstrate that by using a single detector phase at each time sample point, but randomly altering the phase for different points, the sign ambiguity that attends fixed single-phase detection is resolved. Random phase detection enables a reduction in experiment time by a factor of 2 for each indirect dimension, amounting to a factor of 8 for a four-dimensional experiment, albeit at the cost of introducing sampling artifacts. Alternatively, for fixed measuring time, random phase detection can be used to double resolution in each indirect dimension. Random phase detection is complementary to nonuniform sampling methods, and their combination offers the potential for additional benefits. In addition to applications in biomolecular NMR, random phase detection could be useful in magnetic resonance imaging and other signal processing contexts.compressed sensing | MRI

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Natural nanomechanisms such as capillaries, neurotransmitters, and ion channels play a vital role in the living systems. But the design principles developed by nature through evolution are not well understood and, hence, not applicable to engineered nanomachines. Thus, the design of nanoscale mechanisms with prescribed functions remains a challenge. Here, we present a systematic approach based on established kinematics techniques to designing, analyzing, and controlling manufacturable nanomachines with prescribed mobility and function built from a finite but extendable number of available "molecular primitives." Our framework allows the systematic exploration of the design space of irreducibly simple nanomachines, built with prescribed motion specification by combining available nanocomponents into systems having constrained, and consequently controllable motions. We show that the proposed framework has allowed us to discover and verify a molecule in the form of a seven link, seven revolute (7R) close loop spatial linkage with mobility (degree of freedom) of one. Furthermore, our experiments exhibit the type and range of motion predicted by our simulations. Enhancing such a structure into functional nanomechanisms by exploiting and controlling their motions individually or as part of an ensemble could galvanize development of the multitude of engineering, scientific, medical, and consumer applications that can benefit from engineered nanomachines.