Multiwavelength Anomalous Diffraction Research Articles

The magic triangle goes MAD: experimental phasing with a bromine derivative

Experimental phasing is an essential technique for the solution of macromolecular structures. Since many heavy-atom ion soaks suffer from nonspecific binding, a novel class of compounds has been developed that combines heavy atoms with functional groups for binding to proteins. The phasing tool 5-amino-2,4,6-tribromoisophthalic acid (B3C) contains three functional groups (two carboxylate groups and one amino group) that interact with proteins via hydrogen bonds. Three Br atoms suitable for anomalous dispersion phasing are arranged in an equilateral triangle and are thus readily identified in the heavy-atom substructure. B3C was incorporated into proteinase K and a multiwavelength anomalous dispersion (MAD) experiment at the Br K edge was successfully carried out. Radiation damage to the bromine-carbon bond was investigated. A comparison with the phasing tool I3C that contains three I atoms for single-wavelength anomalous dispersion (SAD) phasing was also carried out.

The Structure of the Mammalian RNase H2 Complex Provides Insight into RNA·DNA Hybrid Processing to Prevent Immune Dysfunction

The mammalian RNase H2 ribonuclease complex has a critical function in nucleic acid metabolism to prevent immune activation with likely roles in processing of RNA primers in Okazaki fragments during DNA replication, in removing ribonucleotides misinserted by DNA polymerases, and in eliminating RNA.DNA hybrids during cell death. Mammalian RNase H2 is a heterotrimeric complex of the RNase H2A, RNase H2B, and RNase H2C proteins that are all required for proper function and activity. Mutations in the human RNase H2 genes cause Aicardi-Goutières syndrome. We have determined the crystal structure of the three-protein mouse RNase H2 enzyme complex to better understand the molecular basis of RNase H2 dysfunction in human autoimmunity. The structure reveals the intimately interwoven architecture of RNase H2B and RNase H2C that interface with RNase H2A in a complex ideally suited for nucleic acid binding and hydrolysis coupled to protein-protein interaction motifs that could allow for efficient participation in multiple cellular functions. We have identified four conserved acidic residues in the active site that are necessary for activity and suggest a two-metal ion mechanism of catalysis for RNase H2. An Okazaki fragment has been modeled into the RNase H2 nucleic acid binding site providing insight into the recognition of RNA.DNA junctions by the RNase H2. Further structural and biochemical analyses show that some RNase H2 disease-causing mutations likely result in aberrant protein-protein interactions while the RNase H2A subunit-G37S mutation appears to distort the active site accounting for the demonstrated substrate specificity modification.

Crystal structure of the transcriptional repressor PagR of Bacillus anthracis

PagR is a transcriptional repressor in Bacillus anthracis that controls the chromosomal S-layer genes eag and sap, and downregulates the protective antigen pagA gene by direct binding to their promoter regions. The PagR protein sequence is similar to those of members of the ArsR repressor family involved in the repression of arsenate-resistance genes in numerous bacteria. The crystal structure of PagR was solved using multi-wavelength anomalous diffraction (MAD) techniques and was refined with 1.8 A resolution diffraction data. The PagR molecules form dimers, as observed in all SmtB/ArsR repressor family proteins. In the crystal lattice four PagR dimers pack together to form an inactive octamer. Model-building studies suggest that the dimer binds to a DNA duplex with a bend of around 4 degrees.

Calcium Binds to LipL32, a Lipoprotein from Pathogenic Leptospira, and Modulates Fibronectin Binding

Tubulointerstitial nephritis is a cardinal renal manifestation of leptospirosis. LipL32, a major lipoprotein and a virulence factor, locates on the outer membrane of the pathogen Leptospira. It evades immune response by recognizing and adhering to extracellular matrix components of the host cell. The crystal structure of Ca(2+)-bound LipL32 was determined at 2.3 A resolution. LipL32 has a novel polyD sequence of seven aspartates that forms a continuous acidic surface patch for Ca(2+) binding. A significant conformational change was observed for the Ca(2+)-bound form of LipL32. Calcium binding to LipL32 was determined by isothermal titration calorimetry. The binding of fibronectin to LipL32 was observed by Stains-all CD and enzyme-linked immunosorbent assay experiments. The interaction between LipL32 and fibronectin might be associated with Ca(2+) binding. Based on the crystal structure of Ca(2+)-bound LipL32 and the Stains-all results, fibronectin probably binds near the polyD region on LipL32. Ca(2+) binding to LipL32 might be important for Leptospira to interact with the extracellular matrix of the host cell.

Location of metallic elements in (Co1−xFex)2(OH)2(C8H4O4): use of MAD, neutron diffraction and 57Fe Mössbauer spectroscopy

Four samples of the solid solution (Co1−xFex)2(OH)2(C8H4O4) x = 0.25 (1), x = 0.5 (2), x = 0.75 (3); x = 0.88 (4) (C2/m) were investigated by synchrotron radiation using Multi-wavelength Anomalous Diffraction (MAD) and multipattern refinements in order to locate the two neighbour metallic elements Fe and Co. Although there is no cation ordering, the two metallic sites M1 (0, 0, 0) and M2 (0, 0.5, 0.5) are preferentially occupied by Co and Fe respectively. In this article we demonstrate that this distribution is in agreement with neutron data and 57Fe Mössbauer spectra at 100 K. Consequently, the MAD method can be used to locate close d-metallic elements in metal–organic frameworks.

Expression, Purification, Crystallization and Preliminary X-Ray Crystallographic Analysis of the Resuscitation Promoting Factor Interacting Protein RipA from M. tuberculosis

RipA is an important growth factor of M. tuberculosis. Its depletion produces decreasing bacterial growth and an abnormal phenotype. RipA C-terminal fragment (263-472), containing its predicted catalytic domain has been successfully crystallized using vapor-diffusion methods. The structure has been solved by Multiwavelength Anomalous Dispersion and atomic resolution refinement is in progress.

Structure of the N-terminal Regulatory Domain of a Plant NADPH Oxidase and Its Functional Implications

Plant NADPH oxidases (Rboh, for respiratory burst oxidase homolog) produce reactive oxygen species that are key regulators of various cellular events including plant innate immunity. Rbohs possess a highly conserved cytoplasmic N-terminal region containing two EF-hand motifs that regulate Rboh activity. Rice (Oryza sativa) RbohB (OsRbohB) is regulated by the direct binding of a small GTPase (Rac1) to this regulatory region as well as by Ca(2+) binding to the EF-hands. Here, we present the atomic structure of the N-terminal region of OsRbohB. The structure reveals that OsRbohB forms a unique dimer stabilized by swapping the EF-hand motifs. We identified two additional EF-hand-like motifs that were not predicted from sequence data so far. These EF-hand-like motifs together with the swapped EF-hands form a structure similar to that found in calcineurin B. We observed conformational changes mediated by Ca(2+) binding to only one EF-hand. Structure-based in vitro pulldown assays and NMR titration experiments defined the OsRac1 binding interface within the coiled-coil region created by swapping the EF-hands. In addition, we demonstrate a direct intramolecular interaction between the N and C terminus, and that the complete N-terminal cytoplasmic region is required for this interaction. The structural features and intramolecular interactions characterized here might be common elements shared by Rbohs that contribute to the regulation of reactive oxygen species production.

Crystallization of selenomethionyl exo-β-1,3-galactanase from the basidiomycetePhanerochaete chrysosporium

Exo-beta-1,3-galactanase from Phanerochaete chrysosporium (Pc1,3Gal43A) consists of a glycoside hydrolase family 43 catalytic domain and a substrate-binding domain that belongs to carbohydrate-binding module family 35. It catalyzes the hydrolysis of beta-1,3-galactan, which is the backbone of the arabinogalactan proteins; the C-terminal carbohydrate-binding module family 35 domain increases the local concentration of the enzyme around beta-1,3-galactan by its high affinity for the substrate. To enable phase determination using the multiwavelength anomalous dispersion method, selenomethionyl Pc1,3Gal43A was crystallized at 298 K using the hanging-drop vapour-diffusion method. The presence of selenium in the crystals was confirmed from the X-ray absorption spectrum. The crystals belonged to space group P2(1) and diffracted to 1.8 A resolution.

A case of structure determination using pseudosymmetry.

Here, a case is presented of an unusual structure determination which was facilitated by the use of pseudosymmetry. Group A streptococcus uses cysteine protease Mac-1 (also known as IdeS) to evade the host immune system. Native Mac-1 was crystallized in the orthorhombic space group P2(1)2(1)2. Surprisingly, crystals of the inactive C94A mutant of Mac-1 displayed monoclinic symmetry with space group P2(1), despite the use of native orthorhombic Mac-1 microcrystals for seeding. Attempts to solve the structure of the C94A mutant by MAD phasing in the monoclinic space group did not produce an interpretable map. The native Patterson map of the C94A mutant showed two strong peaks along the (1 0 1) diagonal, indicating possible translational pseudosymmetry in space group P2(1). Interestingly, one-third of the monoclinic reflections obeyed pseudo-orthorhombic P2(1)2(1)2 symmetry similar to that of the wild-type crystals and could be indexed and processed in this space group. The pseudo-orthorhombic and monoclinic unit cells were related by the following vector operations: a(m) = b(o) - c(o), b(m) = a(o) and c(m) = -2c(o) - b(o). The pseudo-orthorhombic subset of data produced good SAD phases, leading to structure determination with one monomer in the asymmetric unit. Subsequently, the structure of the Mac-1 mutant in the monoclinic form was determined by molecular replacement, which showed six molecules forming three translationally related dimers aligned along the (1 0 1) diagonal. Knowing the geometric relationship between the pseudo-orthorhombic and the monoclinic unit cells, all six molecules can be generated in the monoclinic unit cell directly without the use of molecular replacement. The current case provides a successful example of the use of pseudosymmetry as a powerful phase-averaging method for structure determination by anomalous diffraction techniques. In particular, a structure can be solved in a higher pseudosymmetry subcell in which an NCS operator becomes a crystallographic operator. The geometrical relationships between the subcell and parental cell can be used to generate a complete molecular representation of the parental asymmetric unit for refinement.

The PHCCEx domain of Tiam1/2 is a novel protein- and membrane-binding module

Tiam1 and Tiam2 (Tiam1/2) are guanine nucleotide-exchange factors that possess the PH–CC–Ex (pleckstrin homology, coiled coil and extra) region that mediates binding to plasma membranes and signalling proteins in the activation of Rac GTPases. Crystal structures of the PH–CC–Ex regions revealed a single globular domain, PHCCEx domain, comprising a conventional PH subdomain associated with an antiparallel coiled coil of CC subdomain and a novel three-helical globular Ex subdomain. The PH subdomain resembles the β-spectrin PH domain, suggesting non-canonical phosphatidylinositol binding. Mutational and binding studies indicated that CC and Ex subdomains form a positively charged surface for protein binding. We identified two unique acidic sequence motifs in Tiam1/2-interacting proteins for binding to PHCCEx domain, Motif-I in CD44 and ephrinB's and the NMDA receptor, and Motif-II in Par3 and JIP2. Our results suggest the molecular basis by which the Tiam1/2 PHCCEx domain facilitates dual binding to membranes and signalling proteins.

Structural properties of Ge/Si(001) nano-islands and AlGaN nanowires by Diffraction Anomalous Fine Structure and Multiwavelength Anomalous Diffraction

In this paper, we show that combining Multiwavelength Anomalous Diffraction (MAD) and Diffraction Anomalous Fine Structure (DAFS) spectroscopy, in grazing incidence geometry, allows to obtain structural properties (strain and composition) of semiconductor nanostructures. We report results obtained on dome-shaped Ge nano-islands grown on Si(001) surfaces and AlGaN nanowires grown on Si(100). It is shown that, in the case of sharp interfaces, MAD alone can not determine the mean Ge content in the region of the substrate-island interface and needs to be combined with Extented-DAFS measurements.

Structural Insight into Partner Specificity and Phosphoryl Transfer in Two-Component Signal Transduction

The chief mechanism used by bacteria for sensing their environment is based on two conserved proteins: a sensor histidine kinase (HK) and an effector response regulator (RR). The signal transduction process involves highly conserved domains of both proteins that mediate autokinase, phosphotransfer, and phosphatase activities whose output is a finely tuned RR phosphorylation level. Here, we report the structure of the complex between the entire cytoplasmic portion of Thermotoga maritima class I HK853 and its cognate, RR468, as well as the structure of the isolated RR468, both free and BeF(3)(-) bound. Our results provide insight into partner specificity in two-component systems, recognition of the phosphorylation state of each partner, and the catalytic mechanism of the phosphatase reaction. Biochemical analysis shows that the HK853-catalyzed autokinase reaction proceeds by a cis autophosphorylation mechanism within the HK subunit. The results suggest a model for the signal transduction mechanism in two-component systems.

Structural Basis of the Catalytic Mechanism Operating in Open-Closed Conformers of Lipocalin Type Prostaglandin D Synthase

Lipocalin type prostaglandin D synthase (L-PGDS) is a multifunctional protein acting as a somnogen (PGD2)-producing enzyme, an extracellular transporter of various lipophilic ligands, and an amyloid-β chaperone in human cerebrospinal fluid. In this study, we determined the crystal structures of two different conformers of mouse L-PGDS, one with an open cavity of the β-barrel and the other with a closed cavity due to the movement of the flexible E-F loop. The upper compartment of the central large cavity contains the catalytically essential Cys65 residue and its network of hydrogen bonds with the polar residues Ser45, Thr67, and Ser81, whereas the lower compartment is composed of hydrophobic amino acid residues that are highly conserved among other lipocalins. SH titration analysis combined with site-directed mutagenesis revealed that the Cys65 residue is activated by its interaction with Ser45 and Thr67 and that the S45A/T67A/S81A mutant showed less than 10% of the L-PGDS activity. The conformational change between the open and closed states of the cavity indicates that the mobile calyx contributes to the multiligand binding ability of L-PGDS.

Structural basis of typhoid: Salmonella typhi type IVb pilin (PilS) and cystic fibrosis transmembrane conductance regulator interaction

The type IVb pilus of the enteropathogenic bacteria Salmonella typhi is a major adhesion factor during the entry of this pathogen into gastrointestinal epithelial cells. Its target of adhesion is a stretch of 10 residues from the first extracellular domain of cystic fibrosis transmembrane conductance regulator (CFTR). The crystal structure of the N-terminal 25 amino acid deleted S. typhi native PilS protein (DeltaPilS), which makes the pilus, was determined at 1.9 A resolution by the multiwavelength anomalous dispersion method. Also, the structure of the complex of DeltaPilS and a target CFTR peptide, determined at 1.8 A, confirms that residues 113-117 (NKEER) of CFTR are involved in binding with the pilin protein and gives us insight on the amino acids that are essential for binding. Furthermore, we have also explored the role of a conserved disulfide bridge in pilus formation. The subunit structure and assembly architecture are crucial for understanding pilus functions and designing suitable therapeutics against typhoid.

Interpreting a Low Resolution Map of Human U1 snRNP Using Anomalous Scatterers

SummaryWe recently determined the crystal structure of the functional core of human U1 snRNP, consisting of nine proteins and one RNA, based on a 5.5 Å resolution electron density map. At 5–7 Å resolution, α helices and β sheets appear as rods and slabs, respectively, hence it is not possible to determine protein fold de novo. Using inverse beam geometry, accurate anomalous signals were obtained from weakly diffracting and radiation sensitive P1 crystals. We were able to locate anomalous scatterers with positional errors below 2 Å. This enabled us not only to place protein domains of known structure accurately into the map but also to trace an extended polypeptide chain, of previously undetermined structure, using selenomethionine derivatives of single methionine mutants spaced along the sequence. This method of Se-Met scanning, in combination with structure prediction, is a powerful tool for building a protein of unknown fold into a low resolution electron density map.

Crystallization and preliminary X-ray studies of the N-domain of the Wilson disease associated protein

Wilson disease associated protein (ATP7B) is essential for copper transport in human cells. Mutations that affect ATP7B function result in Wilson's disease, a chronic copper toxicosis. Disease-causing mutations within the N-domain of ATP7B (WND) are known to disrupt ATP binding, but a high-resolution X-ray structure of the ATP-binding site has not been reported. The N-domain was modified to delete the disordered loop comprising residues His1115-Asp1138 (WNDDelta(1115-1138)). Unlike the wild-type N-domain, WNDDelta(1115-1138) formed good-quality crystals. Synchrotron diffraction data have been collected from WNDDelta(1115-1138) at the Canadian Light Source. A native WNDDelta(1115-1138) crystal diffracted to 1.7 A resolution and belonged to space group P4(2)2(1)2, with unit-cell parameters a = 39.2, b = 39.2, c = 168.9 A. MAD data were collected to 2.7 A resolution from a SeMet-derivative crystal with unit-cell parameters a = 38.4, b = 38.4, c = 166.7 A. The WNDDelta(1115-1138) structure is likely to be solved by phasing from multiwavelength anomalous diffraction (MAD) experiments.

Pseudosymmetry, high copy number and twinning complicate the structure determination of Desulfovibrio desulfuricans (ATCC 29577) flavodoxin.

The crystal structure of oxidized flavodoxin from Desulfovibrio desulfuricans (ATCC 29577) was determined by molecular replacement in two crystal forms, P3(1)21 and P4(3), at 2.5 and 2.0 A resolution, respectively. Structure determination in space group P3(1)21 was challenging owing to the presence of pseudo-translational symmetry and a high copy number in the asymmetric unit (8). Initial phasing attempts in space group P3(1)21 by molecular replacement using a poor search model (46% identity) and multi-wavelength anomalous dispersion were unsuccessful. It was necessary to solve the structure in a second crystal form, space group P4(3), which was characterized by almost perfect twinning, in order to obtain a suitable search model for molecular replacement. This search model with complementary approaches to molecular replacement utilizing the pseudo-translational symmetry operators determined by analysis of the native Patterson map facilitated the selection and manual placement of molecules to generate an initial solution in the P3(1)21 crystal form. During the early stages of refinement, application of the appropriate twin law, (-h, -k, l), was required to converge to reasonable R-factor values despite the fact that in the final analysis the data were untwinned and the twin law could subsequently be removed. The approaches used in structure determination and refinement may be applicable to other crystal structures characterized by these complicating factors. The refined model shows flexibility of the flavin mononucleotide coordinating loops indicated by the isolation of two loop conformations and provides a starting point for the elucidation of the mechanism used for protein-partner recognition.

The Crystal Structures of Substrate and Nucleotide Complexes of Enterococcus faecium Aminoglycoside-2′′-Phosphotransferase-IIa [APH(2′′)-IIa] Provide Insights into Substrate Selectivity in the APH(2′′) Subfamily

Aminoglycoside-2''-phosphotransferase-IIa [APH(2'')-IIa] is one of a number of homologous bacterial enzymes responsible for the deactivation of the aminoglycoside family of antibiotics and is thus a major component in bacterial resistance to these compounds. APH(2'')-IIa produces resistance to several clinically important aminoglycosides (including kanamycin and gentamicin) in both gram-positive and gram-negative bacteria, most notably in Enterococcus species. We have determined the structures of two complexes of APH(2'')-IIa, the binary gentamicin complex and a ternary complex containing adenosine-5'-(beta,gamma-methylene)triphosphate (AMPPCP) and streptomycin. This is the first crystal structure of a member of the APH(2'') family of aminoglycoside phosphotransferases. The structure of the gentamicin-APH(2'')-IIa complex was solved by multiwavelength anomalous diffraction methods from a single selenomethionine-substituted crystal and was refined to a crystallographic R factor of 0.210 (R(free), 0.271) at a resolution of 2.5 A. The structure of the AMPPCP-streptomycin complex was solved by molecular replacement using the gentamicin-APH(2'')-IIa complex as the starting model. The enzyme has a two-domain structure with the substrate binding site located in a cleft in the C-terminal domain. Gentamicin binding is facilitated by a number of conserved acidic residues lining the binding cleft, with the A and B rings of the substrate forming the majority of the interactions. The inhibitor streptomycin, although binding in the same pocket as gentamicin, is orientated such that no potential phosphorylation sites are adjacent to the catalytic aspartate residue. The binding of gentamicin and streptomycin provides structural insights into the substrate selectivity of the APH(2'') subfamily of aminoglycoside phosphotransferases, specifically, the selectivity between the 4,6-disubstituted and the 4,5-disubstituted aminoglycosides.

Structure of the Noncatalytic Domains and Global Fold of the Protein Disulfide Isomerase ERp72

Protein disulfide isomerases are a family of proteins that catalyze the oxidation and isomerization of disulfide bonds in newly synthesized proteins in the endoplasmic reticulum. The family includes general enzymes such as PDI that recognize unfolded proteins, and others that are selective for specific classes of proteins. Here, we report the X-ray crystal structure of central non-catalytic domains of a specific isomerase, ERp72 (also called CaBP2 and protein disulfide-isomerase A4) from Rattus norvegicus. The structure reveals strong similarity to ERp57, a PDI-family member that interacts with the lectin-like chaperones calnexin and calreticulin but, unexpectedly, ERp72 does not interact with calnexin as shown by isothermal titration calorimetry and nuclear magnetic resonance (NMR) spectroscopy. Small-angle X-ray scattering (SAXS) of ERp72 was used to develop models of the full-length protein using both rigid body refinement and ab initio simulated annealing of dummy atoms. The two methods show excellent agreement and define the relative positions of the five thioredoxin-like domains of ERp72 and potential substrate or chaperone binding sites.

Crystal Structures of Two Archaeal 8-Oxoguanine DNA Glycosylases Provide Structural Insight into Guanine/8-Oxoguanine Distinction

Among the four DNA bases, guanine is particularly vulnerable to oxidative damage and the most common oxidative product, 7,8-dihydro-8-oxoguanine (8-oxoG), is the most prevalent lesion observed in DNA molecules. Fortunately, 8-oxoG is recognized and excised by the 8-oxoguanine DNA glycosylase (Ogg) of the base excision repair pathway. Ogg enzymes are divided into three separate families, namely, Ogg1, Ogg2, and archaeal GO glycosylase (AGOG). To date, structures of members of both Ogg1 and AGOG families are known but no structural information is available for members of Ogg2. Here we describe the first crystal structures of two archaeal Ogg2: Methanocaldococcus janischii Ogg and Sulfolobus solfataricus Ogg. A structural comparison with OGG1 and AGOG suggested that the C-terminal lysine of Ogg2 may play a key role in discriminating between guanine and 8-oxoG. This prediction was substantiated by measuring the glycosylase/lyase activity of a C-terminal deletion mutant of MjaOgg.