The X-ray crystal structure of human endothelin 1, a polypeptide hormone regulator of blood pressure.

Human endothelin is a 21-amino-acid polypeptide, constrained by two intra-chain disulfide bridges, that is made by endothelial cells. It is the most potent vasoconstrictor in the body and is crucially important in the regulation of blood pressure. It plays a major role in a host of medical conditions, including hypertension, diabetes, stroke and cancer. Endothelin was crystallized 28 years ago in the putative space group P6122, but the structure was never successfully solved by X-ray diffraction. Using X-ray diffraction data from 1992, the structure has now been solved. Assuming a unit cell belonging to space group P61 and a twin fraction of 0.28, a solution emerged with two, almost identical, closely associated molecules in the asymmetric unit. Although the data extended to beyond 1.8 Šresolution, a model containing 25 waters was refined to 1.85 Šresolution with an R of 0.216 and an Rfree of 0.284. The disulfide-constrained `core' of the molecule, amino-acid residues 1-15, has a main-chain conformation that is essentially the same as endothelin when bound to its receptor, but many side-chain rotamers are different. The carboxy-terminal `tail' comprising amino-acid residues 16-21 is extended as when receptor-bound, but it exhibits a different conformation with respect to the `core'. The dimer that comprises the asymmetric unit is maintained almost exclusively by hydrophobic interactions and may be stable in an aqueous medium.

Validation of Protein-Ligand Crystal Structure Models: Small Molecule and Peptide Ligands.

Models of target proteins in complex with small molecule ligands or peptide ligands are of significant interest to the biomedical research community. Structure-guided lead discovery and structure-based drug design make extensive use of such models. The bound ligands comprise only a small fraction of the total X-ray scattering mass, and therefore particular care must be taken to properly validate the atomic model of the ligand as experimental data can often be scarce. The ligand model must be validated against both the primary experimental data and the local environment, specifically: (1) the primary evidence in the form of the electron density, (2) examined for reasonable stereochemistry, and (3) the chemical plausibility of the binding interactions must be inspected. Tools that assist the researcher in the validation process are presented.

The utility of comparative models and the local model quality for protein crystal structure determination by Molecular Replacement.

BACKGROUND: Computational models of protein structures were proved to be useful as search models in Molecular Replacement (MR), a common method to solve the phase problem faced by macromolecular crystallography. The success of MR depends on the accuracy of a search model. Unfortunately, this parameter remains unknown until the final structure of the target protein is determined. During the last few years, several Model Quality Assessment Programs (MQAPs) that predict the local accuracy of theoretical models have been developed. In this article, we analyze whether the application of MQAPs improves the utility of theoretical models in MR. RESULTS: For our dataset of 615 search models, the real local accuracy of a model increases the MR success ratio by 101% compared to corresponding polyalanine templates. On the contrary, when local model quality is not utilized in MR, the computational models solved only 4.5% more MR searches than polyalanine templates. For the same dataset of the 615 models, a workflow combining MR with predicted local accuracy of a model found 45% more correct solution than polyalanine templates. To predict such accuracy MetaMQAPclust, a "clustering MQAP" was used. CONCLUSIONS: Using comparative models only marginally increases the MR success ratio in comparison to polyalanine structures of templates. However, the situation changes dramatically once comparative models are used together with their predicted local accuracy. A new functionality was added to the GeneSilico Fold Prediction Metaserver in order to build models that are more useful for MR searches. Additionally, we have developed a simple method, AmIgoMR (Am I good for MR?), to predict if an MR search with a template-based model for a given template is likely to find the correct solution.

Radiation damage in protein crystals is reduced with a micron-sized X-ray beam.

Radiation damage is a major limitation in crystallography of biological macromolecules, even for cryocooled samples, and is particularly acute in microdiffraction. For the X-ray energies most commonly used for protein crystallography at synchrotron sources, photoelectrons are the predominant source of radiation damage. If the beam size is small relative to the photoelectron path length, then the photoelectron may escape the beam footprint, resulting in less damage in the illuminated volume. Thus, it may be possible to exploit this phenomenon to reduce radiation-induced damage during data measurement for techniques such as diffraction, spectroscopy, and imaging that use X-rays to probe both crystalline and noncrystalline biological samples. In a systematic and direct experimental demonstration of reduced radiation damage in protein crystals with small beams, damage was measured as a function of micron-sized X-ray beams of decreasing dimensions. The damage rate normalized for dose was reduced by a factor of three from the largest (15.6 µm) to the smallest (0.84 µm) X-ray beam used. Radiation-induced damage to protein crystals was also mapped parallel and perpendicular to the polarization direction of an incident 1-µm X-ray beam. Damage was greatest at the beam center and decreased monotonically to zero at a distance of about 4 µm, establishing the range of photoelectrons. The observed damage is less anisotropic than photoelectron emission probability, consistent with photoelectron trajectory simulations. These experimental results provide the basis for data collection protocols to mitigate with micron-sized X-ray beams the effects of radiation damage.

De novo sulfur SAD phasing of the lysosomal 66.3 kDa protein from mouse.

The 66.3 kDa protein from mouse is a soluble protein of the lysosomal matrix. It is synthesized as a glycosylated 75 kDa preproprotein which is further processed into 28 and 40 kDa fragments. Despite bioinformatics approaches and molecular characterization of the 66.3 kDa protein, the mode of its maturation as well as its physiological function remained unknown. Therefore, it was decided to tackle this question by means of X-ray crystallography. After expression in a human fibrosarcoma cell line, the C-terminally His-tagged single-chain 66.3 kDa variant and the double-chain form consisting of a 28 kDa fragment and a 40 kDa fragment were purified to homogeneity but could not be separated during the purification procedure. This mixture was therefore used for crystallization. Single crystals were obtained and the structure of the 66.3 kDa protein was solved by means of sulfur SAD phasing using data collected at a wavelength of 1.9 A on the BESSY beamline BL14.2 of Freie Universität Berlin. Based on the anomalous signal, a 22-atom substructure comprising 21 intrinsic S atoms and one Xe atom with very low occupancy was found and refined at a resolution of 2.4 A using the programs SHELXC/D and SHARP. Density modification using SOLOMON and DM resulted in a high-quality electron-density map, enabling automatic model building with ARP/wARP. The initial model contained 85% of the amino-acid residues expected to be present in the asymmetric unit of the crystal. Subsequently, the model was completed and refined to an R(free) factor of 19.8%. The contribution of the single Xe atom to the anomalous signal was analyzed in comparison to that of the S atoms and was found to be negligible. This work should encourage the use of the weak anomalous scattering of intrinsic S atoms in SAD phasing, especially for proteins, which require both expensive and time-consuming expression and purification procedures, preventing extensive screening of heavy-atom crystal soaks.

Crystal structures of the ribosome in complex with release factors RF1 and RF2 bound to a cognate stop codon.

During protein synthesis, translational release factors catalyze the release of the polypeptide chain when a stop codon on the mRNA reaches the A site of the ribosome. The detailed mechanism of this process is currently unknown. We present here the crystal structures of the ribosome from Thermus thermophilus with RF1 and RF2 bound to their cognate stop codons, at resolutions of 5.9 Angstrom and 6.7 Angstrom, respectively. The structures reveal details of interactions of the factors with the ribosome and mRNA, including elements previously implicated in decoding and peptide release. They also shed light on conformational changes both in the factors and in the ribosome during termination. Differences seen in the interaction of RF1 and RF2 with the L11 region of the ribosome allow us to rationalize previous biochemical data. Finally, this work demonstrates the feasibility of crystallizing ribosomes with bound factors at a defined state along the translational pathway.

Crystallographic structure of the T=1 particle of brome mosaic virus.

T=1 icosahedral particles of amino terminally truncated brome mosaic virus (BMV) protein were created by treatment of the wild-type T=3 virus with 1M CaCl2 and crystallized from sodium malonate. Diffraction data were collected from frozen crystals to beyond 2.9 A resolution and the structure determined by molecular replacement and phase extension. The particles are composed of pentameric capsomeres from the wild-type virions which have reoriented with respect to the original particle pentameric axes by rotations of 37 degrees , and formed tenuous interactions with one another, principally through conformationally altered C-terminal polypeptides. Otherwise, the pentamers are virtually superimposable upon those of the original T=3 BMV particles. The T=1 particles, in the crystals, are not perfect icosahedra, but deviate slightly from exact symmetry, possibly due to packing interactions. This suggests that the T=1 particles are deformable, which is consistent with the loose arrangement of pentamers and latticework of holes that penetrate the surface. Atomic force microscopy showed that the T=3 to T=1 transition could occur by shedding of hexameric capsomeres and restructuring of remaining pentamers accompanied by direct condensation. Knowledge of the structures of the BMV wild-type and T=1 particles now permit us to propose a tentative model for that process. A comparison of the BMV T=1 particles was made with the reassembled T=1 particles produced from the coat protein of trypsin treated alfalfa mosaic virus (AlMV), another bromovirus. There is little resemblance between the two particles. The BMV particle, with a maximum diameter of 195 A, is made from distinctive pentameric capsomeres with large holes along the 3-fold axis, while the AlMV particle, of approximate maximum diameter 220 A, has subunits closely packed around the 3-fold axis, large holes along the 5-fold axis, and few contacts within pentamers. In both particles crucial linkages are made about icosahedral dyads.

Why zinc fingers prefer zinc: ligand-field symmetry and the hidden thermodynamics of metal ion selectivity.

The zinc finger, a motif of protein-nucleic acid recognition broadly conserved among eukaryotes, is a globular minidomain containing a tetrahedral metal-binding site. Preferential coordination of Zn(2+) (relative to Co(2+)) is proposed to reflect differences in ligand-field stabilization energies (LFSEs) due to complete or incomplete occupancy of d orbitals. LFSE predicts that the preference for Zn(2+) should be purely enthalpic in accord with calorimetric studies of a high-affinity consensus peptide (CP-1; Blasie, C. A., and Berg, J. (2002) Biochemistry 41, 15068-73). Despite its elegance, the general predominance of LFSE is unclear as (i) the magnitude by which CP-1 prefers Zn(2+) is greater than that expected and (ii) the analogous metal ion selectivity of a zinc metalloenzyme (carbonic anhydrase) is driven by changes in entropy rather than enthalpy. Because CP-1 was designed to optimize zinc binding, we have investigated the NMR structure and metal ion selectivity of a natural finger of lower stability derived from human tumor-suppressor protein WT1. Raman spectroscopy suggests that the structure of the WT1 domain is unaffected by interchange of Zn(2+) and Co(2+). As in CP-1, preferential binding of Zn(2+) (relative to Co(2+)) is driven predominantly by differences in enthalpy, but in this case the enthalpic advantage is less than that predicted by LFSE. A theoretical framework is presented to define the relationship between LFSE and other thermodynamic factors, such as metal ion electroaffinities, enthalpies of hydration, and the topography of the underlying folding landscape. The contribution of environmental coupling to entropy-enthalpy compensation is delineated in a formal thermodynamic cycle. Together, these considerations indicate that LFSE provides an important but incomplete description of the stringency and thermodynamic origin of metal-ion selectivity.

Backbone-backbone geometry of tertiary contacts between alpha-helices.

Knowledge about how secondary-structure elements combine together to form the tertiary structure is crucial for understanding protein folding. We have examined packing solutions for alpha-helices by performing a crystal survey of the underlying backbone-backbone inter-geometry of tertiary contacts. The information content is different from, and complementary to, the results of side-chain to side-chain crystal surveys and studies of helix-helix-crossing angles. Six geometry descriptors were recorded from each tertiary contact in nonredundant data sets of globular and transmembrane proteins. The descriptors included distances, angles, and dihedral angles that together describe completely the underlying geometry of each contact. From the results it is possible to identify differences in the geometry requirements of tertiary contacts between alpha-helices in general and transmembrane alpha-helices. The differences become more apparent when the correlation between the different geometry descriptors is analyzed. The results are compared with those of other types of secondary structure. Finally, an investigation of how the geometry of tertiary contacts changes with the amino-acid types involved in the contact is performed using multivariate techniques. The results of this study provide a well-defined overview of the underlying structural framework of tertiary contacts between alpha-helices, in both globular and TM environments, that will have valuable implications for predicting protein tertiary structure.

MRSAD: using anomalous dispersion from S atoms collected at Cu Kalpha wavelength in molecular-replacement structure determination.

The use of single-wavelength anomalous dispersion (SAD) from S atoms collected in-house to overcome model bias in molecular-replacement (MR) structure determination is demonstrated. The test case considered is a P6(5)22 anti-ssDNA Fab crystal with a theoretical anomalous signal of 0.8% and a diffraction limit of 2.3 A, from which a 360 degrees, 39-fold redundant data set was collected. A nearly complete anomalous scatterer substructure could be quickly built from anomalous difference Fourier analysis based on phases from a full or partial MR solution. The resulting SAD phases were improved with density modification and used to calculate an unbiased electron-density map that could be used for model building. This map displayed clear and continuous density for almost the entire main chain, as well as good density for most side chains. The favorable results obtained from this realistic test case suggest that anomalous differences from S atoms should be routinely collected and used in MR structure determination.

High-phasing-power lanthanide derivatives: taking advantage of ytterbium and lutetium for optimized anomalous diffraction experiments using synchrotron radiation.

Ytterbium and lutetium are well suited for optimized anomalous diffraction experiments using synchrotron radiation. Therefore, two lanthanide complexes Yb-HPDO3A and Lu-HPDO3A have been produced that are similar to the Gd-HPDO3A complex already known to give good derivative crystals. Derivative crystals of hen egg-white lysozyme were obtained by co-crystallization using 100 mM solutions of each lanthanide complex. De novo phasing has been carried out using single-wavelength anomalous diffraction on data sets collected on each derivative crystal at the L(III) absorption edge of the corresponding lanthanide (ff" = 28 e(-)). A third data set was collected on a Lu-HPDO3A derivative crystal at the Se K absorption edge with f"(Lu) = 10 e(-). The structures were refined and compared with the known structure of the Gd-HPDO3A lysozyme derivative. The quality of the experimental electron-density maps allows easy model building. With L(III) absorption edges at shorter wavelengths than the gadolinium absorption edge, lutetium and ytterbium, when chelated by a ligand such as HPDO3A, form lanthanide complexes that are especially interesting for synchrotron-radiation experiments in structural biology.