Summary We show how integrin αVβ6 binds a macromolecular ligand, pro-TGF-β1, in an orientation biologically relevant for force-dependent release of TGF-β from latency. The conformation of the prodomain integrin-binding motif differs in presence and absence of integrin binding; differences extend well outside the interface and illustrate how integrins can remodel extracellular matrix. Remodeled residues outside the interface stabilize the integrin-bound conformation, adopt a conformation similar to earlier evolving family members, and show how macromolecular components outside the binding motif contribute to integrin recognition. Regions in and outside the highly interdigitated interface stabilize a specific integrin-pro-TGF-β orientation that defines the pathway through these macromolecules that actin cytoskeleton-generated tensile force takes when applied through the integrin β-subunit. Simulations of force-dependent activation of TGF-β demonstrate evolutionary specializations for force application through the TGF-β prodomain and through the β and not α-subunit of the integrin.

Circumsporozoite (CS) protein is the major surface component of Plasmodium falciparum sporozoites and is essential for host cell invasion. A vaccine containing tandem repeats, region III, and thrombospondin type-I repeat (TSR) of CS is efficacious in phase III trials but gives only a 35% reduction in severe malaria in the first year postimmunization. We solved crystal structures showing that region III and TSR fold into a single unit, an "αTSR" domain. The αTSR domain possesses a hydrophobic pocket and core, missing in TSR domains. CS binds heparin, but αTSR does not. Interestingly, polymorphic T-cell epitopes map to specialized αTSR regions. The N and C termini are unexpectedly close, providing clues for sporozoite sheath organization. Elucidation of a unique structure of a domain within CS enables rational design of next-generation subunit vaccines and functional and medicinal chemical investigation of the conserved hydrophobic pocket.

Sporozoite gliding motility and invasion of mosquito and vertebrate host cells in malaria is mediated by thrombospondin repeat anonymous protein (TRAP). Tandem von Willebrand factor A (VWA) and thrombospondin type I repeat (TSR) domains in TRAP connect through proline-rich stalk, transmembrane, and cytoplasmic domains to the parasite actin-dependent motility apparatus. We crystallized fragments containing the VWA and TSR domains from Plasmodium vivax and Plasmodium falciparum in different crystal lattices. TRAP VWA domains adopt closed and open conformations, and bind a Mg 2+ ion at a metal ion-dependent adhesion site implicated in ligand binding. Metal ion coordination in the open state is identical to that seen in the open high-affinity state of integrin I domains. The closed VWA conformation associates with a disordered TSR domain. In contrast, the open VWA conformation crystallizes with an extensible β ribbon and ordered TSR domain. The extensible β ribbon is composed of disulfide-bonded segments N-and C-terminal to the VWA domain that are largely drawn out of the closed VWA domain in a 15 Å movement to the open conformation. The extensible β ribbon and TSR domain overlap at a conserved interface. The VWA, extensible β ribbon, and TSR domains adopt a highly elongated overall orientation that would be stabilized by tensile force exerted across a ligand-receptor complex by the actin motility apparatus of the sporozoite. Our results provide insights into regulation of "stick-and-slip" parasite motility and for development of sporozoite subunit vaccines. M osquitoes transmit malaria to humans via sporozoites. Sporozoites are important targets of pre-erythrocytic malaria vaccines. However, we know little about the structure and arrangement of the two most important vaccine targets on sporozoite surfaces, the circumsporozoite (CS) protein (1-3) and thrombospondin repeat anonymous protein (TRAP) (4, 5). CS is a constitutive sporozoite surface protein and has a glycophosphatidylinositol anchor. TRAP mediates sporozoite gliding motility and cell invasion in both mosquito and vertebrate hosts (6).TRAP is mobilized from micronemes to the plasma membrane at the apical end of sporozoites, and is translocated to the posterior end during cell migration and invasion (7,8). TRAP spans the plasma membrane, and its cytoplasmic domain connects to the actin cytoskeleton through aldolase, permitting functional cooperation between extracellular adhesive domains and the intracellular actin/myosin motor (8-10). The TRAP ectodomain contains tandem von Willebrand factor A (VWA) and thrombospondin repeat (TSR) domains. A subset of VWA domains, including the inserted (I) domains in integrins, contain metal ion-dependent adhesion sites (MIDAS), with a Mg 2+ ion at the center of the ligand binding site (11). Conformational change transmitted from neighboring domains regulates affinity of I domains for ligand. The TRAP VWA domain contains the sequence signature of a MIDAS. Mutations of putative TRAP VWA domain MIDAS residues and deletion o...

The Vaccinia virus H1 gene product, VH1, is a dual specificity phosphatase that down-regulates the cellular antiviral response by dephosphorylating STAT1. The crystal structure of VH1, determined at 1.32 Å resolution, reveals a novel dimeric quaternary structure, which exposes two active sites spaced ϳ39 Å away from each other. VH1 forms a stable dimer via an extensive domain swap of the N-terminal helix (residues 1-20). In vitro, VH1 can dephosphorylate activated STAT1, in a reaction that is competed by the nuclear transport adapter importin ␣5. Interestingly, VH1 is inactive with respect to STAT1 bound to DNA, suggesting that the viral phosphatase acts predominantly on the cytoplasmic pool of activated STAT1. We propose that the dimeric quaternary structure of VH1 is essential for specific recognition of activated STAT1, which prevents its nuclear translocation, thus blocking interferon-␥ signal transduction and antiviral response. Dual specificity phosphates (DSPs)2 comprise a growing subclass of protein-tyrosine phosphatases, which dephosphorylate both phosphotyrosine and phosphoserine/threonine residues. The first identified DSP, VH1, is the product of the Vaccinia virus gene H1 (1). To date, the small VH1 (ϳ20 kDa) is the prototype of a family of VH1-like DSPs found in plants, yeast, insects, and higher eukaryotes (2). The human genome encodes at least 38 VH1-like phosphatases, which regulate many critical aspects of the cell cycle (3). VH1-like DSPs share a common catalytic mechanism, which is mediated by a catalytic triad consisting of a cysteine, an arginine, and an aspartic acid, usually present in the context of an extended consensus motif (4). The structural organization of the minimum catalytic core of VH1-like DSPs is known from the crystal structures of several members of the VH1-like family, such as VHZ (5) and VHR (6). All known DSPs share a common topology with members of the classical protein-tyrosine phosphatases, with the most marked structural difference being in the architecture of the active site. To accommodate both phosphotyrosine and phosphothreonine/serine residues, DSPs present a shallow catalytic cleft only ϳ6 Å deep. In contrast, the catalytic cysteine residue of classical protein-tyrosine phosphatases sits at the bottom of a ϳ9-Å-deep pocket, which selectively recognizes bulky phosphotyrosines (6). In vitro, VH1 and many other VH1-like DSPs are characterized by resistance to okadaic acid and sensitivity to sodium vanadate (1). Sodium vanadate acts as a potent inhibitor of cysteine-phosphatases by covalently labeling the cysteine group in the active site (4).The gene encoding VH1 is highly conserved among poxviruses and essential for the viability of Vaccinia virus in tissue cultures (7). VH1 is expressed in the late stage of viral infection, and ϳ200 molecules of VH1 are packaged within the virion (7). The conservation of the VH1 gene in poxviruses as well as its essential role for virus viability emphasize VH1 involvement in a critical step of the virus life cycle. Recent evidenc...

The gene product of Vaccinia virus gene H1, VH1, is the first identified dual specificity phosphatase (DSP). The human genome encodes 38 different VH1-like DSPs, which include major regulators of signaling pathways, highly dysregulated in disease states. VH1 down-regulates cellular antiviral response by dephosphorylating activated STAT1 in the IFN-␥/STAT1 signaling pathway. In this report, we have investigated the molecular basis for VH1 catalytic activity. Using small-angle x-ray scattering (SAXS), we determined that VH1 exists in solution as a boomerang-shaped dimer. Targeted alanine mutations in the dimerization domain (aa 1-27) decrease phosphatase activity while leaving the dimer intact. Deletion of the N-terminal dimer swapped helix (aa 1-20) completely abolishes dimerization and severely reduces phosphatase activity. An engineered chimera of VH1 that contains only one active site retains wild-type levels of catalytic activity. Thus, a dimeric quaternary structure, as opposed to two cooperative active sites within the same dimer is essential for VH1 catalytic activity. Together with laforin, VH1 is the second DSP reported in literature for which dimerization via an N-terminal dimerization domain is necessary for optimal catalytic activity. We propose that dimerization may represent a common mechanism to regulate the activity and substrate recognition of DSPs, often assumed to function as monomers. Dual-specificity phosphatases (DSPs)2 represent a subclass of the protein-tyrosine phosphatase (PTP) superfamily that can dephosphorylate both phosphotyrosine and phosphoserine/ threonine containing substrates (1-5). The first identified DSP, VH1, is encoded by the conserved H1 locus of Vaccinia virus (6). In the past 20 years the list of identified VH1-like DSPs has been greatly expanded and now includes 61 different members (5). Based on sequence similarity and presence of functional/ binding domains, DSPs are usually divided into 7 diverse subgroups (5).Like classical PTPs, VH1-like DSPs contain a catalytic triad consisting of a cysteine, an arginine, and an aspartic acid, usually arranged in the context of an extended consensus motif (7). DSPs employ a similar catalytic mechanism as PTPs, characterized by the formation of a transient enzyme-phosphosubstrate intermediate (1, 2). Similar to PTPs, the DSP catalytic core shows a great degree of substrate specificity. The substrate however, can be non-peptidic for a number of DSPs. For instance, PTEN-like phosphatases dephosphorylate D3-phosphorylated inositol phospholipids (8), or the DSPs PIR (also known as DUSP11) and laforin have been shown to dephosphorylate mRNA (9) and phosphoglucans (10), respectively.The gene encoding VH1 is highly conserved among doublestranded DNA viruses of the Poxviridae family (11). Approximately 200 molecules of VH1 are packaged within the Vaccinia virion and are essential for the viability of Vaccinia virus (12). In vivo, VH1 is required for maturation of two-virion membraneassociated factors, namely A17 (13) and A14 (14). Upon inf...

The underlying mechanisms contributing to injury-induced infection susceptibility remain poorly understood. Here, we describe a rapid increase in neutrophil cell numbers in the lungs following induction of thermal injury. These neutrophils expressed elevated levels of programmed death ligand 1 (PD-L1) and exhibited altered gene expression profiles indicative of a reparative population. Upon injury, neutrophils migrate from the bone marrow to the skin but transiently arrest in the lung vasculature. Arrested neutrophils interact with programmed cell death protein 1 (PD-1) on lung endothelial cells. A period of susceptibility to infection is linked to PD-L1+ neutrophil accumulation in the lung. Systemic treatment of injured animals with an anti–PD-L1 antibody prevented neutrophil accumulation in the lung and reduced susceptibility to infection but augmented skin healing, resulting in increased epidermal growth. This work provides evidence that injury promotes changes to neutrophils that are important for wound healing but contribute to infection susceptibility.

In αI integrins, including leukocyte function-associated antigen 1 (LFA-1), ligand-binding function is delegated to the αI domain, requiring extra steps in the relay of signals that activate ligand binding and coordinate it with cytoplasmic signals. Crystal structures reveal great variation in orientation between the αI domain and the remainder of the integrin head. Here, we investigated the mechanisms involved in signal relay to the αI domain, including whether binding of the ligand intercellular adhesion molecule-1 (ICAM-1) to the αI domain is linked to headpiece opening and engenders a preferred αI domain orientation. Using small-angle X-ray scattering and negative-stain EM, we define structures of ICAM-1, LFA-1, and their complex, and the effect of activation by Mn Headpiece opening was substantially stabilized by substitution of Mg with Mn and became complete upon ICAM-1 addition. These agents stabilized αI-headpiece orientation, resulting in a well-defined orientation of ICAM-1 such that its tandem Ig-like domains pointed in the opposite direction from the β-subunit leg of LFA-1. Mutations in the integrin βI domain α1/α1' helix stabilizing either the open or the closed βI-domain conformation indicated that α1/α1' helix movements are linked to ICAM-1 binding by the αI domain and to the extended-open conformation of the ectodomain. The LFA-1-ICAM-1 orientation described here with ICAM-1 pointing anti-parallel to the LFA-1 β-subunit leg is the same orientation that would be stabilized by tensile force transmitted between the ligand and the actin cytoskeleton and is consistent with the cytoskeletal force model of integrin activation.

Background:Laforin is an essential glycogen phosphatase often mutated in Lafora disease. Results: Human laforin adopts a dimeric quaternary structure. Conclusion: Dimerization generates a unique active site crevice essential to recognize and dephosphorylate glycogen. Significance: We derived a complete structural model of human laforin using hybrid structural methods.