Crystal structure of the extracellular segment of integrin alpha Vbeta3

Integrins are alphabeta heterodimeric receptors that mediate divalent cation-dependent cell-cell and cell-matrix adhesion through tightly regulated interactions with ligands. We have solved the crystal structure of the extracellular portion of integrin alphaVbeta3 at 3.1 A resolution. Its 12 domains assemble into an ovoid "head" and two "tails." In the crystal, alphaVbeta3 is severely bent at a defined region in its tails, reflecting an unusual flexibility that may be linked to integrin regulation. The main inter-subunit interface lies within the head, between a seven-bladed beta-propeller from alphaV and an A domain from beta3, and bears a striking resemblance to the Galpha/Gbeta interface in G proteins. A metal ion-dependent adhesion site (MIDAS) in the betaA domain is positioned to participate in a ligand-binding interface formed of loops from the propeller and betaA domains. MIDAS lies adjacent to a calcium-binding site with a potential regulatory function.     

Crystal structure of the extracellular segment of integrin alpha Vbeta3 in complex with an Arg-Gly-Asp ligand

The structural basis for the divalent cation-dependent binding of heterodimeric alphabeta integrins to their ligands, which contain the prototypical Arg-Gly-Asp sequence, is unknown. Interaction with ligands triggers tertiary and quaternary structural rearrangements in integrins that are needed for cell signaling. Here we report the crystal structure of the extracellular segment of integrin alphaVbeta3 in complex with a cyclic peptide presenting the Arg-Gly-Asp sequence. The ligand binds at the major interface between the alphaV and beta3 subunits and makes extensive contacts with both. Both tertiary and quaternary changes are observed in the presence of ligand. The tertiary rearrangements take place in betaA, the ligand-binding domain of beta3; in the complex, betaA acquires two cations, one of which contacts the ligand Asp directly and the other stabilizes the ligand-binding surface. Ligand binding induces small changes in the orientation of alphaV relative to beta3.     

Atomic structure of ferredoxin-NADP+ reductase: prototype for a structurally novel flavoenzyme family

The three-dimensional structure of spinach ferredoxin-NADP+ reductase (NADP+, nicotinamide adenine dinucleotide phosphate) has been determined by x-ray diffraction at 2.6 angstroms (A) resolution and initially refined to an R factor of 0.226 at 2.2 A resolution. The model includes the flavin-adenine dinucleotide (FAD) prosthetic group and the protein chain from residue 19 through the carboxyl terminus at residue 314 and is composed of two domains. The FAD binding domain (residues 19 to 161) has an antiparallel beta barrel core and a single alpha helix for binding the pyrophosphate of FAD. The NADP binding domain (residues 162 to 314) has a central five-strand parallel beta sheet and six surrounding helices. Binding of the competitive inhibitor 2'-phospho-AMP (AMP, adenosine monophosphate) places the NADP binding site at the carboxyl-terminal edge of the sheet in a manner similar to the nucleotide binding of the dehydrogenase family. The structures reveal the key residues that function in cofactor binding and the catalytic center. With these key residues as a guide, conclusive evidence is presented that the ferredoxin reductase structure is a prototype for the nicotinamide dinucleotide and FAD binding domains of the enzymes NADPH-cytochrome P450 reductase, NADPH-sulfite reductase, NADH-cytochrome b5 reductase, and NADH-nitrate reductase. Thus this structure provides a structural framework for the NADH- or NADPH-dependent flavoenzyme parts of five distinct enzymes involved in photosynthesis, in the assimilation of inorganic nitrogen and sulfur, in fatty-acid oxidation, in the reduction of methemoglobin, and in the metabolism of many pesticides, drugs, and carcinogens.     

Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase

The crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase complexed with a 20-amino acid substrate analog inhibitor has been solved and partially refined at 2.7 A resolution to an R factor of 0.212. The magnesium adenosine triphosphate (MgATP) binding site was located by difference Fourier synthesis. The enzyme structure is bilobal with a deep cleft between the lobes. The cleft is filled by MgATP and a portion of the inhibitor peptide. The smaller lobe, consisting mostly of amino-terminal sequence, is associated with nucleotide binding, and its largely antiparallel beta sheet architecture constitutes an unusual nucleotide binding motif. The larger lobe is dominated by helical structure with a single beta sheet at the domain interface. This lobe is primarily involved in peptide binding and catalysis. Residues 40 through 280 constitute a conserved catalytic core that is shared by more than 100 protein kinases. Most of the invariant amino acids in this conserved catalytic core are clustered at the sites of nucleotide binding and catalysis.     

GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function

The beta2-adrenergic receptor (beta2AR) is a well-studied prototype for heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) that respond to diffusible hormones and neurotransmitters. To overcome the structural flexibility of the beta2AR and to facilitate its crystallization, we engineered a beta2AR fusion protein in which T4 lysozyme (T4L) replaces most of the third intracellular loop of the GPCR ("beta2AR-T4L") and showed that this protein retains near-native pharmacologic properties. Analysis of adrenergic receptor ligand-binding mutants within the context of the reported high-resolution structure of beta2AR-T4L provides insights into inverse-agonist binding and the structural changes required to accommodate catecholamine agonists. Amino acids known to regulate receptor function are linked through packing interactions and a network of hydrogen bonds, suggesting a conformational pathway from the ligand-binding pocket to regions that interact with G proteins.     

The high-affinity E. coli methionine ABC transporter: structure and allosteric regulation

The crystal structure of the high-affinity Escherichia coli MetNI methionine uptake transporter, a member of the adenosine triphosphate (ATP)-binding cassette (ABC) family, has been solved to 3.7 angstrom resolution. The overall architecture of MetNI reveals two copies of the adenosine triphosphatase (ATPase) MetN in complex with two copies of the transmembrane domain MetI, with the transporter adopting an inward-facing conformation exhibiting widely separated nucleotide binding domains. Each MetI subunit is organized around a core of five transmembrane helices that correspond to a subset of the helices observed in the larger membrane-spanning subunits of the molybdate (ModBC) and maltose (MalFGK) ABC transporters. In addition to the conserved nucleotide binding domain of the ABC family, MetN contains a carboxyl-terminal extension with a ferredoxin-like fold previously assigned to a conserved family of regulatory ligand-binding domains. These domains separate the nucleotide binding domains and would interfere with their association required for ATP binding and hydrolysis. Methionine binds to the dimerized carboxyl-terminal domain and is shown to inhibit ATPase activity. These observations are consistent with an allosteric regulatory mechanism operating at the level of transport activity, where increased intracellular levels of the transported ligand stabilize an inward-facing, ATPase-inactive state of MetNI to inhibit further ligand translocation into the cell.     

A beta 3 integrin mutation abolishes ligand binding and alters divalent cation-dependent conformation

The ligand-binding function of integrin adhesion receptors depends on divalent cations. A mutant alpha IIb beta 3 integrin (platelet gpIIb/IIIa) that lacks ligand recognition shows immunologic evidence of a perturbed interaction with divalent cations. This was found to be caused by a G----T mutation that resulted in an Asp119----Tyr119 substitution in the beta 3 subunit. This residue is proximal to bound ligand and is in a conserved region among integrins that are enriched in oxygenated residues. The spacing of these residues aligns with the calcium-binding residues in EF hand proteins, suggesting interaction with receptor-bound divalent cation as a mechanism of ligand binding common to all integrins.     

The structure of interleukin-2 complexed with its alpha receptor

Interleukin-2 (IL-2) is an immunoregulatory cytokine that binds sequentially to the alpha (IL-2Ralpha), beta (IL-2Rbeta), and common gamma chain (gammac) receptor subunits. Here we present the 2.8 angstrom crystal structure of a complex between human IL-2 and IL-2Ralpha, which interact in a docking mode distinct from that of other cytokine receptor complexes. IL-2Ralpha is composed of strand-swapped "sushi-like" domains, unlike the classical cytokine receptor fold. As a result of this domain swap, IL-2Ralpha uses a composite surface to dock into a groove on IL-2 that also serves as a binding site for antagonist drugs. With this complex, we now have representative structures for each class of hematopoietic cytokine receptor-docking modules.     

High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors constitute the largest family of eukaryotic signal transduction proteins that communicate across the membrane. We report the crystal structure of a human beta2-adrenergic receptor-T4 lysozyme fusion protein bound to the partial inverse agonist carazolol at 2.4 angstrom resolution. The structure provides a high-resolution view of a human G protein-coupled receptor bound to a diffusible ligand. Ligand-binding site accessibility is enabled by the second extracellular loop, which is held out of the binding cavity by a pair of closely spaced disulfide bridges and a short helical segment within the loop. Cholesterol, a necessary component for crystallization, mediates an intriguing parallel association of receptor molecules in the crystal lattice. Although the location of carazolol in the beta2-adrenergic receptor is very similar to that of retinal in rhodopsin, structural differences in the ligand-binding site and other regions highlight the challenges in using rhodopsin as a template model for this large receptor family.     

Interaction of the IL-2 receptor with the src-family kinase p56lck: identification of novel intermolecular association

In the interleukin-2 (IL-2) system, intracellular signal transduction is triggered by the beta chain of the IL-2 receptor (IL-2R beta); however, the responsible signaling mechanism remains unidentified. Evidence for the formation of a stable complex of IL-2R beta and the lymphocyte-specific protein tyrosine kinase p56lck is presented. Specific association sites were identified in the tyrosine kinase catalytic domain of p56lck and in the cytoplasmic domain of IL-2R beta. As a result of interaction, IL-2R beta became phosphorylated in vitro by p56lck. Treatment of T lymphocytes with IL-2 promotes p56lck kinase activity. These data suggest the participation of p56lck as a critical signaling molecule downstream of IL-2R via a novel interaction.     

Structural basis of Smad2 recognition by the Smad anchor for receptor activation

The Smad proteins mediate transforming growth factor-beta (TGFbeta) signaling from the transmembrane serine-threonine receptor kinases to the nucleus. The Smad anchor for receptor activation (SARA) recruits Smad2 to the TGFbeta receptors for phosphorylation. The crystal structure of a Smad2 MH2 domain in complex with the Smad-binding domain (SBD) of SARA has been determined at 2.2 angstrom resolution. SARA SBD, in an extended conformation comprising a rigid coil, an alpha helix, and a beta strand, interacts with the beta sheet and the three-helix bundle of Smad2. Recognition between the SARA rigid coil and the Smad2 beta sheet is essential for specificity, whereas interactions between the SARA beta strand and the Smad2 three-helix bundle contribute significantly to binding affinity. Comparison of the structures between Smad2 and a comediator Smad suggests a model for how receptor-regulated Smads are recognized by the type I receptors.     

Structural mechanism of endosome docking by the FYVE domain

The recruitment of trafficking and signaling proteins to membranes containing phosphatidylinositol 3-phosphate [PtdIns(3)P] is mediated by FYVE domains. Here, the solution structure of the FYVE domain of the early endosome antigen 1 protein (EEA1) in the free state was compared with the structures of the domain complexed with PtdIns(3)P and mixed micelles. The multistep binding mechanism involved nonspecific insertion of a hydrophobic loop into the lipid bilayer, positioning and activating the binding pocket. Ligation of PtdIns(3)P then induced a global structural change, drawing the protein termini over the bound phosphoinositide by extension of a hinge. Specific recognition of the 3-phosphate was determined indirectly and directly by two clusters of conserved arginines.     

Interleukin-2 receptor beta chain gene: generation of three receptor forms by cloned human alpha and beta chain cDNA's

Interleukin-2 (IL-2) binds to two distinct receptor molecules, the IL-2 receptor alpha (IL-2R alpha, p55) chain and the newly identified IL-2 receptor beta (IL-2R beta, p70-75) chain. The cDNA encoding the human IL-2R beta chain has now been isolated. The overall primary structure of the IL-2R beta chain shows no apparent homology to other known receptors. Unlike the IL-2R alpha chain, the IL-2R beta chain has a large cytoplasmic region in which a functional domain (or domains) mediating an intracellular signal transduction pathway (or pathways) may be embodied. The cDNA-encoded beta chain binds and internalizes IL-2 when expressed on T lymphoid cells but not fibroblast cells. Furthermore, the cDNA gives rise to the generation of high-affinity IL-2 receptor when co-expressed with the IL-2R alpha chain cDNA.     

A novel, highly stable fold of the immunoglobulin binding domain of streptococcal protein G

The high-resolution three-dimensional structure of a single immunoglobulin binding domain (B1, which comprises 56 residues including the NH2-terminal Met) of protein G from group G Streptococcus has been determined in solution by nuclear magnetic resonance spectroscopy on the basis of 1058 experimental restraints. The average atomic root-mean-square distribution about the mean coordinate positions is 0.27 angstrom (A) for the backbone atoms, 0.65 A for all atoms, and 0.39 A for atoms excluding disordered surface side chains. The structure has no disulfide bridges and is composed of a four-stranded beta sheet, on top of which lies a long helix. The central two strands (beta 1 and beta 4), comprising the NH2- and COOH-termini, are parallel, and the outer two strands (beta 2 and beta 3) are connected by the helix in a +3x crossover. This novel topology (-1, +3x, -1), coupled with an extensive hydrogen-bonding network and a tightly packed and buried hydrophobic core, is probably responsible for the extreme thermal stability of this small domain (reversible melting at 87 degrees C).     

Convergence of Ets- and notch-related structural motifs in a heteromeric DNA binding complex

Analysis of the heteromeric DNA binding protein GABP has revealed the interaction of two distinct peptide sequence motifs normally associated with proteins located in different cellular compartments. The alpha subunit of GABP contains an 85-amino acid segment related to the Ets family of DNA binding proteins. The ETS domain of GABP alpha facilitates weak binding to DNA and, together with an adjacent segment of 37 amino acids, mediates stable interaction with GABP beta. The beta subunit of GABP contains four imperfect repeats of a sequence present in several transmembrane proteins including the product of the Notch gene of Drosophila melanogaster. These amino-terminal repeats of GABP beta mediate stable interaction with GABP alpha and, when complexed with GABP alpha, directly contact DNA. These observations provide evidence for a distinct biochemical role for the 33-amino acid repeats, and suggest that they may serve as a module for the generation of specific dimerization interfaces.     

Role of the ENTH domain in phosphatidylinositol-4,5-bisphosphate binding and endocytosis

Endocytic proteins such as epsin, AP180, and Hip1R (Sla2p) share a conserved modular region termed the epsin NH2-terminal homology (ENTH) domain, which plays a crucial role in clathrin-mediated endocytosis through an unknown target. Here, we demonstrate a strong affinity of the ENTH domain for phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]. With nuclear magnetic resonance analysis of the epsin ENTH domain, we determined that a cleft formed with positively charged residues contributed to phosphoinositide binding. Overexpression of a mutant, epsin Lys76 --> Ala76, with an ENTH domain defective in phosphoinositide binding, blocked epidermal growth factor internalization in COS-7 cells. Thus, interaction between the ENTH domain and PtdIns(4,5)P2 is essential for endocytosis mediated by clathrin-coated pits.     

Solution structures of beta peptide and its constituent fragments: relation to amyloid deposition

The secondary structures in solution of the synthetic, naturally occurring, amyloid beta peptides, residues 1 to 42 [beta (1-42)] and beta (1-39), and related fragments, beta (1-28) and beta (29-42), have been studied by circular dichroism and two-dimensional nuclear magnetic resonance spectroscopy. In patients with Alzheimer's disease, extracellular amyloid plaque core is primarily composed of beta (1-42), whereas cerebrovascular amyloid contains the more soluble beta (1-39). In aqueous trifluoroethanol solution, the beta (1-28), beta (1-39), and beta (1-42) peptides adopt monomeric alpha-helical structures at both low and high pH, whereas at intermediate pH (4 to 7) an oligomeric beta structure (the probable structure in plaques) predominates. Thus, beta peptide is not by itself an insoluble protein (as originally thought), and localized or normal age-related alterations of pH may be necessary for the self-assembly and deposition of beta peptide. The hydrophobic carboxyl-terminal segment, beta(29-42), exists exclusively as an oligomeric beta sheet in solution, regardless of differences in solvent, pH, or temperature, suggesting that this segment directs the folding of the complete beta (1-42) peptide to produce the beta-pleated sheet found in amyloid plaques.     

Unexpected modes of PDZ domain scaffolding revealed by structure of nNOS-syntrophin complex

The PDZ protein interaction domain of neuronal nitric oxide synthase (nNOS) can heterodimerize with the PDZ domains of postsynaptic density protein 95 and syntrophin through interactions that are not mediated by recognition of a typical carboxyl-terminal motif. The nNOS-syntrophin PDZ complex structure revealed that the domains interact in an unusual linear head-to-tail arrangement. The nNOS PDZ domain has two opposite interaction surfaces-one face has the canonical peptide binding groove, whereas the other has a beta-hairpin "finger." This nNOS beta finger docks in the syntrophin peptide binding groove, mimicking a peptide ligand, except that a sharp beta turn replaces the normally required carboxyl terminus. This structure explains how PDZ domains can participate in diverse interaction modes to assemble protein networks.     

BRCT repeats as phosphopeptide-binding modules involved in protein targeting

We used a proteomic approach to identify phosphopeptide-binding modules mediating signal transduction events in the DNA damage response pathway. Using a library of partially degenerate phosphopeptides, we identified tandem BRCT (BRCA1 carboxyl-terminal) domains in PTIP (Pax transactivation domain-interacting protein) and in BRCA1 as phosphoserine- or phosphothreonine-specific binding modules that recognize substrates phosphorylated by the kinases ATM (ataxia telangiectasia-mutated) and ATR (ataxia telangiectasia- and RAD3-related) in response to gamma-irradiation. PTIP tandem BRCT domains are responsible for phosphorylation-dependent protein localization into 53BP1- and phospho-H2AX (gamma-H2AX)-containing nuclear foci, a marker of DNA damage. These findings provide a molecular basis for BRCT domain function in the DNA damage response and may help to explain why the BRCA1 BRCT domain mutation Met1775 --> Arg, which fails to bind phosphopeptides, predisposes women to breast and ovarian cancer.