Integrin activation takes shape.

Integrins are cell surface adhesion receptors that are essential for the development and function of multicellular animals. Here we summarize recent findings on the regulation of integrin affinity for ligand (activation), one mechanism by which cells modulate integrin function. The focus is on the structural basis of integrin activation, the role of the cytoplasmic domain in integrin affinity regulation, and potential mechanisms by which activation signals are propagated from integrin cytoplasmic domains to the extracellular ligand-binding domain.

Dimer formation drives the activation of the cell death protease caspase 9.

A critical step in the induction of apoptosis is the activation of the apoptotic initiator caspase 9. We show that at its normal physiological concentration, caspase 9 is primarily an inactive monomer (zymogen), and that activity is associated with a dimeric species. At the high concentrations used for crystal formation, caspase 9 is dimeric, and the structure reveals two very different active-site conformations within each dimer. One site closely resembles the catalytically competent sites of other caspases, whereas in the second, expulsion of the "activation loop" disrupts the catalytic machinery. We propose that the inactive domain resembles monomeric caspase 9. Activation is induced by dimerization, with interactions at the dimer interface promoting reorientation of the activation loop. These observations support a model in which recruitment by Apaf-1 creates high local concentrations of caspase 9 to provide a pathway for dimer-induced activation.

Crystal structure of the anthrax lethal factor.

Lethal factor (LF) is a protein (relative molecular mass 90,000) that is critical in the pathogenesis of anthrax. It is a highly specific protease that cleaves members of the mitogen-activated protein kinase kinase (MAPKK) family near to their amino termini, leading to the inhibition of one or more signalling pathways. Here we describe the crystal structure of LF and its complex with the N terminus of MAPKK-2. LF comprises four domains: domain I binds the membrane-translocating component of anthrax toxin, the protective antigen (PA); domains II, III and IV together create a long deep groove that holds the 16-residue N-terminal tail of MAPKK-2 before cleavage. Domain II resembles the ADP-ribosylating toxin from Bacillus cereus, but the active site has been mutated and recruited to augment substrate recognition. Domain III is inserted into domain II, and seems to have arisen from a repeated duplication of a structural element of domain II. Domain IV is distantly related to the zinc metalloprotease family, and contains the catalytic centre; it also resembles domain I. The structure thus reveals a protein that has evolved through a process of gene duplication, mutation and fusion, into an enzyme with high and unusual specificity.

Cellular activation of leukocyte function-associated antigen-1 and its affinity are regulated at the I domain allosteric site.

The I domain of the integrin LFA-1 possesses a ligand binding interface that includes the metal ion-dependent adhesion site. Binding of the LFA-1 ligand, ICAM-1 to the metal ion-dependent adhesion site is regulated by the I domain allosteric site (IDAS). We demonstrate here that intracellular signaling leading to activation of LFA-1 binding to ICAM-1 is regulated at the IDAS. Inhibitory mutations in or proximal to the IDAS are dominant to cytoplasmic signals that activate binding to ICAM-1. In addition, mutational activation at the IDAS greatly increases the binding of lymphocyte-expressed LFA-1 to ICAM-1 in response to PMA, but does not result in constitutive binding. Binding of a novel CD18 activation epitope mAb to LFA-1 in response to soluble ICAM-1 binding was also blocked by inhibitory and was enhanced by activating IDAS mutations. Surface plasmon resonance using soluble wild-type LFA-1 and an IDAS mutant of LFA-1 indicate that the IDAS can regulate a 6-fold change in the K(d) of ICAM-1 binding. The K(d) of wild-type LFA-1 (1.2 x 10(-1) s(-1)) differed with that of the activating IDAS mutant (1.9 x 10(-2) s(-1)), but their K(a) values were identical (2.2 x 10(5) M(-1)s(-1)). We propose that IDAS regulates the binding of LFA-1 to ICAM-1 activated by intracellular signals. IDAS can control the affinity state of LFA-1 with concomitant I domain and CD18 conformational changes.

Structural basis for the inhibition of caspase-3 by XIAP.

The molecular mechanism(s) that regulate apoptosis by caspase inhibition remain poorly understood. The main endogenous inhibitors are members of the IAP family and are exemplified by XIAP, which regulates the initiator caspase-9, and the executioner caspases-3 and -7. We report the crystal structure of the second BIR domain of XIAP (BIR2) in complex with caspase-3, at a resolution of 2.7 A, revealing the structural basis for inhibition. The inhibitor makes limited contacts through its BIR domain to the surface of the enzyme, and most contacts to caspase-3 originate from the N-terminal extension. This lies across the substrate binding cleft, but in reverse orientation compared to substrate binding. The mechanism of inhibition is due to a steric blockade prohibitive of substrate binding, and is distinct from the mechanism utilized by synthetic substrate analog inhibitors.

Role of simian virus 40 Vp1 cysteines in virion infectivity.

We have developed a new nonoverlapping infectious viral genome (NO-SV40) in order to facilitate structure-based analysis of the simian virus 40 (SV40) life cycle. We first tested the role of cysteine residues in the formation of infectious virions by individually mutating the seven cysteines in the major capsid protein, Vp1. All seven cysteine mutants-C9A, C49A, C87A, C104A, C207S, C254A, and C267L-retained viability. In the crystal structure of SV40, disulfide bridges are formed between certain Cys104 residues on neighboring pentamers. However, our results show that none of these disulfide bonds are required for virion infectivity in culture. We also introduced five different mutations into Cys254, the most strictly conserved cysteine across the polyomavirus family. We found that C254L, C254S, C254G, C254Q, and C254R mutants all showed greatly reduced (around 100,000-fold) plaque-forming ability. These mutants had no apparent defect in viral DNA replication. Mutant Vp1's, as well as wild-type Vp2/3, were mostly localized in the nucleus. Further analysis of the C254L mutant revealed that the mutant Vp1 was able to form pentamers in vitro. DNase I-resistant virion-like particles were present in NO-SV40-C254L-transfected cell lysate, but at about 1/18 the amount in wild-type-transfected lysate. An examination of the three-dimensional structure reveals that Cys254 is buried near the surface of Vp1, so that it cannot form disulfide bonds, and is not involved in intrapentamer interactions, consistent with the normal pentamer formation by the C254L mutant. It is, however, located at a critical junction between three pentamers, on a conserved loop (G2H) that packs against the dual interpentamer Ca(2+)-binding sites and the invading C-terminal helix of an adjacent pentamer. The substitution by the larger side chains is predicted to cause a localized shift in the G2H loop, which may disrupt Ca(2+) ion coordination and the packing of the invading helix, consistent with the defect in virion assembly. Our experimental system thus allows dissection of structure-function relationships during the distinct steps of the SV40 life cycle.

Crystal structure of the apoptotic suppressor CrmA in its cleaved form.

BACKGROUND: Cowpox virus expresses the serpin CrmA (cytokine response modifier A) in order to avoid inflammatory and apoptotic responses of infected host cells. The targets of CrmA are members of the caspase family of proteases that either initiate the extrinsic pathway of apoptosis (caspases 8 and 10) or trigger activation of the pro-inflammatory cytokines interleukin-1beta and interleukin-18 (caspase 1). RESULTS: We have determined the structure of a cleaved form of CrmA to 2.26 A resolution. CrmA has the typical fold of a cleaved serpin, even though it lacks the N-terminal half of the A helix, the entire D helix, and a portion of the E helix that are present in all other known serpins. The reactive-site loop of CrmA was mutated to contain the optimal substrate recognition sequence for caspase 3; however, the mutation only marginally increased the ability of CrmA to inhibit caspase 3. Superposition of the reactive-site loop of alpha1-proteinase inhibitor on the cleaved CrmA structure provides a model for virgin CrmA that can be docked to caspase 1, but not to caspase 3. CONCLUSIONS: CrmA exemplifies viral economy, selective pressure having resulted in a 'minimal' serpin that lacks the regions not needed for structural integrity or inhibitory activity. The docking model provides an explanation for the selectivity of CrmA. Our demonstration that engineering optimal substrate recognition sequences into the CrmA reactive-site loop fails to generate a good caspase 3 inhibitor is consistent with the docking model.

Structural basis of collagen recognition by integrin alpha2beta1.

We have determined the crystal structure of a complex between the I domain of integrin alpha2beta1 and a triple helical collagen peptide containing a critical GFOGER motif. Three loops on the upper surface of the I domain that coordinate a metal ion also engage the collagen, with a collagen glutamate completing the coordination sphere of the metal. Comparison with the unliganded I domain reveals a change in metal coordination linked to a reorganization of the upper surface that together create a complementary surface for binding collagen. Conformational changes propagate from the upper surface to the opposite pole of the domain, suggesting both a basis for affinity regulation and a pathway for signal transduction. The structural features observed here may represent a general mechanism for integrin-ligand recognition.

Mapping the collagen-binding site in the I domain of the glycoprotein Ia/IIa (integrin alpha(2)beta(1)).

The I domain present within the alpha2 chain of the integrin alpha(2)beta(1) (GPIa/IIa) contains the principal collagen-binding site. Based on the crystal structure of the alpha2-I domain, a hypothetical model was proposed in which collagen binds to a groove on the upper surface of the I domain (Emsley, J., King, S. L., Bergelson, J. M., and Liddington, R. C. (1997) J. Biol. Chem. 272, 28512-28517). We have introduced point mutations into 13 residues on the upper surface of the domain. Recombinant mutant proteins were assayed for binding to monoclonal antibodies 6F1 and 12F1, to collagen under static conditions, and for the ability to retain adhesive activity under flow conditions. The mutations to residues surrounding the metal ion-dependent adhesion site that caused the greatest loss of collagen binding under both static and flow conditions are N154S in the betaA-alpha1 turn, N190D in the betaB-betaC turn, D219R in the alpha3-alpha4 turn, and E256V and H258V in the betaD-alpha5 turn. Mutation in one of the residues that coordinate the metal binding, S155A, completely lost the adhesive activity under flow but bound normally under static conditions, whereas the mutation Y285F had the converse effect. We conclude that the upper surface of the domain, including the metal ion-dependent adhesion site motif, defines the collagen recognition site.

Crystal structure of the vinculin tail suggests a pathway for activation.

Vinculin plays a dynamic role in the assembly of the actin cytoskeleton. A strong interaction between its head and tail domains that regulates binding to other cytoskeletal components is disrupted by acidic phospholipids. Here, we present the crystal structure of the vinculin tail, residues 879-1066. Five amphipathic helices form an antiparallel bundle that resembles exchangeable apolipoproteins. A C-terminal arm wraps across the base of the bundle and emerges as a hydrophobic hairpin surrounded by a collar of basic residues, adjacent to the N terminus. We show that the C-terminal arm is required for binding to acidic phospholipids but not to actin, and that binding either ligand induces conformational changes that may represent the first step in activation.

Interaction between collagen and the alpha(2) I-domain of integrin alpha(2)beta(1). Critical role of conserved residues in the metal ion-dependent adhesion site (MIDAS) region.

A docking model of the alpha(2) I-domain and collagen has been proposed based on their crystal structures (Emsley, J., King, S., Bergelson, J., and Liddington, R. C. (1997) J. Biol. Chem. 272, 28512-28517). In this model, several amino acid residues in the I-domain make direct contact with collagen (Asn-154, Asp-219, Leu-220, Glu-256, His-258, Tyr-285, Asn-289, Leu-291, Asn-295, and Lys-298), and the protruding C-helix of alpha(2) (residues 284-288) determines ligand specificity. Because most of the proposed critical residues are not conserved, different I-domains are predicted to bind to collagen differently. We found that deleting the entire C-helix or mutating the predicted critical residues had no effect on collagen binding to whole alpha(2)beta(1), with the exception that mutating Asn-154, Asp-219, and His-258 had a moderate effect. We performed further studies and found that mutating the conserved surface-exposed residues in the metal ion-dependent adhesion site (MIDAS) (Tyr-157 and Gln-215) significantly blocks collagen binding. We have revised the docking model based on the mutagenesis data. In the revised model, conserved Tyr-157 makes contact with collagen in addition to the previously proposed Asn-154, Asp-219, His-258, and Tyr-285 residues. These results suggest that the collagen-binding I-domains (e.g. alpha(1), alpha(2), and alpha(10)) bind to collagen in a similar fashion.

Crystal structure of a tandem pair of fibronectin type III domains from the cytoplasmic tail of integrin alpha6beta4.

The integrin alpha6beta4 is an essential component of hemidesmosomes but it also plays a dynamic role in invasive carcinoma cells. The cytoplasmic tail of the beta4 subunit is uniquely large among integrins and includes two pairs of fibronectin type III domains separated by a connecting segment. Here we describe the crystal structure of the first tandem domain pair, a module that is critical for alpha6beta4 function. The structure reveals a novel interdomain interface and candidate protein-binding sites, including a large acidic cleft formed from the surfaces of both domains and a prominent loop that is reminiscent of the RGD integrin-binding loop of fibronectin. This is the first crystal structure of either a hemidesmosome component or an integrin cytoplasmic domain, and it will enable the intracellular functions of alpha6beta4 to be dissected at the atomic level.

14-3-3zeta binds a phosphorylated Raf peptide and an unphosphorylated peptide via its conserved amphipathic groove.

14-3-3 proteins bind a variety of molecules involved in signal transduction, cell cycle regulation and apoptosis. 14-3-3 binds ligands such as Raf-1 kinase and Bad by recognizing the phosphorylated consensus motif, RSXpSXP, but must bind unphosphorylated ligands, such as glycoprotein Ib and Pseudomonas aeruginosa exoenzyme S, via a different motif. Here we report the crystal structures of the zeta isoform of 14-3-3 in complex with two peptide ligands: a Raf-derived phosphopeptide (pS-Raf-259, LSQRQRSTpSTPNVHMV) and an unphosphorylated peptide derived from phage display (R18, PHCVPRDLSWLDLEANMCLP) that inhibits binding of exoenzyme S and Raf-1. The two peptides bind within a conserved amphipathic groove on the surface of 14-3-3 at overlapping but distinct sites. The phosphoserine of pS-Raf-259 engages a cluster of basic residues (Lys49, Arg56, Arg60, and Arg127), whereas R18 binds via the amphipathic sequence, WLDLE, with its two acidic groups coordinating the same basic cluster. 14-3-3 is dimeric, and its two peptide-binding grooves are arranged in an antiparallel fashion, 30 A apart. The ability of each groove to bind different peptide motifs suggests how 14-3-3 can act in signal transduction by inducing either homodimer or heterodimer formation in its target proteins.

Crystal structure of the I domain from integrin alpha2beta1.

We have determined the high resolution crystal structure of the I domain from the alpha-subunit of the integrin alpha2beta1, a cell surface adhesion receptor for collagen and the human pathogen echovirus-1. The domain, as expected, adopts the dinucleotide-binding fold, and contains a metal ion-dependent adhesion site motif with bound Mg2+ at the top of the beta-sheet. Comparison with the crystal structures of the leukocyte integrin I domains reveals a new helix (the C-helix) protruding from the metal ion-dependent adhesion site face of the domain which creates a groove centered on the magnesium ion. Modeling of a collagen triple helix into the groove suggests that a glutamic acid side chain from collagen can coordinate the metal ion, and that the C-helix insert is a major determinant of binding specificity. The binding site for echovirus-1 maps to a distinct surface of the alpha2-I domain (one edge of the beta-sheet), consistent with data showing that virus and collagen binding occur by different mechanisms. Comparison with the homologous von Willebrand factor A3 domain, which also binds collagen, suggests that the two domains bind collagen in different ways.

Echovirus 1 interaction with the human very late antigen-2 (integrin alpha2beta1) I domain. Identification of two independent virus contact sites distinct from the metal ion-dependent adhesion site.

The human integrin very late antigen (VLA)-2 (CD49b/CD29) mediates interactions with collagen and is the receptor for echovirus 1. Binding sites for both collagen and echovirus 1 have been mapped to the I domain within the alpha2 subunit of the VLA-2 alpha2beta1 heterodimer. Although murine VLA-2 interacts with collagen, it does not bind virus. We have used isolated human-murine chimeric I domains expressed as glutathione S-transferase fusion proteins in Escherichia coli to identify two groups of amino acids, 199-201 and 212-216, independently involved in virus attachment. These residues are distinct from the metal ion-dependent adhesion site previously demonstrated to be essential for VLA-2 interactions with collagen. Mutations in three metal ion-dependent adhesion site residues that abolish adhesion to collagen had no effect on virus binding. These results confirm that different sites within the I domain are responsible for VLA-2 interaction with extracellular matrix proteins and with viral ligands.

Tension in haemoglobin revealed by Fe-His(F8) bond rupture in the fully liganded T-state.

In 1972, Perutz proposed that the low affinity of T-state haemoglobin is caused by tension in the bond between the iron and the proximal histidine, restraining the Fe from moving into the porphyrin plane on binding oxygen. This proposal has often been disputed. If such tension does exist, it will be manifest in the liganded T-state. Here we describe the structure of the fully liganded T-state cyanide complex of haemoglobin, in which the Fe-proximal histidine bond in the alpha-subunits, but not in the beta-subunits, is ruptured. This rupture uncouples the structural changes at the alpha-haem from those in the globin and the beta-haem, and demonstrates unequivocally the existence of tension and its transmission through this bond.

Crystal structure of the breakage-reunion domain of DNA gyrase.

DNA gyrase is a type II DNA topoisomerase from bacteria that introduces supercoils into DNA. It catalyses the breakage of a DNA duplex (the G segment), the passage of another segment (the T segment) through the break, and then the reunification of the break. This activity involves the opening and dosing of a series of molecular 'gates' which is coupled to ATP hydrolysis. Here we present the crystal structure of the 'breakage-reunion' domain of the gyrase at 2.8 A resolution. Comparison of the structure of this 59K (relative molecular mass, 59,000) domain with that of a 92K fragment of yeast topoisomerase II reveals a very different quaternary organization, and we propose that the two structures represent two principal conformations that participate in the enzymatic pathway. The gyrase structure reveals a new dimer contact with a grooved concave surface for binding the G segment and a cluster of conserved charged residues surrounding the active-site tyrosines. It also shows how breakage of the G segment can occur and, together with the topoisomerase II structure, suggests a pathway by which the T segment can be released through the second gate of the enzyme. Mutations that confer resistance to the quinolone antibacterial agents cluster at the new dimer interface, indicating how these drugs might interact with the gyrase-DNA complex.