@article {546736, title = {Application of encoded library technology (ELT) to a protein-protein interaction target: discovery of a potent class of integrin lymphocyte function-associated antigen 1 (LFA-1) antagonists.}, journal = {Bioorg Med Chem. }, volume = {22}, number = {7}, year = {2014}, pages = {2353-65}, abstract = { The inhibition of\ protein-protein\ interactions remains a challenge for traditional small molecule drug\ discovery. Here we describe the use of DNA-encoded\ library\ technology\ for the\ discovery\ of small molecules that are\ potent\ inhibitors of the\ interaction\ betweenlymphocyte\ function-associated\ antigen\ 1\ and its ligand intercellular adhesion molecule\ 1. A DNA-encoded\ library\ with a potential complexity of 4.1\ billion compounds was exposed to the I-domain of the\ target\ protein and the bound ligands were affinity selected, yielding an enriched small-molecule hit family. Compounds representing this family were synthesized without their DNA encoding moiety and found to inhibit the\ lymphocyte\ function-associated\ antigen\ 1/intercellular adhesion molecule-1\ interaction\ with submicromolar potency in both ELISA and cell adhesion assays. Re-synthesized compounds conjugated to DNA or a fluorophore were demonstrated to bind to cells expressing the\ target\ protein. Copyright {\textcopyright} 2014 Elsevier Ltd. All rights reserved. }, author = {Kollman, CS and Bai, X and Tsai, CH and Yang, H and Lind, KE and Skinner, SR and Zhu, Z. and Israel, DI and Cuozzo, JW and Morgan, BA and Yuki, K and Xie, C. and Springer, T.A. and Shimaoka, M. and Evindar, G} } @article {546731, title = {Highly reinforced structure of a C-terminal dimerization domain in von Willebrand factor.}, journal = {Blood}, volume = {123}, number = {12}, year = {2014}, pages = {1785-93}, abstract = {The\ C-terminal\ cystine knot (CK) (CTCK)\ domain\ in\ von\ Willebrand\ factor\ (VWF) mediates\ dimerization\ of proVWF in the endoplasmic reticulum and is essential for long multimers required for hemostatic function. The CTCK dimer crystal\ structure\ revealshighly\ elongated monomers with 2 β-ribbons and 4 intra-chain disulfides, including 3 in the CK.\ Dimerization\ buries an extensive interface of 1500 {\r A}(2) corresponding to 32\% of the surface of each monomer and forms a super β-sheet and 3 inter-chain disulfides. The shape, dimensions, and N-terminal connections of the crystal\ structure\ agree perfectly with previous electron microscopic images of VWF dimeric bouquets with the CTCK dimer forming a down-curved base. The dimer interface is suited to resist hydrodynamic force and disulfide reduction. CKs in each monomer flank the 3 inter-chain disulfides, and their presence in β-structures with dense backbone hydrogen bonds creates a rigid,\ highly\ crosslinked interface. The\ structure\ reveals the basis for\ vonWillebrand\ disease phenotypes and the fold and disulfide linkages for CTCK domains in diverse protein families involved in barrier function, eye and inner ear development, insect coagulation and innate immunity, axon guidance, and signaling in extracellular matrices.}, author = {Zhou, Y.F. and Springer, T.A.} } @article {546746, title = {Metal ion and ligand binding of integrin α5β1.}, journal = {Proc Natl Acad Sci USA}, volume = {111}, number = {50}, year = {2014}, pages = {17863-8}, abstract = {Integrin\ α5β1 binds to an Arg-Gly-Asp (RGD) motif in its\ ligand\ fibronectin. We report high-resolution crystal structures of a four-domain α5β1 headpiece fragment, alone or with RGD peptides soaked into crystals, and RGD peptide affinity measurements. The headpiece crystallizes in a closed conformation essentially identical to that seen previously for α5β1 complexed with a Fab that allosterically inhibits\ ligand\ binding\ by stabilizing the closed conformation. Soaking experiments show that\ binding\ of cyclic RGD peptide with 20-fold higher affinity than a linear RGD peptide induces conformational change in the β1-subunit βI domain to a state that is intermediate between closed (low affinity) and open (high affinity). In contrast,\ binding\ of a linear RGD peptide induces no shape shifting. However, linear peptide\ binding\ induces shape shifting when Ca(2+) is depleted during soaking. Ca(2+) bound to the adjacent to\ metal\ ion-dependent adhesion site (ADMIDAS), at the locus of shape shifting, moves and decreases in occupancy, correlating with an increase in affinity for RGD measured when Ca(2+) is depleted. The results directly demonstrate that Ca(2+)binding\ to the ADMIDAS stabilizes integrins in the low-affinity, closed conformation. Comparisons in affinity between four-domain and six-domain headpiece constructs suggest that flexible\ integrin\ leg domains contribute to conformational equilibria. High-resolution views of the hybrid domain interface with the plexin-semaphorin-integrin\ (PSI) domain in different orientations show a ball-and-socket joint with a hybrid domain Arg side chain that rocks in a PSI domain socket lined with carbonyl oxygens.}, author = {Xia W and Springer TA} } @article {546721, title = {Structural basis of regulation of von Willebrand factor binding to glycoprotein Ib.}, journal = {J Biol Chem.}, volume = {289}, number = {9}, year = {2014}, pages = {5565-79}, abstract = {Activation by elongational flow of\ von Willebrand factor\ (VWF) is critical for primary hemostasis. Mutations causing type 2B\ vonWillebrand\ disease (VWD),\ platelet-type VWD (PT-VWD), and tensile force each increase affinity of the VWF A1 domain and\ plateletglycoprotein Ibα (GPIbα) for one another; however, the\ structural\ basis\ for these observations remains elusive. Directed evolution was used to discover a further gain-of-function mutation in A1 that shifts the long range disulfide bond by one residue. We solved multiple crystal structures of this mutant A1 and A1 containing two VWD mutations complexed with GPIbα containing two PT-VWD mutations. We observed a gained interaction between A1 and the central leucine-rich repeats (LRRs) of GPIbα, previously shown to be important at high shear stress, and verified its importance mutationally. These findings suggest that\ structural\ changes, including central GPIbα LRR-A1 contact, contribute to VWF affinity\ regulation. Among the mutant complexes, variation in contacts and poor complementarity between the GPIbα β-finger and the region of A1 harboring VWD mutations lead us to hypothesize that the structures are on a pathway to, but have not yet reached, a force-induced super high affinity state.}, author = {Blenner, M.A. and Dong, X. and Springer, T.A.} } @article {546751, title = {Structural determinants of integrin β-subunit specificity for latent TGF-β.}, journal = {Nat Struct Mol Biol.}, volume = {21}, number = {12}, year = {2014}, pages = {1091-6}, abstract = {Eight\ integrin\ α-β heterodimers recognize ligands with an Arg-Gly-Asp (RGD) motif. However, the\ structural\ mechanism by which integrins differentiate among extracellular proteins with RGD motifs is not understood. Here, crystal structures, mutations and peptide-affinity measurements show that αVβ6 binds with high affinity to a RGDLXXL/I motif within the prodomains of TGF-β1 and TGF-β3. The LXXL/I motif forms an amphipathic α-helix that binds in a hydrophobic pocket in the β6 subunit. Elucidation of the basis for ligand binding\ specificity\ by the\ integrin\ β subunit reveals contributions by three different βI-domain loops, which we designatespecificity-determining loops (SDLs) 1, 2 and 3. Variation in a pair of single key residues in SDL1 and SDL3 correlates with the variation of the entire β subunit in\ integrin\ evolution, thus suggesting a paradigmatic role in overall β-subunit function.}, author = {Dong, X. and Hudson, N.E. and Lu, C. and Springer, T.A.} } @article {546726, title = {Structures of the Toxoplasma gliding motility adhesin}, journal = {Proc Natl Acad Sci USA}, volume = {111}, number = {13}, year = {2014}, pages = {4862-7}, abstract = {Micronemal protein 2 (MIC2) is the key\ adhesin\ that supports\ gliding\ motility\ and host cell invasion by\ Toxoplasma\ gondii. With a von Willebrand factor A (VWA) domain and six thrombospondin repeat domains (TSR1-6) in its ectodomain, MIC2 connects to the parasite actomyosin system through its cytoplasmic tail. MIC2-associated protein (M2AP) binds noncovalently to the MIC2 ectodomain. MIC2 and M2AP are stored in micronemes as proforms. We find that the MIC2-M2AP ectodomain complex is a highly elongated 1:1 monomer with M2AP bound to the TSR6 domain. Crystal\ structures\ of N-terminal fragments containing the VWA and TSR1 domains for proMIC2 and MIC2 reveal a closed conformation of the VWA domain and how it associates with the TSR1 domain. A long, proline-rich, disulfide-bonded pigtail loop in TSR1 overlaps the VWA domain. Mannose α-C-linked to Trp-276 in TSR1 has an unusual (1)C4 chair conformation. The MIC2 VWA domain includes a mobile α5-helix and a 22-residue disordered region containing two disulfide bonds in place of an α6-helix. A hydrophobic residue in the prodomain binds to a pocket adjacent to the α7-helix that pistons in opening of the VWA domain to a putative high-affinity state.}, author = {Song, G. and Springer, TA.} } @article {546741, title = {von Willebrand Factor, Jedi Knight of the Bloodstream.}, journal = {Blood}, volume = {124}, number = {9}, year = {2014}, pages = {1412-25}, abstract = { When blood vessels are cut, the forces in the\ bloodstream\ increase and change character. The dark side of these forces causes hemorrhage and death. However,\ von\ Willebrand\ factor\ (VWF), with help from our circulatory system and platelets, harnesses the same forces to form a hemostatic plug. Force and VWF function are so closely intertwined that, like members of the\ Jedi\ Order in the movie Star Wars who learn to use "the Force" to do good, VWF may be considered the\ Jedi\ knight\ of the\ bloodstream. The long length of VWF enables responsiveness to flow. The shape of VWF is predicted to alter from irregularly coiled to extended thread-like in the transition from shear to elongational flow at sites of hemostasis and thrombosis. Elongational force propagated through the length of VWF in its thread-like shape exposes its monomers for multimeric binding to platelets and subendothelium and likely also increases affinity of the A1 domain for platelets. Specialized domains concatenate and compact VWF during biosynthesis. A2 domain unfolding by hydrodynamic force enables postsecretion regulation of VWF length. Mutations in VWF in\ von\ Willebranddisease contribute to and are illuminated by VWF biology. I attempt to integrate classic studies on the physiology of hemostatic plug formation into modern molecular understanding, and point out what remains to be learned. {\textcopyright} 2014 by The American Society of Hematology. }, author = {Springer, T.A.} }