The ternary MalP-GSG4-P complex shows the phosphate group poised to attack the glycosidic bond and promote phosphorolysis. In all three complexes the pentasaccharide exhibits an altered conformation across sub-sites -1 and ⍣1, the site of catalysis, from the preferred conformation for α(1-4)-linked glucosyl polymers.

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Tensin is a cytoskeletal protein that links integrins to the actin cytoskeleton at sites of cell-matrix adhesion. Here we describe the crystal structure of the phosphotyrosine-binding (PTB) domain of tensin1, and show that it binds integrins in an NPxY-dependent fashion. Alanine mutagenesis of both the PTB domain and integrin tails supports a model of integrin binding similar to that of the PTB-like domain of talin. However, we also show that phosphorylation of the NPxY tyrosine, which disrupts talin binding, has a negligible effect on tensin binding. This suggests that tyrosine phosphorylation of integrin, which occurs during the maturation of focal adhesions, could act as a switch to promote the migration of tensin-integrin complexes into fibronectin-mediated fibrillar adhesions.Keywords: structure/function studies; crystallography; calorimetry; mutagenesis (site-directed and general); docking proteins; surface plasmon resonance Supplemental material: see www.proteinscience.org Cells bind to the extracellular matrix (ECM) by means of the integrin family of plasma membrane receptors (Hynes 2002). Integrins make direct bonds to ECM proteins and indirect bonds to the actin cytoskeleton via a number of cytoskeletal proteins, including tensin, talin, vinculin, and paxillin (Zamir et al. 1999). These complexes provide a scaffold for signaling enzymes that regulate many aspects of cellular behavior. Geiger (Zaidel-Bar et al. 2003) has described three stages in the development of cell-ECM contacts: ''focal contacts'' are short-lived structures containing integrin aVb3, which transform into classical ''focal adhesions'' upon activation of RhoA, and contain both integrins aVb3 and a5b1; a third type of contact, ''fibrillar adhesions,'' is formed when integrin a5b1 (the fibronectin receptor) translocates from focal adhesions (Pankov et al. 2000).The cytoskeletal protein tensin is absent from focal contacts but is recruited to focal adhesions and plays a key role in the establishment of fibrillar adhesions (Zaidel-Bar et al. 2003). Tensin contains binding sites for actin in the N terminus that crosslink actin filaments, and a central domain that caps the barbed ends of F-actin (Lo et al. 1994;Chuang et al. 1995). The C-terminal region contains a Src homology 2 (SH2) domain that mediates binding to tyrosine-phosphorylated proteins, such as phosphoinositide 3-kinase, Cas, and focal adhesion kinase (Auger et al. 1996), and a PTB domain that is required for focal adhesion targeting (Chen and Lo 2003 Abbreviations: PTB, phosphotyrosine-binding (domain); ECM, extracellular matrix; SH2, Src homology 2; PIPKIg, phosphatidylinositol phosphate kinase type Ig ; EDC, N-ethyl-N9-(3-dimethylaminopropyl)carbodiimide hydrochloride; NHS, N-hydroxysuccinimide; RMSD, root mean square deviation; SPR, surface plasmon resonance; DSC, differential scanning calorimetry.Article published online ahead of print. Article and publication date are at

TRPV channels are important polymodal integrators of noxious stimuli mediating thermosensation and nociception. An ankyrin repeat domain (ARD), which is a common protein-protein recognition domain, is conserved in the N-terminal intracellular domain of all TRPV channels and predicted to contain three to four ankyrin repeats. Here we report the first structure from the TRPV channel subfamily, a 1.7 Å resolution crystal structure of the human TRPV2 ARD. Our crystal structure reveals a six ankyrin repeat stack with multiple insertions in each repeat generating several unique features compared with a canonical ARD. The surface typically used for ligand recognition, the ankyrin groove, contains extended loops with an exposed hydrophobic patch and a prominent kink resulting from a large rotational shift of the last two repeats. The TRPV2 ARD provides the first structural insight into a domain that coordinates nociceptive sensory transduction and is likely to be a prototype for other TRPV channel ARDs.Keywords: TRPV channel; ankyrin repeat; crystal structure Supplemental material: see www.proteinscience.org Transient receptor potential (TRP) channels are biological sensors essential for processing numerous environmental stimuli (Clapham 2003). The TRPV subfamily is comprised of nonselective cation channels important for nociception, osmosensation, mechanosensation, maintenance of calcium homeostasis, and temperature sensing, with TRPV1 through 4 each exhibiting a distinct thermal activation threshold (Patapoutian et al. 2003). TRPV channels can be polymodally activated as demonstrated by TRPV1, which is activated by heat ($43°C), acidification, vanilloid compounds such as capsaicin, the active ingredient of hot chili peppers, and several proinflammatory mediators (Caterina and Julius 2001). TRPV channels are also emerging as multifunctional receptors as shown by TRPV2, which in addition to responding to noxious thermal stimuli ($52°C) (Caterina et al. 1999), neuropeptides (Boels et al. 2001, and growth factors (Kanzaki et al. 1999), is proposed to play a role in proinflammatory degranulation events in mast cells (Stokes et al. 2004) and mechanosensation in vascular smooth muscle (Muraki et al. 2003).Although TRPV channels mediate a range of physiological and sensory responses, they share a common structural architecture. Their general topology consists of an intracellular N-and C-terminal domain and six transmembrane helices (TM1 to TM6) with TM5, TM6, and the connecting loop forming the cation-conducting pore (Voets et al. 2004). The channels assemble as tetramers (Kedei et al. 2001) and in some cases form heteromeric channels (Hoenderop et al. 2003).The intracellular N-terminal domain of TRPV channels contains multiple ankyrin (ANK) repeats. This is a common motif utilized in protein-protein recognition, mediating a diverse range of functions, including ion transport, cell-cell Little is known about the function of ANK repeats in TRPV channels, although a role in cell surface trafficking has been proposed. An...

The alpha-I domain, found in the alpha-subunit of the leucocyte integrins such as alphaMbeta2 and alphaLbeta2, switches between the open and closed tertiary conformations, reflecting the high- and low-affinity ligand-binding states of the integrin that are required for regulated cell adhesion and migration. In the present study we show, by using point mutations and engineered disulphide bonds, that ligand affinity can be reduced or increased allosterically by altering the equilibrium between the closed and open states. We determined equilibrium constants for the binding of two ligands, fibrinogen and intercellular cell-adhesion molecule 1, to the alphaM-I domain by surface plasmon resonance, and determined crystal structures of a low-affinity mutant. Locking the domain in the open conformation increases affinity by a factor of no greater than 10, consistent with a closely balanced equilibrium between the two conformations in the absence of ligand. This behaviour contrasts with that of the unliganded alphaL-I domain, for which the equilibrium lies strongly in favour of the closed conformation. These results suggest significant differences in the way the parent integrins regulate I domain conformation and hence ligand affinity.