Targeting SxIP-EB1 interaction: An integrated approach to the discovery of small molecule modulators of dynamic binding sites

End binding protein 1 (EB1) is a key element in the complex network of protein-protein interactions at microtubule (MT) growing ends, which has a fundamental role in MT polymerisation. EB1 is an important protein target as it is involved in regulating MT dynamic behaviour, and has been associated with several disease states, such as cancer and neuronal diseases. Diverse EB1 binding partners are recognised through a conserved four amino acid motif, (serine-X-isoleucine-proline) which exists within an intrinsically disordered region. Here we report the use of a multidisciplinary computational and experimental approach for the discovery of the first small molecule scaffold which targets the EB1 recruiting domain. This approach includes virtual screening (structure- and ligand-based design) and multiparameter compound selection. Subsequent studies on the selected compounds enabled the elucidation of the NMR structures of the C-terminal domain of EB1 in the free form and complexed with a small molecule. These structures show that the binding site is not preformed in solution, and ligand binding is fundamental for the binding site formation. This work is a successful demonstration of the combination of modelling and experimental methods to enable the discovery of compounds which bind to these challenging systems

LD Motif Recognition by Talin: Structure of the Talin-DLC1 Complex

Cell migration requires coordination between integrin-mediated cell adhesion to the extracellular matrix and force applied to adhesion sites. Talin plays a key role in coupling integrin receptors to the actomyosin contractile machinery, while deleted in liver cancer 1 (DLC1) is a Rho GAP that binds talin and regulates Rho, and therefore actomyosin contractility. We show that the LD motif of DLC1 forms a helix that binds to the four-helix bundle of the talin R8 domain in a canonical triple-helix arrangement. We demonstrate that the same R8 surface interacts with the paxillin LD1 and LD2 motifs. We identify key charged residues that stabilize the R8 interactions with LD motifs and demonstrate their importance in vitro and in cells. Our results suggest a network of competitive interactions in adhesion complexes that involve LD motifs, and identify mutations that can be used to analyze the biological roles of specific protein-protein interactions in cell migration

A Further Unique Chondroitin Sulfate from the shrimp Litopenaeus vannamei with Antithrombin Activity that Modulates Acute Inflammation

The detailed structure of a further Chondroitin Sulfate from Litopenaeus vannamei shrimp (sCS) is described. The backbone structure was established by 1H/13C NMR, which identified 3-O-sulfated GlcA, 4-O-sulfated GalNAc, 6-O-sulfated GalNAc, and 4,6-di-O-sulfated GalNAc residues. GlcA is linked to GalNAc 4,6 di S and GlcA 3S is linked to GalNAc 4S, GalNAc 4,6 di-S and GalNAc6S residues. The anticoagulant properties of this sCS were evaluated by activated partial thromboplastin time, anti-IIa, anti-Xa and anti-heparin cofactor II-mediated activities, and sCS failed to stabilise antithrombin in a fluoresence shift assay. The anti-inflammatory effect of sCS was explored using a model of acute peritonitis, followed by leukocyte count and measurement of the cytokines, IL-1β, IL-6 and TNF-α. The compound showed low clotting effects, but high anti-IIa activity and HCII-mediated thrombin inhibition. Its anti-inflammatory effect was shown by leukocyte recruitment inhibition and a decrease in pro-inflammatory cytokine levels. Although the biological role of sCS remains unknown, its properties indicate that it is suitable for studies of multi-potent molecules obtained from natural sources

Structure of a double ubiquitin-like domain in the talin head: a role in integrin activation

Talin is a 270-kDa protein that activates integrins and couples them to cytoskeletal actin. Talin contains an N-terminal FERM domain comprised of F1, F2 and F3 domains, but it is atypical in that F1 contains a large insert and is preceded by an extra domain F0. Although F3 contains the binding site for β-integrin tails, F0 and F1 are also required for activation of β1-integrins. Here, we report the solution structures of F0, F1 and of the F0F1 double domain. Both F0 and F1 have ubiquitin-like folds joined in a novel fixed orientation by an extensive charged interface. The F1 insert forms a loop with helical propensity, and basic residues predicted to reside on one surface of the helix are required for binding to acidic phospholipids and for talin-mediated activation of β1-integrins. This and the fact that basic residues on F2 and F3 are also essential for integrin activation suggest that extensive interactions between the talin FERM domain and acidic membrane phospholipids are required to orientate the FERM domain such that it can activate integrins

A computational analysis of the dynamic roles of talin, Dok1, and PIPKI for integrin activation

Integrin signaling regulates cell migration and plays a pivotal role in developmental processes and cancer metastasis. Integrin signaling has been studied extensively and much data is available on pathway components and interactions. Yet the data is fragmented and an integrated model is missing. We use a rule-based modeling approach to integrate available data and test biological hypotheses regarding the role of talin, Dok1 and PIPKI in integrin activation. The detailed biochemical characterization of integrin signaling provides us with measured values for most of the kinetics parameters. However, measurements are not fully accurate and the cellular concentrations of signaling proteins are largely unknown and expected to vary substantially across different cellular conditions. By sampling model behaviors over the physiologically realistic parameter range we find that the model exhibits only two different qualitative behaviours and these depend mainly on the relative protein concentrations, which offers a powerful point of control to the cell. Our study highlights the necessity to characterize model behavior not for a single parameter optimum, but to identify parameter sets that characterize different signaling modes

Structural and biophysical properties of the integrin-associated cytoskeletal protein talin

Talin is a large cytoskeletal protein (2541 amino acid residues) which plays a key role in integrin-mediated events that are crucial for cell adhesion, migration, proliferation and survival. This review summarises recent work on the structure of talin and on some of the structurally better defined interactions with other proteins. The N-terminal talin head (approx. 50 kDa) consists of an atypical FERM domain linked to a long flexible rod (approx. 220 kDa) made up of a series of amphipathic helical bundle domains. The F3 FERM subdomain in the head binds the cytoplasmic tail of integrins, but this interaction can be inhibited by an interaction of F3 with a helical bundle in the talin rod, the so-called “autoinhibited form” of the molecule. The talin rod contains a second integrin-binding site, at least two actin-binding sites and a large number of binding sites for vinculin, which is important in reinforcing the initial integrin–actin link mediated by talin. The vinculin binding sites are defined by hydrophobic residues buried within helical bundles, and these must unfold to allow vinculin binding. Recent experiments suggest that this unfolding may be mediated by mechanical force exerted on the talin molecule by actomyosin contraction

Quantitative Evaluation of Cross-Peak Volumes in Multidimensional Spectra by Nonlinear-Least-Squares Curve Fitting

A procedure for quantitative evaluation of cross-peak volumes in spectra of any order of dimensions is described; this is based on a generalized algorithm for combining appropriate one-dimensional integrals obtained by nonlinear-least-squares curve-fitting techniques. This procedure is embodied in a program, NDVOL, which has three modes of operation: a fully automatic mode, a manual mode for interactive selection of fitting parameters, and a fast reintegration mode. The procedures used in the NDVOL program to obtain accurate volumes for overlapping cross peaks are illustrated using various simulated overlapping cross-peak patterns. The precision and accuracy of the estimates of cross-peak volumes obtained by application of the program to these simulated cross peaks and to a back-calculated 2D NOESY spectrum of dihydrofolate reductase are presented. Examples are shown of the use of the program with real 2D and 3D data. It is shown that the program is able to provide excellent estimates of volume even for seriously overlapping cross peaks with minimal intervention by the user. © 1995 Academic Press. All rights reserved.link_to_subscribed_fulltex

Pressure-Dependent Chemical Shifts in the R3 Domain of Talin Show that It Is Thermodynamically Poised for Binding to Either Vinculin or RIAM

Talin mediates attachment of the cell to the extracellular matrix. It is targeted by the Rap1 effector RIAM to focal adhesion sites and subsequently undergoes force-induced conformational opening to recruit the actin-interacting protein vinculin. The conformational switch involves the talin R3 domain, which binds RIAM when closed and vinculin when open. Here, we apply pressure to R3 and measure ¹H, ¹⁵N, and ¹³C chemical shift changes, which are fitted using a simple model, and indicate that R3 is only 50% closed: the closed form is a four-helix bundle, while in the open state helix 1 is twisted out. Strikingly, a mutant of R3 that binds RIAM with an affinity similar to wild-type but more weakly to vinculin is shown to be 0.84 kJ mol‾¹ more stable when closed. These results demonstrate that R3 is thermodynamically poised to bind either RIAM or vinculin, and thus constitutes a good mechanosensitive switch