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Death receptor signaling is initiated by the assembly of the death-inducing signaling complex, which culminates in the activation of the initiator caspase, either caspase-8 or caspase-10. A family of viral and cellular proteins, known as FLIP, plays an essential role in the regulation of death receptor signaling. Viral FLIP (v-FLIP) and short cellular FLIP (c-FLIPS) inhibit apoptosis by interfering with death receptor signaling. The structure and mechanisms of v-FLIP and c-FLIPS remain largely unknown. Here we report a high resolution crystal structure of MC159, a v-FLIP derived from the molluscum contagiosum virus, which is a member of the human poxvirus family. Unexpectedly, the two tandem death effector domains (DEDs) of MC159 rigidly associate with each other through a hydrophobic interface. Structure-based sequence analysis suggests that this interface is conserved in the tandem DEDs from other v-FLIP, c-FLIPS, and caspase-8 and -10. Strikingly, the overall packing arrangement between the two DEDs of MC159 resembles that between the caspase recruitment domains of Apaf-1 and caspase-9. In addition, each DED of MC159 contains a highly conserved binding motif on the surface, to which loss-of-function mutations in MC159 map. These observations, in conjunction with published evidence, reveal significant insights into the function of v-FLIP and suggest a mechanism by which v-FLIP and c-FLIPS inhibit death receptor signaling.

The ubiquitin-specific processing protease (UBP) family of deubiquitinating enzymes plays an essential role in numerous cellular processes. Mammalian USP14 (Ubp6 in yeast) is unique among known UBP enzymes in that it is activated catalytically upon specific association with the 26S proteasome. Here, we report the crystal structures of the 45-kDa catalytic domain of USP14 in isolation and in a complex with ubiquitin aldehyde, which reveal distinct structural features. In the absence of ubiquitin binding, the catalytic cleft leading to the active site of USP14 is blocked by two surface loops. Binding by ubiquitin induces a significant conformational change that translocates the two surface loops thereby allowing access of the ubiquitin C-terminus to the active site. These structural observations, in conjunction with biochemical characterization, identify important regulatory mechanisms for USP14.

Most gastrointestinal stromal tumors (GIST) have an activating mutation in either KIT or PDGFRA. Imatinib is a selective tyrosine kinase inhibitor and achieves a partial response or stable disease in about 80% of patients with metastatic GIST. It is now clear that some patients with GIST develop resistance to imatinib during chronic therapy. To identify the mechanism of resistance, we studied 31 patients with GIST who were treated with imatinib and then underwent surgical resection. There were 13 patients who were nonresistant to imatinib, 3 with primary resistance, and 15 with acquired resistance after initial benefit from the drug. There were no secondary mutations in KIT or PDGFRA in the nonresistant or primary resistance groups. In contrast, secondary mutations were found in 7 of 15 (46%) patients with acquired resistance, each of whom had a primary mutation in KIT exon 11. Most secondary mutations were located in KIT exon 17. KIT phosphorylation was heterogeneous and did not correlate with clinical response to imatinib or mutation status. That acquired resistance to imatinib in GIST commonly occurs via secondary gene mutation in the KIT kinase domain has implications for strategies to delay or prevent imatinib resistance and to employ newer targeted therapies.

The Mob protein family comprises a group of highly conserved eukaryotic proteins whose founding member functions in the mitotic exit network. At the molecular level, Mob proteins act as kinase-activating subunits. We cloned a human Mob1 family member, Mob1A, and determined its three-dimensional structure by X-ray crystallography. The core of Mob1A consists of a four-helix bundle that is stabilized by a bound zinc atom. The N-terminal helix of the bundle is solvent exposed and together with adjacent secondary structure elements forms an evolutionarily conserved surface with a strong negative electrostatic potential. Several conditional mutant alleles of S. cerevisiae MOB1 target this surface and decrease its net negative charge. Interestingly, the kinases with which yeast Mob proteins interact have two conserved basic regions within their N-terminal lobe. Thus, Mob proteins may regulate their target kinases through electrostatic interactions mediated by conserved charged surfaces.

The myelin-derived proteins Nogo, MAG and OMgp limit axonal regeneration after injury of the spinal cord and brain. These cell-surface proteins signal through multi-subunit neuronal receptors that contain a common ligand-binding glycosylphosphatidylinositol-anchored subunit termed the Nogo-66 receptor (NgR). By deletion analysis, we show that the binding of soluble fragments of Nogo, MAG and NgR to cell-surface NgR requires the entire leucine-rich repeat (LRR) region of NgR, but not other portions of the protein. Despite sharing extensive sequence similarity with NgR, two related proteins, NgR2 and NgR3, which we have identified, do not bind Nogo, MAG, OMgp or NgR. To investigate NgR specificity and multi-ligand binding, we determined the crystal structure of the biologically active ligand-binding soluble ectodomain of NgR. The molecule is banana shaped with elongation and curvature arising from eight LRRs flanked by an N-terminal cap and a small C-terminal subdomain. The NgR structure analysis, as well as a comparison of NgR surface residues not conserved in NgR2 and NgR3, identifies potential protein interaction sites important in the assembly of a functional signaling complex.

The SCF ubiquitin ligases catalyze protein ubiquitination in diverse cellular processes. SCFs bind substrates through the interchangeable F box protein subunit, with the70 human F box proteins allowing the recognition of a wide range of substrates. The F box protein beta-TrCP1 recognizes the doubly phosphorylated DpSGphiXpS destruction motif, present in beta-catenin and IkappaB, and directs the SCF(beta-TrCP1) to ubiquitinate these proteins at specific lysines. The 3.0 A structure of a beta-TrCP1-Skp1-beta-catenin complex reveals the basis of substrate recognition by the beta-TrCP1 WD40 domain. The structure, together with the previous SCF(Skp2) structure, leads to the model of SCF catalyzing ubiquitination by increasing the effective concentration of the substrate lysine at the E2 active site. The model's prediction that the lysine-destruction motif spacing is a determinant of ubiquitination efficiency is confirmed by measuring ubiquitination rates of mutant beta-catenin peptides, solidifying the model and also providing a mechanistic basis for lysine selection.

Antibody 26–10, obtained in a secondary immune response, binds digoxin with high affinity (K a = 1.3 × 1010 m −1) because of extensive shape complementarity. We demonstrated previously that mutations of the hapten contact residue HTrp-100 to Arg (where H refers to the heavy chain) resulted in increased specificity for digoxin analogs substituted at the cardenolide 16 position. However, mutagenesis of H:CDR1 did not result in such a specificity change despite the proximity of the H:CDR1 hapten contact residue Asn-35 to the cardenolide 16 position. Here we constructed a bacteriophage-displayed library containing randomized mutations at H chain residues 30–35 in a 26–10 mutant containing Arg-100 (26–10-RRALD). Phage were selected by panning against digoxin, gitoxin (16-OH), and 16-acetylgitoxin coupled to bovine serum albumin. Clones that retained wild-type Asn at position 35 showed preferred binding to gitoxin, like the 26–10-RRALD parent. In contrast, clones containing Val-35 selected mainly on digoxin-bovine serum albumin demonstrated a shift back to wild-type specificity. Several clones containing Val-35 bound digoxin with increased affinity, approaching that of the wild type in a few instances, in contrast to the mutation Val-35 in the wild-type 26–10 background, which reduces affinity for digoxin 90-fold. It has therefore proven possible to reorder the 26–10 binding site by mutations including two major contact residues on opposite sides of the site and yet to retain high affinity for binding for digoxin. Thus, even among antibodies that have undergone affinity maturation in vivo, different structural solutions to high affinity binding may be revealed.

We constructed Fab libraries of bacteriophage-displayed H:CDR3 mutants in the high-affinity anti-digoxin antibody 26-10 to determine structural constraints on affinity and specificity for digoxin. Libraries of mutant Fabs randomized at five or 10 contiguous positions were panned against digoxin and three C16-substituted analogs, gitoxin (16-OH), 16-formylgitoxin and 16-acetylgitoxin. The sequence data from 83 different mutant Fabs showed highly restricted consensus patterns at positions H:100, 100a and 100b for binding to digoxin; these residues contact digoxin in the 26-10:digoxin co-crystal structure. Several mutant Fabs obtained following panning on digoxin-BSA showed increased affinity for digoxin compared with 26-10 and retained the wild-type (wt) Trp at position 100. Those Fabs selected following panning on C16-substituted analogs showed enhanced binding to the analogs. Replacement of H:Trp100 by Arg resulted in mutants that bound better to the analogs than to digoxin. This specificity change was unexpected, as C16 lies on the opposite side of digoxin from H:CDR3. Substitution of wt Trp by Arg appears to alter specificity by allowing the hapten to shift toward H:CDR3, thereby providing room for C16 substituents in the region of H:CDR1.

The cyclin-dependent kinases 4 and 6 (Cdk4/6) that drive progression through the G1 phase of the cell cycle play a central role in the control of cell proliferation, and CDK deregulation is a frequent event in cancer. Cdk4/6 are regulated by the D-type cyclins, which bind to CDKs and activate the kinase, and by the INK4 family of inhibitors. INK4 proteins can bind both monomeric CDK, preventing its association with a cyclin, and also the CDK-cyclin complex, forming an inactive ternary complex. In vivo, binary INK4–Cdk4/6 complexes are more abundant than ternary INK4–Cdk4/6–cyclinD complexes, and it has been suggested that INK4 binding may lead to the eventual dissociation of the cyclin. Here we present the 2.9-Å crystal structure of the inactive ternary complex between Cdk6, the INK4 inhibitor p18INK4c, and a D-type viral cyclin. The structure reveals that p18INK4c inhibits the CDK–cyclin complex by distorting the ATP binding site and misaligning catalytic residues. p18INK4c also distorts the cyclin-binding site, with the cyclin remaining bound at an interface that is substantially reduced in size. These observations support the model that INK4 binding weakens the cyclin's affinity for the CDK. This structure also provides insights into the specificity of the D-type cyclins for Cdk4/6.

F-box proteins are members of a large family that regulates the cell cycle, the immune response, signalling cascades and developmental programmes by targeting proteins, such as cyclins, cyclin-dependent kinase inhibitors, IkappaBalpha and beta-catenin, for ubiquitination (reviewed in refs 1-3). F-box proteins are the substrate-recognition components of SCF (Skp1-Cullin-F-box protein) ubiquitin-protein ligases. They bind the SCF constant catalytic core by means of the F-box motif interacting with Skp1, and they bind substrates through their variable protein-protein interaction domains. The large number of F-box proteins is thought to allow ubiquitination of numerous, diverse substrates. Most organisms have several Skp1 family members, but the function of these Skp1 homologues and the rules of recognition between different F-box and Skp1 proteins remain unknown. Here we describe the crystal structure of the human F-box protein Skp2 bound to Skp1. Skp1 recruits the F-box protein through a bipartite interface involving both the F-box and the substrate-recognition domain. The structure raises the possibility that different Skp1 family members evolved to function with different subsets of F-box proteins, and suggests that the F-box protein may not only recruit substrate, but may also position it optimally for the ubiquitination reaction.

Serum transferrin binds ferric ions in the bloodstream and transports them to cells, where they are released in a process involving receptor-mediated endocytosis. Iron release is believed to be pH dependent and is coupled with a large conformational change. To help define the steps in iron release, we have determined the three-dimensional structure of the iron-free (apo) form of the recombinant N-lobe half-molecule of human serum transferrin (ApoTfN) by X-ray crystallography. Two crystal forms were obtained, form 1 with four molecules in the asymmetric unit and form 2 with two molecules in the asymmetric unit. The structures of both forms were determined by molecular replacement and were refined at 2.2 and 3.2 A resolution, respectively. Final R-factors were 0.203 (free R = 0. 292) for form 1 and 0.217 (free R = 0.312) for form 2. All six copies of the ApoTfN structure are essentially identical. Comparison with the holo form (FeTfN) shows that a large rigid-body domain movement of 63 degrees has occurred in ApoTfN, to give an open binding cleft. The extent of domain opening is the same as in the N-lobe of human lactoferrin, showing that it depends on internal constraints that are conserved in both proteins, and that it is unaffected by the presence or absence of the C-lobe. Although the conformational change is primarily a rigid-body motion, several local adjustments occur. In particular, two iron ligands, Asp 63 and His 249, change conformation to form salt bridges, with Lys 296 and Glu 83, respectively, in the binding cleft of the apo protein. Both salt bridges would have to break for iron coordination to occur. Most importantly, the structure, determined at a pH (5.3) that is close to the pH of physiological iron release, indicates that protonation of His 249 is a key step in iron release.

The large family of Gram-positive quorum-sensing receptors known as the RNPP proteins consists of receptors homologous to the Rap, NprR, PlcR, and PrgX proteins that are regulated by imported oligopeptide autoinducers. Rap proteins are phosphatases and transcriptional anti-activators, and NprR, PlcR, and PrgX proteins are DNA binding transcription factors. Despite their obvious importance, the mechanistic basis of oligopeptide receptor regulation is largely unknown. Here, we report the X-ray crystal structure of the Bacillus subtilis quorum-sensing receptor RapJ in complex with the centrally important oligopeptide autoinducer competence and sporulation factor (CSF, also termed PhrC), a member of the Phr family of quorum-sensing signals. Furthermore, we present the crystal structure of RapI. Comparison of the RapJ-PhrC, RapI, RapH-Spo0F, and RapF-ComA(C) crystal structures reveals the mechanistic basis of Phr activity. More specifically, when complexed with target proteins, Rap proteins consist of a C-terminal tetratricopeptide repeat (TPR) domain connected by a flexible helix-containing linker to an N-terminal 3-helix bundle. In the absence of a target protein or regulatory peptide, the Rap protein 3-helix bundle adopts different conformations. However, in the peptide-bound conformation, the Rap protein N-terminal 3-helix bundle and linker undergo a radical conformational change, form TPR-like folds, and merge with the existing C-terminal TPR domain. To our knowledge, this is the first example of conformational change-induced repeat domain expansion. Furthermore, upon Phr binding, the entire Rap protein is compressed along the TPR superhelical axis, generating new intramolecular contacts that lock the Rap protein in an inactive state. The fact that Rap proteins are conformationally flexible is surprising considering that it is accepted dogma that TPR proteins do not undergo large conformational changes. Repeat proteins are widely used as scaffolds for the development of designed affinity reagents, and we propose that Rap proteins could be used as scaffolds for engineering novel ligand-switchable affinity reagents.

Cyclin-dependent kinase (CDK)-cyclin complexes require phosphorylation on the CDK subunit for full activation of their Ser/Thr protein kinase activity. The crystal structure of the phosphorylated CDK2-CyclinA-ATP gamma S complex has been determined at 2.6 A resolution. The phosphate group, which is on the regulatory T-loop of CDK2, is mostly buried, its charge being neutralized by three Arg side chains. The arginines help extend the influence of the phosphate group through a network of hydrogen bonds to both CDK2 and cyclinA. Comparison with the unphosphorylated CDK2-CyclinA complex shows that the T-loop moves by as much as 7 A, and this affects the putative substrate binding site as well as resulting in additional CDK2-CyclinA contacts. The phosphate group thus acts as a major organizing centre in the CDK2-CyclinA complex.

The crystal structures of the murine BR96 Fab and its human chimera have been determined in complex with the nonoate methyl ester derivative of Lewis Y (nLey) at 2.8 A and 2.5 A resolution, respectively. BR96 binds the carbohydrate in a large pocket which is formed by residues of all CDR loops except L2. The binding of the carbohydrate is mediated predominantly by aromatic residues in BR96. Analysis of the structure suggests that BR96 is capable of recognizing a structure larger than the Le(y) tetrasaccharide, providing a possible explanation for its high tumour selectivity. The structure provides a rationale for mutagenesis experiments that have resulted in BR96 CDR loop mutants with increased affinity for nLey and/or tumour cells.

The p53 protein is a tetrameric transcription factor that plays a central role in the prevention of neoplastic transformation. Oligomerization appears to be essential for the tumor suppressing activity of p53 because oligomerization-deficient p53 mutants cannot suppress the growth of carcinoma cell lines. The crystal structure of the tetramerization domain of p53 (residues 325 to 356) was determined at 1.7 angstrom resolution and refined to a crystallographic R factor of 19.2 percent. The monomer, which consists of a beta strand and an alpha helix, associates with a second monomer across an antiparallel beta sheet and an antiparallel helix-helix interface to form a dimer. Two of these dimers associate across a second and distinct parallel helix-helix interface to form the tetramer.

The Fab fragments of several monoclonal antibodies that bind digoxin and other cardiac glycosides have been screened for crystallization conditions. We have crystallized two of these in forms suitable for X-ray analysis. The anti-digoxin antibody 40-50 in complex with ouabain crystallizes with symmetry consistent with space group C2, with a = 92.6, b = 85.0, c = 73.0 A and beta = 131.7 degrees. This crystal form shows considerable non-isomorphism between crystals. A second anti-digoxin antibody, 49-10, crystallizes with symmetry consistent with space group P2(1)2(1)2, with a = 95.3, b = 147.1 and c = 76.2 A.

The monoclonal anti-tumor antibody BR96 binds a tetrasaccharide, Lewis y (Le y ), in vitro and recognizes a Le y -bearing or Le y -related tumor-associated antigen in vivo . The Fab of the murine monoclonal antibody, mBR96 (IgG3, κ ), and the Fab' of its human chimera, cBR96 (IgG1, κ ), and their complexes with Le y have been screened for crystallization conditions. Crystals suitable for X-ray diffraction have been obtained for uncomplexed cBR96 Fab', cBR96 Fab' in complex with Le y and mBR96 Fab in complex with Le y . The symmetry of the cBR96 Fab' crystals is consistent with space group P 2 1 2 1 2, a = 61·1 A; b = 174·3 A; c = 45·6 A; the symmetry of the cBR96 Fab'-Le y complex crystals with space group P 4 3 2 1 2 (or its enantiomorph), a = b = 82·2 A; c = 167·1 A and the symmetry of the mBR96 Fab-Le y complex crystals with space group P 2 1 2 1 2 1 , a = 69·4 A; b = 84·9 A; c = 86·8 A.

Procedures for separating and recombining the light-chain variable (VL) and heavy-chain variable (VH) domains of antibody Fv fragments have been applied to produce a recombinant Fv of the anti-digoxin antibody 26-10 that is 15N-labeled exclusively in the VL domain. Comparison of a two-dimensional 1H-15N heteronuclear single-quantum correlation (HSQC) spectrum of the reconstituted Fv with a HSQC spectrum of a fully 15N-labeled Fv sample reveals that all 1H-15N correlations of the VL domain align precisely in both spectra. Assignments for 105 of the 106 backbone HN groups of the VL domain within the reconstituted Fv have been obtained by analysis of three-dimensional nuclear Overhauser effect spectroscopy-HSCQ and total correlation spectroscopy-HSQC spectra with reference to assignments previously reported for the isolated VL domain. Chemical shift differences between the isolated VL domain and the VL domain within the Fv are moderately correlated with proximity to the surfaces of the VH domain and bound hapten (ouabain) as defined by X-ray crystallography and molecular modeling. These results demonstrate that nuclear magnetic resonance studies of reconstituted antibody Fv fragments, in conjunction with investigations of isolated antibody domains, can yield extensive resonance assignments for the Fv. This will facilitate detailed studies of antigen-antibody and domain-domain interactions.

The antigen-binding fragments of four monoclonal antibodies that cross-react with both the "loop" peptide of hen egg-white lysozyme (residues 57 to 84) against which they were raised, and with the native protein (HEL) have been crystallized. One of these fragments also crystallizes as a complex with the peptide antigen.

Herpesvirus-associated ubiquitin-specific protease (HAUSP, also known as USP7), a deubiquitylating enzyme of the ubiquitin-specific processing protease family, specifically deubiquitylates both p53 and MDM2, hence playing an important yet enigmatic role in the p53–MDM2 pathway. Here we demonstrate that both p53 and MDM2 specifically recognize the N-terminal tumor necrosis factor–receptor associated factor (TRAF)–like domain of HAUSP in a mutually exclusive manner. HAUSP preferentially forms a stable HAUSP–MDM2 complex even in the presence of excess p53. The HAUSP-binding elements were mapped to a peptide fragment in the carboxy-terminus of p53 and to a short-peptide region preceding the acidic domain of MDM2. The crystal structures of the HAUSP TRAF-like domain in complex with p53 and MDM2 peptides, determined at 2.3-Å and 1.7-Å resolutions, respectively, reveal that the MDM2 peptide recognizes the same surface groove in HAUSP as that recognized by p53 but mediates more extensive interactions. Structural comparison led to the identification of a consensus peptide-recognition sequence by HAUSP. These results, together with the structure of a combined substrate-binding-and-deubiquitylation domain of HAUSP, provide important insights into regulation of the p53–MDM2 pathway by HAUSP., HAUSP specifically deubiquitinates both p53 and MDM2, to unknown effect. Structural and biochemical analysis shows that HAUSP binds these targets in a mutually exclusive manner, binding MDM2 preferentially.

SummaryBacteria sense their environment using receptors of the histidine sensor kinase family, but how kinase activity is regulated by ligand binding is not well understood. Autoinducer-2 (AI-2), a secreted signaling molecule originally identified in studies of the marine bacterium Vibrio harveyi, regulates quorum-sensing responses and allows communication between different bacterial species. AI-2 signal transduction in V. harveyi requires the integral membrane receptor LuxPQ, comprised of periplasmic binding protein (LuxP) and histidine sensor kinase (LuxQ) subunits. Combined X-ray crystallographic and functional studies show that AI-2 binding causes a major conformational change within LuxP, which in turn stabilizes a quaternary arrangement in which two LuxPQ monomers are asymmetrically associated. We propose that formation of this asymmetric quaternary structure is responsible for repressing the kinase activity of both LuxQ subunits and triggering the transition of V. harveyi into quorum-sensing mode.

Background: Arginase is a manganese-dependent enzyme that catalyzes the hydrolysis of L-arginine to L-ornithine and urea. In ureotelic animals arginase is the final enzyme of the urea cycle, but in many species it has a wider role controlling the use of arginine for other metabolic purposes, including the production of creatine, polyamines, proline and nitric oxide. Arginase activity is regulated by various small molecules, including the product L-ornithine. The aim of these structural studies was to test aspects of the catalytic mechanism and to investigate the structural basis of arginase inhibition. Results: We report here the crystal structures of arginase from Bacillus caldovelox at pH 5.6 and pH 8.5, and of binary complexes of the enzyme with L-arginine, L-ornithine and L-lysine at pH 8.5. The arginase monomer comprises a single compact α / β domain that further associates into a hexameric quaternary structure. The binary complexes reveal a common mode of ligand binding, which places the substrate adjacent to the dimanganese centre. We also observe a conformational change that impacts on the active site and is coupled with the occupancy of an external site by guanidine or arginine. Conclusions: The structures reported here clarify aspects of the active site and indicate key features of the catalytic mechanism, including substrate coordination to one of the manganese ions and an orientational role for a neighboring histidine residue. Stereospecificity for L-amino acids is found to depend on their precise recognition at the active-site rim. Identification of a second arginine-binding site, remote from the active site, and associated conformational changes lead us to propose a regulatory role for this site in substrate hydrolysis.