Craig M. Ogata

Structural analysis of substrate binding by the molecular chaperone DnaK.

DnaK and other members of the 70-kilodalton heat-shock protein (hsp70) family promote protein folding, interaction, and translocation, both constitutively and in response to stress, by binding to unfolded polypeptide segments. These proteins have two functional units: a substrate-binding portion binds the polypeptide, and an adenosine triphosphatase portion facilitates substrate exchange. The crystal structure of a peptide complex with the substrate-binding unit of DnaK has now been determined at 2.0 Å resolution. The structure consists of a β-sandwich subdomain followed by α-helical segments. The peptide is bound to DnaK in an extended conformation through a channel defined by loops from the β sandwich. An α-helical domain stabilizes the complex, but does not contact the peptide directly. This domain is rotated in the molecules of a second crystal lattice, which suggests a model of conformation-dependent substrate binding that features a latch mechanism for maintaining long lifetime complexes.

[28] Phase determination from multiwavelength anomalous diffraction measurements

Publisher Summary This chapter discusses phase determination from multiwavelength anomalous diffraction (MAD) measurements. The MAD approach to macromolecular structure determination has potential advantages for accuracy and convenience in phase evaluation. Isomorphism is intrinsically perfect; an algebraically exact analysis is possible; relative scattering strength and phasing power increase with scattering angle; and all required diffraction data can be measured from a single crystal. The virtual immortalization of crystals through freezing makes these advantages real and practical. Although many of the procedures in MAD phasing are in common with crystallographic practice, there are also a number of practical steps unique to this methodology. The chapter discusses the basic foundations of the method, presents the design and execution of experiments, and describes the steps and alternatives for data analysis.

Crystal structure of a complement control protein that regulates both pathways of complement activation and binds heparan sulfate proteoglycans

Vaccinia virus complement control protein (VCP) inhibits both pathways of complement activation through binding the third and fourth components. A homolog of mammalian regulators of complement activation, its ability to bind heparin endows VCP with additional activities of significance to viral infectivity. The structure of VCP reveals a highly extended molecule with a putative heparin recognition site at its C-terminal end. A second cluster of positive charges provides a possibly overlapping binding site for both heparin and complement components. Experiments suggested by the structure indicate that VCP can bind heparin and control complement simultaneously. This, the structure of any intact regulator of complement activation, along with attendant functional insights, will stimulate the design of new therapeutic inhibitors of complement.

Crystal Structure of Recombinant Human Interleukin-22

Interleukin-22 (IL-10-related T cell-derived inducible factor/IL-TIF/IL-22) is a novel cytokine belonging to the IL-10 family. Recombinant human IL-22 (hIL-22) was found to activate the signal transducers and activators of transcription factors 1 and 3 as well as acute phase reactants in several hepatoma cell lines, suggesting its involvement in the inflammatory response. The crystallographic structure of recombinant hIL-22 has been solved at 2.0 A resolution using the SIRAS method. Contrary to IL-10, the hIL-22 dimer does not present an interpenetration of the secondary-structure elements belonging to the two distinct polypeptide chains but results from interface interactions between monomers. Structural differences between these two cytokines, revealed by the crystallographic studies, clearly indicate that, while a homodimer of IL-10 is required for signaling, hIL-22 most probably interacts with its receptor as a monomer.

Crystal structure of a sweet tasting protein thaumatin I, at 1.65 Å resolution

The crystal structure of thaumatin I, a potently sweet protein isolated from the fruits of the West African shrub, Thaumatococcus danielli Benth, has been refined at a resolution better than 1.65 A using a combination of energy minimization and stereochemically restrained least-squares methods. The final model consists of all 207 amino acids, 28 alternate amino acid conformers and 236 waters, with a crystallographic R-factor of 0.145 for 19,877 reflections having F > 4 sigma F between 10.0 A and 1.65 A (R = 0.167 for all 24,022 reflections). The model has good stereochemistry, with root-mean-square deviations from ideal values for bond and angle distances of 0.014 A and 0.029 A, respectively. The estimated root-mean-square co-ordinate error is 0.15 A. The current model confirms the previously reported 3.1 A C alpha trace in both main chain connectivity and disulfide topology, including two disulfide bonds, that differed from the earlier reported biochemical determination. The structure contains three domains. The core of the molecule consists of an eleven-stranded, flattened beta-sandwich folded into two Greek key motifs. All beta-strands in this sandwich are antiparallel except the parallel N-terminal and the C-terminal strands. The average hydrogen bond length in this sandwich is 2.89 A, with an angle of 155.1 degrees. Two beta-bulges are found in one of the sheets. The second domain consists of two beta-strands forming a beta-ribbon and connected by an omega-loop, and contains a proline residue in cis conformation. This structural motif folds back against the main sandwich to form a smaller sandwich-like structure. The third domain is a disulfide-rich region stretching away from the sandwich portion of the molecule. It contains one alpha-helix and three short helical fragments. Two of the helical segments are connected by an unusually sharp turn, stabilized by a disulfide bridge. One of the three disulfide bonds in this domain takes on two conformations.

MAD phasing grows up

Multiwavelength anomalous diffraction (MAD) phasing, which relies on synchrotron radiation, has grown from a novelty technique to a mainstream method that has been used to determine macromolecule structures of up to 200,000 Mr.

Crystal structures of two intensely sweet proteins

Abstract Three-dimensional structures of two intensely sweet proteins (thaumatin and monellin) have been determined by X-ray crystallographic methods. Despite their resemblance in taste, the two proteins have no significant similarities in their crystal structures or amino acid sequences; and yet the two proteins are immunologically cross-reactive.

Multiwavelength anomalous diffraction analysis at the M absorption edges of uranium

The multiwavelength anomalous diffraction (MAD) method for phase evaluation is now widely used in macromolecular crystallography. Successful MAD structure determinations have been carried out at the K or L absorption edges of a variety of elements. In this study, we investigate the anomalous scattering properties of uranium at its MIV (3.326 Å) and MV (3.490 Å) edge. Fluorescence spectra showed remarkably strong anomalous scattering at these edges (f′ = −70e, f′′ = 80e at the MIV edge and f′ = −90e, f′′ = 105e at the MV edge), many times higher than from any anomalous scatterers used previously for MAD phasing. However, the large scattering angles and high absorption at the low energies of these edges present some difficulties not found in typical crystallographic studies. We conducted test experiments at the MIV edge with crystals of porcine elastase derivatized with uranyl nitrate. A four-wavelength MAD data set complete to 3.2-Å Bragg spacings was collected from a single small frozen crystal. Analysis of the data yielded satisfactory phase information (average difference of 0ϕT − 0ϕA for replicated determinations is 32°) and produced an interpretable electron-density map. Our results demonstrate that it is practical to measure macromolecular diffraction data at these edges with current instrumentation and that phase information of good accuracy can be extracted from such experiments. We show that such experiments have potential for the phasing of very large macromolecular assemblages.

Structure of monellin refined to 2.3 a resolution in the orthorhombic crystal form.

The structure of orthorhombic crystals of monellin, a sweet protein extracted from African serendipity berries, has been solved by molecular replacement and refined to 2.3 A resolution. The final R factor was 0.150 for a model with excellent geometry. A monellin molecule consists of two peptides that are non-covalently bound, with chain A composed of three beta-strands interconnected by loop regions and chain B composed of two beta-strands interconnected by an alpha-helix. The N terminus of chain A is in close proximity to the C terminus of chain B. The two molecules in the asymmetric unit are related by a non-crystallographic twofold axis and form a dimer, similar to those previously observed in other crystal forms of both natural and single-chain monellin. The r.m.s, deviation between the Calpha atoms in the two independent molecules is 0.60 A, while the deviations from the individual molecules in the previously reported monoclinic crystals are 0.50-0.57 A. This result proves that the structure of monellin is not significantly influenced by crystal packing forces.