Eight-stranded Antiparallel Β-barrel Research Articles

Proposed lipocalin fold for apolipoprotein M based on bioinformatics and site-directed mutagenesis

Apolipoprotein M (apoM) is a novel apolipoprotein that is predominantly present in high-density lipoprotein. Sensitive sequence searches, threading and comparative model building experiments revealed apoM to be structurally related to the lipocalin protein family. In a 3D model, characterized by an eight-stranded anti-parallel β-barrel, a segment including Asn135 could adopt a closed or open conformation. Using site-directed mutagenesis, we demonstrated Asn135 in wild-type apoM to be glycosylated, suggesting that the segment is solvent exposed. ApoM displays two strong acidic patches of potential functional importance, one around the N-terminus and the other next to the opening of the β-barrel.

The three-dimensional structure of a Plasmodium falciparum cyclophilin in complex with the potent anti-malarial cyclosporin A

Cyclosporin A (CsA) is a potent anti-malarial compound in vitro and in vivo in mice though better known for its immunosuppressive properties in humans. Crystal structures of wild-type and a double mutant Plasmodium falciparum cyclophilin (PfCyP19 and mPfCyP19) complexed with CsA have been determined using diffraction terms to a resolution of 2.1 Å (1 Å=0.1 nm). The wild-type has a single PfCyP19/CsA complex per asymmetric unit in space group P1 and refined to an R-work of 0.15 and R-free of 0.19. An altered cyclophilin, with two accidental mutations, Phe120 to Leu in the CsA binding pocket and Leu171 to Trp at the C terminus, presents two complexes per asymmetric unit in the orthorhombic space group P2 12 12. This refined to an R-work of 0.18 and R-free 0.21. The mutations were identified from the crystallographic analysis and the C-terminal alteration helps to explain the different crystal forms obtained. PfCyP19 shares approximately 61 % sequence identity with human cyclophilin A (hCyPA) and the structures are similar, consisting of an eight-stranded antiparallel β-barrel core capped by two α-helices. The fold creates a hydrophobic active-site, the floor of which is formed by side-chains of residues from four antiparallel β-strands and the walls from loops and turns. We identified C—H…O hydrogen bonds between the drug and protein that may be an important feature of cyclophilins and suggest a general mode of interaction between hydrophobic molecules. Comparisons with cyclophilin-dipeptide complexes suggests that a specific C—H…O hydrogen bonding interaction may contribute to ligand binding. Residues Ser106, His99 and Asp130, located close to the active site and conserved in most cyclophilins, are arranged in a manner reminiscent of a serine protease catalytic triad. A Ser106Ala mutant was engineered to test the hypothesis that this triad contributes to CyP function. Mutant and wild-type enzymes were found to have similar catalytic properties.

The X-ray three-dimensional structure of avidin

Avidin is a basic, highly stable, homotetrameric protein, isolated from bird egg-white, binding up to four molecules of d-biotin with extremely high affinity ( K d∼10 −15 M). The protein has been the object of different crystallographic investigations. In all the crystal structures, the four avidin subunits display almost exact 222 symmetry. Each avidin chain (128 amino acids) is arranged in a eight-stranded antiparallel β-barrel, whose inner region defines the d-biotin binding site. The molecular bases of d-biotin affinity can be recognised in a fairly rigid binding site, which is sterically complementary to the shape and polarity of the incoming vitamin, and is readily accessible in the apoprotein structure. Avidin displays remarkable structural and functional relationships to the acidic protein sretpavidin, isolated from Streptomyces avidinii.

The solution structure and dynamics of human neutrophil gelatinase-associated lipocalin

Human neutrophil gelatinase-associated lipocalin (HNGAL) is a member of the lipocalin family of extracellular proteins that function as transporters of small, hydrophobic molecules. HNGAL, a component of human blood granulocytes, binds bacterially derived formyl peptides that act as chemotactic agents and induce leukocyte granule discharge. HNGAL also forms a complex with the proenzyme form of matrix metalloproteinase-9 (pro-MMP-9, or progelatinase B) via an intermolecular disulphide bridge. This association allows the subsequent formation of ternary and quaternary metalloproteinase/inhibitor complexes that vary greatly in their metalloproteinase activities. The structure and dynamics of apo-HNGAL have been determined by NMR spectroscopy. Simulated annealing calculations yielded a set of 20 convergent structures with an average backbone RMSD from mean coordinate positions of 0.79(±0.13) Å over secondary structure elements. The overall rotational correlation time (13.3 ns) derived from 15N relaxation data is consistent with a monomeric protein of the size of HNGAL (179 residues) under the experimental conditions (1.4 mM protein, pH 6.0, 24.5 °C). The structure features an eight-stranded antiparallel β-barrel, typical of the lipocalin family. One end of the barrel is open, providing access to the binding site within the barrel cavity, while the other is closed by a short 310-helix. The free cysteine residue required for association with pro-MMP-9 lies in an inter-strand loop at the closed end of the barrel. The structure provides a detailed model of the ligand-binding site and has led to the proposal of a site for pro-MMP-9 association. Dynamic data correlate well with structural features, which has allowed us to investigate a mechanism by which a cell-surface receptor might distinguish between apo and holo-HNGAL through conformational changes at the open end of the barrel.

Membrane assembly of the Escherichia coli outer membrane protein OmpA: exploring sequence constraints on transmembrane β-strands

The eight-stranded antiparallel β-barrel domain of the OmpA protein from Escherichia coli serves as a paradigm for the study of membrane assembly of integral β-structured membrane proteins. Previous studies have shown that neither the periplasmic turns nor the surface-exposed loops contain topogenic information. Consequently, the question of whether any structural constraint is imposed onto individual transmembrane β-strands is now addressed. To this end, amino acid sequences of β-strands 4, 6 and 8 were randomized. In vivo membrane assembly of mutant proteins was assayed and 288 variants were sequenced. Three parameters were found to be important for efficient membrane assembly. (i) At least four of five randomized residues with side-chains pointing towards the lipid bilayer must be hydrophobic and none of the three central residues must be charged. (ii) Side-chains pointing into the β-barrel interior must not be enlarged too much, possibly because of packing constraints. (iii) Proline residues are, in general, hardly tolerated in the transmembrane β-strands.

The NMR solution conformation of unligated human cyclophilin A

The nuclear magnetic resonance (NMR) solution structure of free, unligated cyclophilin A (CypA), which is an 18 kDa protein from human T-lymphocytes that was expressed in Escherichia coli for the present study, was determined using multidimensional heteronuclear NMR techniques. Sequence-specific resonance assignments for 99.5% of all backbone amide protons and non-labile hydrogen atoms provided the basis for collection of an input of 4101 nuclear Overhauser enhancement (NOE) upper distance constraints and 371 dihedral angle constraints for distance geometry calculations and energy minimization with the programs DIANA and OPAL. The average RMSD values of the 20 best energy-refined NMR conformers relative to the mean coordinates are 0.49 Å for the backbone atoms and 0.88 Å for all heavy atoms of residues 2 to 165. The molecular architecture includes an eight-stranded antiparallel β-barrel that is closed by two amphipathic α-helices. Detailed comparisons with the crystal structure of free CypA revealed subtle but significant conformational differences that can in most cases be related to lattice contacts in the crystal structure. 15N spin relaxation times and NMR lineshape analyses for CypA in the free form and complexed with cyclosporin A (CsA) revealed transitions of polypeptide loops surrounding the ligand-binding site from locally flexible conformations in the free protein, some of which include well-defined conformational equilibria, to well-defined spatial arrangements in the CypA-CsA complex. Compared to the crystal structure of free CypA, where the ligand-binding area is extensively involved in lattice contacts, the NMR structure presents a highly relevant reference for studies of changes in structure and internal mobility of the binding pocket upon ligand binding, and possible consequences of this conformational variability for calcineurin recognition by the CypA-CsA complex.

Crystal Structure of a Complex BetweenSerratia marcescensMetallo-protease and an Inhibitor fromErwinia chrysanthemi

The crystal structure of the complex between the 50 kDa metallo-endo-proteinase fromSerratia marcescens(SMP), a member of the metzincin superfamily, and an inhibitor fromErwinia chrysanthemi (Inh)was solved by molecular replacement using the known structure of SMP, and refined at 2.30 Å resolution to a crystallographicR-factor of 0.195. TheE. chrysanthemiinhibitor folds into a compact eight-stranded antiparallel β-barrel of simple up-down topology such as is found for members of the retinol binding protein family. It mainly interacts with the proteaseviaits five N-terminal residues, which insert into the active site cleft, occupying the S′ sites. The first N-terminal residue, SerI1, is partially cleaved off by the protease, while SerI2 makes a hydrogen bond with the catalytically active glutamic acid, Glu177, of the protease. Further interactions are made between one face of the inhibitor formed by the strands s3, s4 and s5 and the protease segment 218 to 228, which is located immediately after the characteristic “Met-turn” of the metzincins.

The Structure of Human Parvovirus B19 at 8 Å; Resolution

Empty capsids of the human parvovirus B19, self-assembled in a baculovirus expression system, have been crystallized in a cubic space group P2,3 with a = 362 Å. In spite of extensive purifications, the crystals diffract X-rays to only 8.0 Å resolution. Diffraction data were collected using oscillation photography with synchrotron radiation. The orientations of the particles in the unit cell were determined with a self-rotation function and their positions were obtained with an R-factor search using the known homologous canine parvovirus (CPV) structure. The resultant phases were improved by electron density averaging and solvent flattening to include all the terms between 23.0 and 8.0 Å resolution. The central eight-stranded antiparallel β-barrel, common to many viruses, is situated similarly in B19 with respect to the icosaheral symmetry axes to that observed for feline panleukopenia virus (FPV) and CPV. However, the surface structure of B19 is significantly different from the other known parvoviruses. The most striking difference is that B19 lacks the prominent spikes on the threefold icosahedral axes observed in FPV and CPV. This spike region contains residues involved in host recognition and antigenicity for the latter viruses, showing that there are major differences between subgroups of autonomous parvoviruses.

The Canine Parvovirus Empty Capsid Structure

The structure of empty canine parvovirus capsids shows that residues 37 to the carboxy-terminal residue 584 (VP2 numbering) are ordered in each of the 60 subunits. The central structural motif of each subunit is the eight-stranded antiparallel β-barrel that has been found in many other virus structures. Five β-hairpin turns form β-cylindrical structure at each icosahedral 5-fold axis. The N-terminal glycine-rich sequence can be accommodated within this cylinder without excessive steric hindrance, consistent with the electron density distribution. By far the largest conformational differences between the full and empty virus were found in the region where some ordered DNA has been observed to bind in canine parvovirus full particles. Extensive interactions among 3-fold related subunits indicate that a trimeric subunit might be a viral assembly intermediate.

The structure of antiviral agents that inhibit uncoating when complexed with viral capsides

The tertiary structure of most icosahedral viral capsid proteins consists of an eight-stranded antiparallel β-barrel with a hydrophobic interior. In a group of picornaviruses, this hydrophobic pocket can be filled by suitable organic molecules, which stop viral uncoating after attachment and penetration into the host cell. The antiviral activity of these agents is probably due to increased rigidity of the capsid protein, thereby inhibiting disassembly. The hydrophobic pocket may be an essential functional component of the protein and may have been conserved in the evolution of many viruses from a common precursor. Since eight-stranded antiparallel β-barrels, with a topology as in viral capsid proteins, are not generally found in other proteins involved in cell metabolism, antiviral agents that bind in the interior of viral capsid proteins are likely to be more virus-specific and less cytotoxic. Furthermore, the greatest conservation of viral capsid proteins occurs within this pocket, whereas the least conserved part is the antigenic exterior. Thus, compounds that bind to such a pocket are likely to be effective against a broader group of serologically distinct viruses. Discovery of antiviral agents of this type depends on designing compounds that can enter and fit snugly into the hydrophobic pocket of a particular viral capsid protein.