Multiple-wavelength Anomalous Dispersion Data Research Articles

Purification, crystallization and preliminary X-ray analysis of β-glucosidase fromKluyveromyces marxianusNBRC1777

The intracellular beta-glucosidase from Kluyveromyces marxianus NBRC1777 (KmBglI) belongs to glycoside hydrolase family 3 and has a unique domain architecture. Selenomethionine-labelled KmBglI was purified and crystallized by the hanging-drop vapour-diffusion method using the purified enzyme at 30 mg ml(-1), 0.04 M potassium dihydrogen phosphate pH 5.1, 16%(w/v) PEG 8000 and 20%(v/v) glycerol. The crystal belonged to space group C2, with unitcell parameters a = 245.8, b = 148.7, c = 119.9 angstrom, beta = 112.9 degrees. Multiple-wavelength anomalous dispersion data were collected at 2.4 and 2.5 angstrom resolution. A tetramer was assumed to be present in the asymmetric unit, which gave a Matthews coefficient of 2.6 angstrom(3) Da(-1).

First Structure of a Eukaryotic Phosphohistidine Phosphatase

Phosphatases are a diverse group of enzymes that regulate numerous cellular processes. Much of what is known relates to the tyrosine, threonine, and serine phosphatases, whereas the histidine phosphatases have not been studied as much. The structure of phosphohistidine phosphatase (PHPT1), the first identified eukaryotic-protein histidine phosphatase, has been determined to a resolution of 1.9A using multiple-wavelength anomalous dispersion methods. This enzyme can dephosphorylate a variety of proteins (e.g. ATP-citrate lyase and the beta-subunit of G proteins). A putative active site has been identified by its electrostatic character, ion binding, and conserved protein residues. Histidine 53 is proposed to play a major role in histidine dephosphorylation based on these observations and previous mutational studies. Models of peptide binding are discussed to suggest possible mechanisms for substrate recognition.

Activation of the LicT Transcriptional Antiterminator Involves a Domain Swing/Lock Mechanism Provoking Massive Structural Changes

The transcriptional antiterminator protein LicT regulates the expression of Bacillus subtilis operons involved in beta-glucoside metabolism. It consists of an N-terminal RNA-binding domain (co-antiterminator (CAT)) and two phosphorylatable phosphotransferase system regulation domains (PRD1 and PRD2). In the activated state, each PRD forms a dimeric unit with the phosphorylation sites totally buried at the dimer interface. Here we present the 1.95 A resolution structure of the inactive LicT PRDs as well as the molecular solution structure of the full-length protein deduced from small angle x-ray scattering. Comparison of native (inactive) and mutant (constitutively active) PRD crystal structures shows massive tertiary and quaternary rearrangements of the entire regulatory domain. In the inactive state, a wide swing movement of PRD2 results in dimer opening and brings the phosphorylation sites to the protein surface. This movement is accompanied by additional structural rearrangements of both the PRD1-PRD1 ' interface and the CAT-PRD1 linker. Small angle x-ray scattering experiments indicate that the amplitude of the PRD2 swing might even be wider in solution than in the crystals. Our results suggest that PRD2 is highly mobile in the native protein, whereas it is locked upon activation by phosphorylation.

The Structure of the AXH Domain of Spinocerebellar Ataxin-1

Spinocerebellar ataxia type 1 is a late-onset neurodegenerative disease caused by the expansion of a CAG triplet repeat in the SCA1 gene. This results in the lengthening of a polyglutamine tract in the gene product ataxin-1. This produces a toxic gain of function that results in specific neuronal death. A region in ataxin-1, the AXH domain, exhibits significant sequence similarity to the transcription factor HBP1. This region of the protein has been implicated in RNA binding and self-association. We have determined the crystal structure of the AXH domain of ataxin-1. The AXH domain is dimeric and contains an OB-fold, a structural motif found in many oligonucleotide-binding proteins, supporting its proposed role in RNA binding. By structure comparison with other proteins that contain an OB-fold, a putative RNA-binding site has been identified. We also identified a cluster of charged surface residues that are well conserved among AXH domains. These residues may constitute a second ligand-binding surface, suggesting that all AXH domains interact with a common yet unidentified partner.

Crystallization and X-ray diffraction analysis of the sensor domain of the HemAT aerotactic receptor.

HemAT is a 432-amino-acid protein with two structural domains identified from Bacillus subtilis. It is responsible for sensing oxygen and delivering the signal to the downstream signal transduction cascade through a two-component system [Hou et al. (2000), Nature (London), 403, 540-544]. The consequence of such events is to change the flagellar movement and alter the swimming behavior of bacteria. To elucidate the molecular mechanism of oxygen sensing, the sensor domain of HemAT from B. subtilis was cloned, expressed and crystallized. Multiple-wavelength anomalous dispersion (MAD) data were collected from the intrinsic anomalous scatterer, iron, using synchrotron radiation. Three-wavelength iron MAD data sets were collected to 2.8 A resolution. The native data set was collected to 2.15 A resolution. Initial crystallographic analysis revealed the crystals to belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 50.00, b = 80.12, c = 85.95 A. There is one dimer in the asymmetric unit, with 40% solvent content. Structure determination using MAD methods and model building are currently under way.

Use of multiple anomalous dispersion to phase highly merohedrally twinned crystals of interleukin-1beta.

The crystal structure at 1.54 A resolution of a double mutant of interleukin-1beta (F42W/W120F), a cytokine secreted by macrophages, was determined by multiple-wavelength anomalous dispersion (MAD) using data from highly twinned selenomethionine-modified crystals. The space group is P4(3), with unit-cell parameters a = b = 53.9, c = 77.4 A. Self-rotation function analysis and various intensity statistics revealed the presence of merohedral twinning in crystals of both the native (twinning fraction alpha approximately 0.35) and SeMet (alpha approximately 0.40) forms. Structure determination and refinement are discussed with emphasis on the possible reasons for successful phasing using untreated twinned MAD data.

Crystallization of a nickel-containing superoxide dismutase and preliminary phase determination by MAD at the Ni K edge.

Superoxide dismutases are metalloenzymes which catalyse the disproportion of superoxide radicals and thus play an important role in the protection of biomolecules from oxidative damage. Redox-active metal ions known to act as the catalytic centre of these enzymes are Cu, Mn or Fe. Recently, enzymes containing Ni have been found in various Streptomyces species, introducing a fourth type of metal ion to the superoxide dismutase family. NiSOD has been crystallized for the purpose of structure determination by X-ray crystallography using Ni as an anomalous scatterer in multiple-wavelength anomalous dispersion (MAD) experiments. Two crystal forms belonging to space group P2(1)2(1)2(1) and one belonging to space group R3 were obtained using ammonium sulfate as a precipitant. Patterson maps of one of the orthorhombic forms revealed the presence of pseudo-translation, which could be removed for the other orthorhombic form by using 10% glycerol in its crystallization conditions. In addition, this reduced the unit cell by half. Phase information which led to interpretable electron-density maps was derived from MAD data to 2.0 A resolution after density modification applying solvent flattening, histogram matching and NCS averaging. Phases were extended to 1.68 A resolution with a data set collected at a wavelength of 1 A. Model building based on the resulting electron-density maps is in progress.

The method of joint probability distribution functions applied to MAD techniques. The centric case

Traditional probabilistic approaches consider MAD (multiple-wavelength anomalous-dispersion) data as a special MIR (multiple isomorphous replacement) case. The rigorous method of the joint probability distribution functions has been applied to solve the phase problem, with the assumption that the anomalous scatterers' substructure is a priori known. The probabilistic approach is able to handle measurement errors: it has been applied to symmetry-restricted phases and provides simple and efficient formulas.

Trichromatic Concept at SPring-8 RIKEN Beamline I.

SPring-8 RIKEN beamline I has been designed and developed for structural biology research by the Institute of Physical and Chemical Research (RIKEN). The beamline consists of two experimental stations for protein crystallography and small-angle X-ray scattering. Both types of experiments can be carried out simultaneously, with dichromatic synchrotron radiation emitted from two coaxial undulators with vertical polarization. The branched beams are generated by a transparent diamond crystal. With synchrotron radiation, the multiple-wavelength anomalous-dispersion (MAD) method, which gives phases from a single anomalous scatterer, has been developed. Anomalous scattering contributes a small proportion of the diffraction intensity so that the accuracy of intensity data is important. The protein crystallography branch of RIKEN beamline I has been designed based on a 'trichromatic concept' to optimize MAD data collection. This concept requires the quasi-simultaneous collection, by use of a 'trichromator', of three intensity data sets at three different wavelengths from a single protein crystal without changing any settings. The main feature of the concept is the minimization of systematic errors in the measurement of anomalous diffraction for the MAD method. Initial commissioning of the beamline has provided three different monochromated undulator beams, which were successfully observed on the phosphor screen located at the near end of the trichromator.

Crystal structure of troponin C in complex with troponin I fragment at 2.3-A resolution.

Troponin (Tn), the complex of three subunits (TnC, TnI, and TnT), plays a key role in Ca2+-dependent regulation of muscle contraction. To elucidate the interactions between the Tn subunits and the conformation of TnC in the Tn complex, we have determined the crystal structure of TnC (two Ca2+ bound state) in complex with the N-terminal fragment of TnI (TnI1-47). The structure was solved by the single isomorphous replacement method in combination with multiple wavelength anomalous dispersion data. The refinement converged to a crystallographic R factor of 22.2% (Rfree = 32.6%). The central, connecting alpha-helix observed in the structure of uncomplexed TnC (TnCfree) is unwound at the center (residues Ala-87, Lys-88, Gly-89, Lys-90, and Ser-91) and bent by 90 degrees. As a result, TnC in the complex has a compact globular shape with direct interactions between the N- and C-terminal lobes, in contrast to the elongated dumb-bell shaped molecule of uncomplexed TnC. The 31-residue long TnI1-47 alpha-helix stretches on the surface of TnC and stabilizes its compact conformation by multiple contacts with both TnC lobes. The amphiphilic C-end of the TnI1-47 alpha-helix is bound in the hydrophobic pocket of the TnC C-lobe through 38 van der Waals interactions. The results indicate the major difference between Ca2+ receptors integrated with the other proteins (TnC in Tn) and isolated in the cytosol (calmodulin). The TnC/TnI1-47 structure implies a mechanism of how Tn regulates the muscle contraction and suggests a unique alpha-helical regulatory TnI segment, which binds to the N-lobe of TnC in its Ca2+ bound conformation.

A general phasing algorithm for multiple MAD and MIR data.

A phasing algorithm is presented for combining multiple wavelength anomalous dispersion (MAD) data from multiple types of anomalous scatterers, either in the same or in different derivative crystals, as well as for combining MAD data with multiple isomorphous replacement (MIR) data from different derivative crystals. A heavy-atom phasing and refinement program originally written by Rossmann [(1967) HATOMLSQ program, Purdue University, West Lafayette, Indiana, USA] has been modified to refine the parameters that define the anomalous and isomorphous scatterers and to determine protein phases by using all MAD and MIR derivatives simultaneously. The technique allows for appropriate weighting of every data set, including the native data, which contains neither an anomalous nor an isomorphous component. This method is a generalization of currently used heavy-atom methods. Numerical tests are presented for different experimental scenarios, including a double MAD experiment on the same crystal (diffraction data at two absorption edges), combination of two MAD experiments on different crystals, and combination of MAD data with MIR data from multiple crystals. An appendix shows how the Karle equations used in MAD phasing can be reformulated as a particular case of this algorithm.