The crystal structure of the globular head of complement protein C1q provides a basis for its versatile recognition properties

C1q is a versatile recognition protein that binds to an amazing variety of immune and non-immune ligands and triggers activation of the classical pathway of complement. The crystal structure of the C1q globular domain responsible for its recognition properties has now been solved and refined to 1.9 A of resolution. The structure reveals a compact, almost spherical heterotrimeric assembly held together mainly by non-polar interactions, with a Ca2+ ion bound at the top. The heterotrimeric assembly of the C1q globular domain appears to be a key factor of the versatile recognition properties of this protein. Plausible three-dimensional models of the C1q globular domain in complex with two of its physiological ligands, C-reactive protein and IgG, are proposed, highlighting two of the possible recognition modes of C1q. The C1q/human IgG1 model suggests a critical role for the hinge region of IgG and for the relative orientation of its Fab domain in C1q binding.     

The crystal structure of the DNase domain of colicin E7 in complex with its inhibitor Im7 protein

Background: Colicin E7 (ColE7) is one of the bacterial toxins classified as a DNase-type E-group colicin. The cytotoxic activity of a colicin in a colicin-producing cell can be counteracted by binding of the colicin to a highly specific immunity protein. This biological event is a good model system for the investigation of protein recognition.Results: The crystal structure of a one-to-one complex between the DNase domain of colicin E7 and its cognate immunity protein Im7 has been determined at 2.3 Å resolution. Im7 in the complex is a varied four-helix bundle that is identical to the structure previously determined for uncomplexed Im7. The structure of the DNase domain of ColE7 displays a novel α/β fold and contains a Zn²⁺ ion bound to three histidine residues and one water molecule in a distorted tetrahedron geometry. Im7 has a V-shaped structure, extending two arms to clamp the DNase domain of ColE7. One arm (α1¹–loop12–α2¹; where ¹ represents helices in Im7) is located in the region that displays the greatest sequence variation among members of the immunity proteins in the same subfamily. This arm mainly uses acidic sidechains to interact with the basic sidechains in the DNase domain of ColE7. The other arm (loop 23–α3¹–loop 34) is more conserved and it interacts not only with the sidechain but also with the mainchain atoms of the DNase domain of ColE7.Conclusions: The protein interfaces between the DNase domain of ColE7 and Im7 are charge-complementary and charge interactions contribute significantly to the tight and specific binding between the two proteins. The more variable arm in Im7 dominates the binding specificity of the immunity protein to its cognate colicin. Biological and structural data suggest that the DNase active site for ColE7 is probably near the metal-binding site.     

The nucleocapsid protein of coronavirus infectious bronchitis virus: crystal structure of its N-terminal domain and multimerization properties

The coronavirus nucleocapsid (N) protein packages viral genomic RNA into a ribonucleoprotein complex. Interactions between N proteins and RNA are thus crucial for the assembly of infectious virus particles. The 45 kDa recombinant nucleocapsid N protein of coronavirus infectious bronchitis virus (IBV) is highly sensitive to proteolysis. We obtained a stable fragment of 14.7 kDa spanning its N-terminal residues 29-160 (IBV-N29-160). Like the N-terminal RNA binding domain (SARS-N45-181) of the severe acute respiratory syndrome virus (SARS-CoV) N protein, the crystal structure of the IBV-N29-160 fragment at 1.85 A resolution reveals a protein core composed of a five-stranded antiparallel beta sheet with a positively charged beta hairpin extension and a hydrophobic platform that are probably involved in RNA binding. Crosslinking studies demonstrate the formation of dimers, tetramers, and higher multimers of IBV-N. A model for coronavirus shell formation is proposed in which dimerization of the C-terminal domain of IBV-N leads to oligomerization of the IBV-nucleocapsid protein and viral RNA condensation.     

The crystal structure of the BAR domain from human Bin1/amphiphysin II and its implications for molecular recognition

BAR domains are found in proteins that bind and remodel membranes and participate in cytoskeletal and nuclear processes. Here, we report the crystal structure of the BAR domain from the human Bin1 protein at 2.0 A resolution. Both the quaternary and tertiary architectures of the homodimeric Bin1BAR domain are built upon "knobs-into-holes" packing of side chains, like those found in conventional left-handed coiled-coils, and this packing governs the curvature of a putative membrane-engaging concave face. Our calculations indicate that the Bin1BAR domain contains two potential sites for protein-protein interactions on the convex face of the dimer. Comparative analysis of structural features reveals that at least three architectural subtypes of the BAR domain are encoded in the human genome, represented by the Arfaptin, Bin1/Amphiphysin, and IRSp53 BAR domains. We discuss how these principal groups may differ in their potential to form regulatory heterotypic interactions.     

The crystal structure of a fusagenic sperm protein reveals extreme surface properties

Sp18 is an 18 kDa protein that is released from abalone sperm during the acrosome reaction. It coats the acrosomal process where it is thought to mediate fusion between sperm and egg cell membranes. Sp18 is evolutionarily related to lysin, a 16 kDa abalone sperm protein that dissolves the vitelline envelope surrounding the egg. The two proteins were generated by gene duplication followed by rapid divergence by positive selection. Here, we present the crystal structure of green abalone sp18 resolved to 1.86 A. Sp18 is composed of a bundle of five alpha-helices with surface clusters of basic and hydrophobic residues, giving it a large dipole moment and making it extremely amphipathic. The large clusters of hydrophobic surface residues and domains of high positive electrostatic surface charge explain sp18's ability as a potent fusagen of liposomes. The overall fold of sp18 is similar to that of green abalone lysin; however, the surface features of the proteins are quite different, accounting for their different roles in fertilization. This is the first crystal structure of a protein implicated in sperm-egg fusion during animal fertilization.     

The crystal structure of odorant binding protein 7 from Anopheles gambiae exhibits an outstanding adaptability of its binding site

Anopheles gambiae (Agam) targets human and animals by using its olfactory system, leading to the spread of Plasmodium falciparum, the malaria vector. Odorant binding proteins (OBPs) participate to the first event in odorant recognition and constitute an interesting target for insect control. OBPs interact with olfactory receptors to which they deliver the odorant molecule. We have undertaken a large-scale study of proteins belonging to the olfactory system of Agam with in mind of designing strong olfactory repellants. Here, we report the expression, three-dimensional structures and binding properties of AgamOBP07, a member of a new structural class of OBPs, characterized by the occurrence of eight cysteines. We showed that AgamOBP07 possesses seven α-helices and four disulfide bridges, instead of six α-helices and three disulfide bridges in classical OBPs. The extra seventh helix is located at the surface of the protein, locked by the fourth disulfide bridge, and forms a wall of the internal cavity. The binding site of the protein is mainly hydrophobic, elongated and open and is able to accommodate elongated ligands, linear or polycyclic, as suggested also by binding experiments. An elongated electron density was observed in the internal cavity of the purified protein, belonging to a serendipitous ligand. The structure of AgamOBP07 in complex with an azo-bicyclic model compound reveals that a large conformational change in the protein has reshaped its binding site, provoking helix 4 unfolding and doubling of the cavity volume.     

The structure of a centrosymmetric protein crystal

Crystals of racemic rubredoxin, prepared by independent chemical synthesis of the two enantiomers, have been grown and characterized. The unit cell contains two molecules, one of each enantiomer. Examination of the intensity distribution in the diffraction pattern revealed that the crystals are centrosymmetric. This was confirmed by solution of thestructure to 2 Å resolution via molecular replacement methods. The electron density maps are of very high quality due to the fact that the phaseof each reflection must be exactly 0° or exactly 180°. These results demonstrate the feasibility of using synthetic racemic proteins to yield centrosymmetric protein crystals with electron density maps that have very low phase error and model bias. © 1993 Wiley-Liss, Inc.     

PDZ tandem of human syntenin: crystal structure and functional properties

Syntenin, a 33 kDa protein, interacts with several cell membrane receptors and with merlin, the product of the causal gene for neurofibromatosis type II. We report a crystal structure of the functional fragment of human syntenin containing two canonical PDZ domains, as well as binding studies for full-length syntenin, the PDZ tandem, and isolated PDZ domains. We show that the functional properties of syntenin are a result of independent interactions with target peptides, and that each domain is able to bind peptides belonging to two different classes: PDZ1 binds peptides from classes I and III, while PDZ2 interacts with classes I and II. The independent binding of merlin by PDZ1 and syndecan-4 by PDZ2 provides direct evidence for the coupling of syndecan-mediated signaling to actin regulation by merlin.     

The crystal structure of human receptor protein tyrosine phosphatase kappa phosphatase domain 1

The receptor-type protein tyrosine phosphatases (RPTPs) are integral membrane proteins composed of extracellular adhesion molecule-like domains, a single transmembrane domain, and a cytoplasmic domain. The cytoplasmic domain consists of tandem PTP domains, of which the D1 domain is enzymatically active. RPTPkappa is a member of the R2A/IIb subfamily of RPTPs along with RPTPmu, RPTPrho, and RPTPlambda. Here, we have determined the crystal structure of catalytically active, monomeric D1 domain of RPTPkappa at 1.9 A. Structural comparison with other PTP family members indicates an overall classical PTP architecture of twisted mixed beta-sheets flanked by alpha-helices, in which the catalytically important WPD loop is in an unhindered open conformation. Though the residues forming the dimeric interface in the RPTPmu structure are all conserved, they are not involved in the protein-protein interaction in RPTPkappa. The N-terminal beta-strand, formed by betax association with betay, is conserved only in RPTPs but not in cytosolic PTPs, and this feature is conserved in the RPTPkappa structure forming a beta-strand. Analytical ultracentrifugation studies show that the presence of reducing agents and higher ionic strength are necessary to maintain RPTPkappa as a monomer. In this family the crystal structure of catalytically active RPTPmu D1 was solved as a dimer, but the dimerization was proposed to be a consequence of crystallization since the protein was monomeric in solution. In agreement, we show that RPTPkappa is monomeric in solution and crystal structure.     

The high-resolution crystal structure of human LCAT

LCAT is intimately involved in HDL maturation and is a key component of the reverse cholesterol transport (RCT) pathway which removes excess cholesterol molecules from the peripheral tissues to the liver for excretion. Patients with loss-of-function LCAT mutations exhibit low levels of HDL cholesterol and corneal opacity. Here we report the 2.65 Å crystal structure of the human LCAT protein. Crystallization required enzymatic removal of N-linked glycans and complex formation with a Fab fragment from a tool antibody. The crystal structure reveals that LCAT has an α/β hydrolase core with two additional subdomains that play important roles in LCAT function. Subdomain 1 contains the region of LCAT shown to be required for interfacial activation, while subdomain 2 contains the lid and amino acids that shape the substrate binding pocket. Mapping the naturally occurring mutations onto the structure provides insight into how they may affect LCAT enzymatic activity.     

The crystal structure and thermotropic liquid-crystal properties of N-n-undecyl-D-gluconamide

N-n-Undecyl-D-gluconamide, C17H35O6, crystallizes in space group P1, with one molecule in a unit cell a = 5.2267(6), b = 19.628(9), c = 4.7810(4) A, alpha = 93.23(2), beta = 95.60(1), gamma = 89.58(2) degrees, V = 487.35 A3, Dx = 1.19 g.cm-3. The crystal lattice is isostructural with N-n-heptyl-D-gluconamide having monolayer head-to-tail molecular packing. The molecules have a V-shaped conformation. The hydrogen bonding of the gluconamide moieties includes a four-link homodromic cycle. The transition to a smectic A liquid-crystal phase at 156.7 degrees is preceded by two crystal-to-crystal phase transitions at 77.2 degrees and 99.4 degrees. The long d-spacing of the intermediate crystal phase of 39 A, and the d-spacing of the liquid-crystal phase of 32 A, are consistent with a transition to a bilayer head-to-head molecular packing.     

The crystal structure of Escherichia coli MoeA and its relationship to the multifunctional protein gephyrin

BACKGROUND: Molybdenum cofactor (Moco) biosynthesis is an evolutionarily conserved pathway present in archaea, eubacteria, and eukaryotes. In humans, genetic abnormalities in the biosynthetic pathway result in Moco deficiency, which is accompanied by severe neurological symptoms and death shortly after birth. The Escherichia coli MoeA and MogA proteins are involved in the final step of Moco biosynthesis: the incorporation of molybdenum into molybdopterin (MPT), the organic pyranopterin moiety of Moco. RESULTS: The crystal structure of E. coli MoeA has been refined at 2 A resolution and reveals that the highly elongated MoeA monomer consists of four clearly separated domains, one of which is structurally related to MogA, indicating a divergent evolutionary relationship between both proteins. The active form of MoeA is a dimer, and a putative active site appears to be localized to a cleft formed between domain II of the first monomer and domains III and IV of the second monomer. CONCLUSIONS: In eukaryotes, MogA and MoeA are fused into a single polypeptide chain. The corresponding mammalian protein gephyrin has also been implicated in the anchoring of glycinergic receptors to the cytoskeleton at inhibitory synapses. Based on the structures of MoeA and MogA, gephyrin is surmised to be a highly organized molecule containing at least five domains. This multidomain arrangement could provide a structural basis for its functional diversity. The oligomeric states of MoeA and MogA suggest how gephyrin could assemble into a hexagonal scaffold at inhibitory synapses.     

The crystal structure of human eukaryotic release factor eRF1--mechanism of stop codon recognition and peptidyl-tRNA hydrolysis

The release factor eRF1 terminates protein biosynthesis by recognizing stop codons at the A site of the ribosome and stimulating peptidyl-tRNA bond hydrolysis at the peptidyl transferase center. The crystal structure of human eRF1 to 2.8 A resolution, combined with mutagenesis analyses of the universal GGQ motif, reveals the molecular mechanism of release factor activity. The overall shape and dimensions of eRF1 resemble a tRNA molecule with domains 1, 2, and 3 of eRF1 corresponding to the anticodon loop, aminoacyl acceptor stem, and T stem of a tRNA molecule, respectively. The position of the essential GGQ motif at an exposed tip of domain 2 suggests that the Gln residue coordinates a water molecule to mediate the hydrolytic activity at the peptidyl transferase center. A conserved groove on domain 1, 80 A from the GGQ motif, is proposed to form the codon recognition site.     

The crystal structure of yeast protein disulfide isomerase suggests cooperativity between its active sites

Protein disulfide isomerase plays a key role in catalyzing the folding of secretory proteins. It features two catalytically inactive thioredoxin domains inserted between two catalytically active thioredoxin domains and an acidic C-terminal tail. The crystal structure of yeast PDI reveals that the four thioredoxin domains are arranged in the shape of a twisted "U" with the active sites facing each other across the long sides of the "U." The inside surface of the "U" is enriched in hydrophobic residues, thereby facilitating interactions with misfolded proteins. The domain arrangement, active site location, and surface features strikingly resemble the Escherichia coli DsbC and DsbG protein disulfide isomerases. Biochemical studies demonstrate that all domains of PDI, including the C-terminal tail, are required for full catalytic activity. The structure defines a framework for rationalizing the differences between the two active sites and their respective roles in catalyzing the formation and rearrangement of disulfide bonds.     

The structure and biochemical properties of the human spliceosomal protein U1C

The spliceosomal U1C protein is critical to the initiation and regulation of precursor messenger RNA (pre-mRNA) splicing, as part of the U1 small nuclear ribonucleoprotein particle (snRNP). We have produced full-length and 61 residue constructs of human U1C in soluble form in Escherichia coli. Atomic absorption spectroscopy and mass spectrometry show that both constructs contain one Zn atom and are monomeric. Gelmobility-shift assays showed that one molecule of recombinant U1C, either full-length or 61 residue construct, can be incorporated into the U1 snRNP core domain in the presence of U1 70k. This result is in perfect agreement with the previous experiment with U1C isolated from the HeLa U1 snRNP showing that the recombinant U1C is functionally active. We have determined the solution structure of the N-terminal 61 residue construct of U1C by NMR. A Cys(2)His(2)-type zinc finger, distinct from the TFIIIA-type, is extended at its C terminus by two additional helices. The two Zn-coordinating histidine residues are separated by a five residue loop. The conserved basic residues in the first two helices and the intervening loop may be involved in RNA binding. The opposite beta-sheet face with two surface-exposed Tyr residues may be involved in protein contacts. Both the full-length and 61 residue constructs of human U1C fail to bind RNA containing the 5' splice site sequence, in contrast to what has been reported for the Saccharomyces cerevisiae orthologue.     

The X-ray crystal structure of full-length human plasminogen

Plasminogen is the proenzyme precursor of the primary fibrinolytic protease plasmin. Circulating plasminogen, which comprises a Pan-apple (PAp) domain, five kringle domains (KR1-5), and a serine protease (SP) domain, adopts a closed, activation-resistant conformation. The kringle domains mediate interactions with fibrin clots and cell-surface receptors. These interactions trigger plasminogen to adopt an open form that can be cleaved and converted to plasmin by tissue-type and urokinase-type plasminogen activators. Here, the structure of closed plasminogen reveals that the PAp and SP domains, together with chloride ions, maintain the closed conformation through interactions with the kringle array. Differences in glycosylation alter the position of KR3, although in all structures the loop cleaved by plasminogen activators is inaccessible. The ligand-binding site of KR1 is exposed and likely governs proenzyme recruitment to targets. Furthermore, analysis of our structure suggests that KR5 peeling away from the PAp domain may initiate plasminogen conformational change.     

The crystal and molecular structure of the anticancer drug actinomycin D--some explanations for its unusual properties

The crystal structure of the antitumor antibiotic actinomycin D has been determined by the methods of x-ray crystallography. There are three independent molecules in the asymmetric unit. Two of the molecules form a hydrogen-bonded dimer. The peptides within all three molecules have very similar conformations. The greatest conformational variability in the drug molecules occurs in the torsion angles of the bonds connecting the phenoxazone ring to the peptide rings. The results of this analysis has allowed us to explain the unusual physical properties of ActD as well as to provide an explanation for the thermodynamics of its interactions with DNA.     

A critical switch in the enzymatic properties of the Cid1 protein deciphered from its product-bound crystal structure

The addition of uridine nucleotide by the poly(U) polymerase (PUP) enzymes has a demonstrated impact on various classes of RNAs such as microRNAs (miRNAs), histone-encoding RNAs and messenger RNAs. Cid1 protein is a member of the PUP family. We solved the crystal structure of Cid1 in complex with non-hydrolyzable UMPNPP and a short dinucleotide compound ApU. These structures revealed new residues involved in substrate/product stabilization. In particular, one of the three catalytic aspartate residues explains the RNA dependence of its PUP activity. Moreover, other residues such as residue N165 or the β-trapdoor are shown to be critical for Cid1 activity. We finally suggest that the length and sequence of Cid1 substrate RNA influence the balance between Cid1''s processive and distributive activities. We propose that particular processes regulated by PUPs require the enzymes to switch between the two types of activity as shown for the miRNA biogenesis where PUPs can either promote DICER cleavage via short U-tail or trigger miRNA degradation by adding longer poly(U) tail. The enzymatic properties of these enzymes may be critical for determining their particular function in vivo.     

The physiological structure of human C-reactive protein and its complex with phosphocholine.

BACKGROUND: Human C-reactive protein (CRP) is the classical acute phase reactant, the circulating concentration of which rises rapidly and extensively in a cytokine-mediated response to tissue injury, infection and inflammation. Serum CRP values are routinely measured, empirically, to detect and monitor many human diseases. However, CRP is likely to have important host defence, scavenging and metabolic functions through its capacity for calcium-dependent binding to exogenous and autologous molecules containing phosphocholine (PC) and then activating the classical complement pathway. CRP may also have pathogenic effects and the recent discovery of a prognostic association between increased CRP production and coronary atherothrombotic events is of particular interest. RESUTLS: The X-ray structures of fully calcified C-reactive protein, in the presence and absence of bound PC, reveal that although the subunit beta-sheet jellyroll fold is very similar to that of the homologous pentameric protein serum amyloid P component, each subunit is tipped towards the fivefold axis. PC is bound in a shallow surface pocket on each subunit, interacting with the two protein-bound calcium ions via the phosphate group and with Glu81 via the choline moiety. There is also an unexpected hydrophobic pocket adjacent to the ligand. CONCLUSIONS: The structure shows how large ligands containing PC may be bound by CRP via a phosphate oxygen that projects away from the surface of the protein. Multipoint attachment of one planar face of the CRP molecule to a PC-bearing surface would leave available, on the opposite exposed face, the recognition sites for C1q, which have been identified by mutagenesis. This would enable CRP to target physiologically and/or pathologically significant complement activation. The hydrophobic pocket adjacent to bound PC invites the design of inhibitors of CRP binding that may have therapeutic relevance to the possible role of CRP in atherothrombotic events.