Multiwavelength Anomalous Diffraction Method Research Articles

Structural basis of the cystein protease inhibitor Clonorchis sinensis Stefin-1

Cystein protease plays a critical role as a virulence factor in the development and progression of various diseases. Cystatin is a superfamily of cysteine protease inhibitors that participates in various physiological and pathological processes. The cysteine protease inhibitor CsStein-1 isolated from Clonorchis sinensis belongs to the type 1 stefin of cystatins. This inhibitor regulates the activity and processing of CsCF (Cathepsin F of Clonorchis sienesis), which plays an important role in parasite nutrition and host-parasite interaction. CsStefin-1 has also been proposed as a host immune modulator and a participant in the mechanism associated with anti-inflammatory ability. Here, we report the first crystal structure of CsStefin-1 determined by the multi-wavelength anomalous diffraction (MAD) method to 2.3 Å. There are six molecules of CsStefin-1 per asymmetric unit, with a solvent content of 36.5%. The structure of CsStefin-1 is composed of twisted four-stranded antiparallel β-sheets, a central α-helix, and a short α-helix. We also demonstrate that CsStefin-1 binds to CsCF-8 cysteine protease and inhibits its activity. In addition, a molecular docking model of CsStefin-1 and CsCF-8 was developed using homology modeling based on their structures. The structural information regarding CsStefin-1 and molecular insight into its interaction with CsCF-8 are important to understanding their biological function and to design of inhibitors that modulate cysteine protease activity.

Crystallization and preliminary X-ray crystallographic studies of dipeptidyl peptidase 11 from Porphyromonas gingivalis.

Dipeptidyl peptidase 11 from Porphyromonas gingivalis (PgDPP11) preferentially cleaves substrate peptides with Asp and Glu at the P1 position [NH2-P2-P1(Asp/Glu)-P1'-P2'...]. For crystallographic studies, PgDPP11 was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data to 1.82 Å resolution were collected from an orthorhombic crystal form belonging to space group C2221, with unit-cell parameters a = 99.33, b = 103.60, c = 177.33 Å. Structural analysis by the multi-wavelength anomalous diffraction method is in progress.

Crystal Structure analyses of dipeptidyl aminopeptidase BII fromP. mexicanaWO24.

The peptidase family S46 that contains the dipeptidyl aminopeptidase BII (DAP BII) fromPseudoxanthomonas mexicanaWO24 is the only exopeptidase family in clan PA peptidases. Our present phylogenetic and experimental studies indicated that the catalytic triad of DAP BII is composed of His 86, Asp 224 and Ser 657 and implied that unknown large helical domains involved in exopeptidase activity[1]. However, three-dimensional structure of a family S46 peptidase has not yet been reported. Thus, the crystal structure of DAP BII is essential not only to understand the catalytic mechanism of family S46 peptidases but also to clarify the structural origin of the exo-type peptidase activities of these enzymes. Recently, we have successfully crystallized the DAP BII and collected X-ray diffraction data to 2.3 Å resolution from the crystal. This crystal belonging to space groupP212121, with unit-cell parametersa= 76.55 Å,b= 130.86 Å,c= 170.87 Å[2]. Structural analysis by the multi-wavelength anomalous diffraction method is underway[3]. Here, we report the first crystallization and structural analysis of the DAP BII fromP. mexicanaWO24 as family S46 peptidase. Other enzymes that belong to this family are DPP7 and DPP11 fromPorphyromonas gingivalis, DPP11 fromPorphyromonas endodontalis(periodontal pathogen) and DPP11 fromShewanella putrefaciens(multidrug resistance associated opportunistic pathogen). These gram-negative bacterial pathogens are known to asaccharolytic. Especially,Porphyromonas gingivalisis known to utilize dipeptides, instead of free amino acids, as energy source and cellular material. Since S46 peptidases are not found in mammals, we expect our study will be useful for the discovery of specific inhibitors to S46 peptidases from these pathogens.

Crystallization and preliminary X-ray diffraction analysis of YidC, a membrane-protein chaperone and insertase from Bacillus halodurans.

YidC, a member of the YidC/Oxa1/Alb3 family, inserts proteins into the membrane and facilitates membrane-protein folding in bacteria. YidC plays key roles in both Sec-mediated integration and Sec-independent insertion of membrane proteins. Here, Bacillus halodurans YidC2, which has five transmembrane helices conserved among the other family members, was identified as a target protein for structure determination by a fluorescent size-exclusion chromatography analysis. The protein was overexpressed, purified and crystallized in the lipidic cubic phase. The crystals diffracted X-rays to 2.4 Å resolution and belonged to space group P21, with unit-cell parameters a = 43.9, b = 60.6, c = 58.9 Å, β = 100.3°. The experimental phases were determined by the multiwavelength anomalous diffraction method using a mercury-derivatized crystal.

Combining secondary-structure and protein solvent-accessibility predictions in methionine substitution for anomalous dispersion.

In X-ray crystallographic analysis, the single-wavelength and multi-wavelength anomalous diffraction (SAD and MAD) methods have been widely used in order to solve the phase problem. Selenium-labelled methionine has been shown to be very effective for anomalous dispersion phasing, and at least one selenomethionine is required for every 100 amino acids. Some proteins, such as the Arabidopsis thaliana thylakoid lumen protein AtTLP18.3, can be overexpressed in an Escherichia coli system and high-quality protein crystals can be obtained. However, AtTLP18.3 contains no methionine residues, and site-directed mutagenesis was required in order to introduce methionine residues into the protein. A criterion for the mutated residues is that they should avoid affecting the structure and function. In this study, several leucine and isoleucine residues were selected for methionine substitution by combining secondary-structure and solvent-accessibility predictions. From the secondary-structure prediction, mutated residues were first determined in the coil or loop regions at the junction of two secondary structures. Since leucine and isoleucine residues are hydrophobic and are normally buried within the protein core, these residues should have a higher solvent-accessibility prediction so that they would be partially buried or exposed in the protein. In addition, five residues (Leu107, Leu202, Ile133, Leu128 and Ile159) of AtTLP18.3 were mutated to methionine residues. After overexpression and purification, only two single-mutant lines, L128M and I159M, could be crystallized. Finally, a double-mutation line of truncated AtTLP18.3 with L128M and I159M mutations was constructed. The structure of the double mutant AtTLP18.3 protein was resolved using the single-wavelength anomalous diffraction method at 2.6 Å resolution. The results indicated that a combination of secondary-structure and solvent-accessibility prediction for methionine substitution is a useful method in SAD and MAD phasing.

Crystallization and preliminary X-ray crystallographic studies of dipeptidyl aminopeptidase BII from Pseudoxanthomonas mexicana WO24.

Dipeptidyl aminopeptidase BII from Pseudoxanthomonas mexicana WO24 (DAP BII) is able to cleave a variety of dipeptides from the amino-terminus of substrate peptides. For crystallographic studies, DAP BII was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data to 2.3 Å resolution were collected using an orthorhombic crystal form belonging to space group P2(1)2(1)2(1), with unit-cell parameters a = 76.55, b = 130.86, c = 170.87 Å. Structural analysis by the multi-wavelength anomalous diffraction method is in progress.

Multiwavelength Anomalous Diffraction at High X-Ray Intensity

The multiwavelength anomalous diffraction (MAD) method is used to determine phase information in x-ray crystallography by employing anomalous scattering from heavy atoms. X-ray free-electron lasers (FELs) show promise for revealing the structure of single molecules or nanocrystals, but the phase problem remains largely unsolved. Because of the ultrabrightness of x-ray FEL, samples experience severe electronic radiation damage, especially to heavy atoms, which hinders direct implementation of MAD with x-ray FELs. Here, we propose a generalized version of MAD phasing at high x-ray intensity. We demonstrate the existence of a Karle-Hendrickson-type equation in the high-intensity regime and calculate relevant coefficients with detailed electronic damage dynamics of heavy atoms. The present method offers a potential for ab initio structural determination in femtosecond x-ray nanocrystallography.

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.

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.

Structural Analysis of a Periplasmic Binding Protein in the Tripartite ATP-independent Transporter Family Reveals a Tetrameric Assembly That May Have a Role in Ligand Transport

Several bacterial solute transport mechanisms involve members of the periplasmic binding protein (PBP) superfamily that bind and deliver ligand to integral membrane transport proteins in the ATP-binding cassette, tripartite tricarboxylate transporter, or tripartite ATP-independent (TRAP) families. PBPs involved in ATP-binding cassette transport systems have been well characterized, but only a few PBPs involved in TRAP transport have been studied. We have measured the thermal stability, determined the oligomerization state by small angle x-ray scattering, and solved the x-ray crystal structure to 1.9 A resolution of a TRAP-PBP (open reading frame tm0322) from the hyperthermophilic bacterium Thermotoga maritima (TM0322). The overall fold of TM0322 is similar to other TRAP transport related PBPs, although the structural similarity of backbone atoms (2.5-3.1 A root mean square deviation) is unusually low for PBPs within the same group. Individual monomers within the tetrameric asymmetric unit of TM0322 exhibit high root mean square deviation (0.9 A) to each other as a consequence of conformational heterogeneity in their binding pockets. The gel filtration elution profile and the small angle x-ray scattering analysis indicate that TM0322 assembles as dimers in solution that in turn assemble into a dimer of dimers in the crystallographic asymmetric unit. Tetramerization has been previously observed in another TRAP-PBP (the Rhodobacter sphaeroides alpha-keto acid-binding protein) where quaternary structure formation is postulated to be an important requisite for the transmembrane transport process.

Crystal structure and function of C-terminal Sau3AI domain

Sau3AI is a type II restriction enzyme that recognizes the 5′-GATC-3′ sequence in double-strand DNA and cleaves at 5′ to the G residue. The C-terminal domain of Sau3AI (Sau3AI-C), which contains amino acids from 233 to 489, was crystallized and its structure was solved by using the Multi-wavelength Anomalous Diffraction method. The Sau3AI-C structure at 1.9 Å resolution is similar to the structure of MutH, a DNA mismatch repair protein that shares high sequence similarity with the N-terminal Sau3AI domain. The functional analysis shows that Sau3AI-C can bind DNA with one recognition sequence but has no cleavage activity. These results indicate that Sau3AI is a pseudo-dimer belonging to the type IIe restriction enzymes and the Sau3AI-C is the allosteric effector domain that assists DNA binding and cleavage.

Crystal Structures of NADH:FMN Oxidoreductase (EmoB) at Different Stages of Catalysis

EDTA has become a major organic pollutant in the environment because of its extreme usage and resistance to biodegradation. Recently, two critical enzymes, EDTA monooxygenase (EmoA) and NADH:FMN oxidoreductase (EmoB), belonging to the newly established two-component flavin-diffusible monooxygenase family, were identified in the EDTA degradation pathway in Mesorhizobium sp. BNC1. EmoA is an FMNH2-dependent enzyme that requires EmoB to provide FMNH2 for the conversion of EDTA to ethylenediaminediacetate. To understand the molecular basis of this FMN-mediated reaction, the crystal structures of the apo-form, FMN.FMN complex, and FMN.NADH complex of EmoB were determined at 2.5 angstroms resolution. The structure of EmoB is a homotetramer consisting of four alpha/beta-single-domain monomers of five parallel beta-strands flanked by five alpha-helices, which is quite different from those of other known two-component flavin-diffusible monooxygenase family members, such as PheA2 and HpaC, in terms of both tertiary and quaternary structures. For the first time, the crystal structures of both the FMN.FMN and FMN.NADH complexes of an NADH:FMN oxidoreductase were determined. Two stacked isoalloxazine rings and nicotinamide/isoalloxazine rings were at a proper distance for hydride transfer. The structures indicated a ping-pong reaction mechanism, which was confirmed by activity assays. Thus, the structural data offer detailed mechanistic information for hydride transfer between NADH to an enzyme-bound FMN and between the bound FMNH2 and a diffusible FMN.

Crystal Structures of F420-dependent Glucose-6-phosphate Dehydrogenase FGD1 Involved in the Activation of the Anti-tuberculosis Drug Candidate PA-824 Reveal the Basis of Coenzyme and Substrate Binding

The modified flavin coenzyme F(420) is found in a restricted number of microorganisms. It is widely distributed in mycobacteria, however, where it is important in energy metabolism, and in Mycobacterium tuberculosis (Mtb) is implicated in redox processes related to non-replicating persistence. In Mtb, the F(420)-dependent glucose-6-phosphate dehydrogenase FGD1 provides reduced F(420) for the in vivo activation of the nitroimidazopyran prodrug PA-824, currently being developed for anti-tuberculosis therapy against both replicating and persistent bacteria. The structure of M. tuberculosis FGD1 has been determined by x-ray crystallography both in its apo state and in complex with F(420) and citrate at resolutions of 1.90 and 1.95 A(,) respectively. The structure reveals a highly specific F(420) binding mode, which is shared with several other F(420)-dependent enzymes. Citrate occupies the substrate binding pocket adjacent to F(420) and is shown to be a competitive inhibitor (IC(50) 43 microm). Modeling of the binding of the glucose 6-phosphate (G6P) substrate identifies a positively charged phosphate binding pocket and shows that G6P, like citrate, packs against the isoalloxazine moiety of F(420) and helps promote a butterfly bend conformation that facilitates F(420) reduction and catalysis.

Molecular Basis of Prodrug Activation by Human Valacyclovirase, an α-Amino Acid Ester Hydrolase

Chemical modification to improve biopharmaceutical properties, especially oral absorption and bioavailability, is a common strategy employed by pharmaceutical chemists. The approach often employs a simple structural modification and utilizes ubiquitous endogenous esterases as activation enzymes, although such enzymes are often unidentified. This report describes the crystal structure and specificity of a novel activating enzyme for valacyclovir and valganciclovir. Our structural insights show that human valacyclovirase has a unique binding mode and specificity for amino acid esters. Biochemical data demonstrate that the enzyme hydrolyzes esters of alpha-amino acids exclusively and displays a broad specificity spectrum for the aminoacyl moiety similar to tricorn-interacting aminopeptidase F1. Crystal structures of the enzyme, two mechanistic mutants, and a complex with a product analogue, when combined with biochemical analysis, reveal the key determinants for substrate recognition; that is, a flexible and mostly hydrophobic acyl pocket, a localized negative electrostatic potential, a large open leaving group-accommodating groove, and a pivotal acidic residue, Asp-123, after the nucleophile Ser-122. This is the first time that a residue immediately after the nucleophile has been found to have its side chain directed into the substrate binding pocket and play an essential role in substrate discrimination in serine hydrolases. These results as well as a phylogenetic analysis establish that the enzyme functions as a specific alpha-amino acid ester hydrolase. Valacyclovirase is a valuable target for amino acid ester prodrug-based oral drug delivery enhancement strategies.

Evidence of Cholesterol Accumulated in High Curvature Regions: Implication to the Curvature Elastic Energy for Lipid Mixtures

Recent experiments suggested that cholesterol and other lipid components of high negative spontaneous curvature facilitate membrane fusion. This is taken as evidence supporting the stalk-pore model of membrane fusion in which the lipid bilayers go through intermediate structures of high curvature. How do the high-curvature lipid components lower the free energy of the curved structure? Do the high-curvature lipid components modify the average spontaneous curvature of the relevant monolayer, thereby facilitate its bending, or do the lipid components redistribute in the curved structure so as to lower the free energy? This question is fundamental to the curvature elastic energy for lipid mixtures. Here we investigate the lipid distribution in a monolayer of a binary lipid mixture before and after bending, or more precisely in the lamellar, hexagonal, and distorted hexagonal phases. The lipid mixture is composed of 2:1 ratio of brominated di18:0PC and cholesterol. Using a newly developed procedure for the multiwavelength anomalous diffraction method, we are able to isolate the bromine distribution and reconstruct the electron density distribution of the lipid mixture in the three phases. We found that the lipid distribution is homogenous and uniform in the lamellar and hexagonal phases. But in the distorted hexagonal phase, the lipid monolayer has nonuniform curvature, and cholesterol almost entirely concentrates in the high curvature region. This finding demonstrates that the association energies between lipid molecules vary with the curvature of membrane. Thus, lipid components in a mixture may redistribute under conditions of nonuniform curvature, such as in the stalk structure. In such cases, the spontaneous curvature depends on the local lipid composition and the free energy minimum is determined by lipid distribution as well as curvature.

Structural and Mutational Studies of Anthocyanin Malonyltransferases Establish the Features of BAHD Enzyme Catalysis

The BAHD family is a class of acyl-CoA-dependent acyltransferases that are involved in plant secondary metabolism and show a diverse range of specificities for acyl acceptors. Anthocyanin acyltransferases make up an important class of the BAHD family and catalyze the acylation of anthocyanins that are responsible for most of the red-to-blue colors of flowers. Here, we describe crystallographic and mutational studies of three similar anthocyanin malonyltransferases from red chrysanthemum petals: anthocyanidin 3-O-glucoside-6''-O-malonyltransferase (Dm3MaT1), anthocyanidin 3-O-glucoside-3'', 6''-O-dimalonyltransferase (Dm3MaT2), and a homolog (Dm3MaT3). Mutational analyses revealed that seven amino acid residues in the N- and C-terminal regions are important for the differential acyl-acceptor specificity between Dm3MaT1 and Dm3MaT2. Crystallographic studies of Dm3MaT3 provided the first structure of a BAHD member, complexed with acyl-CoA, showing the detailed interactions between the enzyme and acyl-CoA molecules. The structure, combined with the results of mutational analyses, allowed us to identify the acyl-acceptor binding site of anthocyanin malonyltransferases, which is structurally different from the corresponding portion of vinorine synthase, another BAHD member, thus permitting the diversity of the acyl-acceptor specificity of BAHD family to be understood.

Crystal structure of a transcription regulator (TM1602) from Thermotoga maritima at 2.3 Å resolution

Crystal structure of a transcription regulator (TM1602) from <i>Thermotoga maritima</i> at 2.3 Å resolution

Structure ofEscherichia coliUDP-N-acetylmuramoyl:L-alanine ligase (MurC)

The bacterial cell wall provides essential protection from the external environment and confers strength and rigidity to counteract internal osmotic pressure. Without this layer the cell would be easily ruptured and it is for this reason that biosynthetic pathways leading to the formation of peptidoglycan have for many years been a prime target for effective antibiotics. Central to this pathway are four similar ligase enzymes which add peptide groups to glycan moieties. As part of a program to better understand the structure-function relationships in these four enzymes, the crystal structure of Escherichia coli UDP-N-acetylmuramoyl:L-alanine ligase (MurC) has been determined to 2.6 A resolution. The structure was solved by multiwavelength anomalous diffraction methods from a single selenomethionine-substituted crystal and refined to a crystallographic R factor of 0.212 (R(free) = 0.259). The enzyme has a modular multi-domain structure very similar to those of other members of the mur family of ATP-dependent amide-bond ligases. Detailed comparison of these four enzymes shows that considerable conformational changes are possible. These changes, together with the recruitment of two different N-terminal domains, allow this family of enzymes to bind a substrate which is identical at one end and at the other has the growing peptide tail which will ultimately become part of the rigid bacterial cell wall. Comparison of the E. coli and Haemophilus influenzae structures and analysis of the sequences of known MurC enzymes indicate the presence of a ;dimerization' motif in almost 50% of the MurC enzymes and points to a highly conserved loop in domain 3 that may play a key role in amino-acid ligand specificity.

Crystal structure of a substrate complex of myo -inositol oxygenase, a di-iron oxygenase with a key role in inositol metabolism

Altered metabolism of the inositol sugars myo-inositol (MI) and d-chiro-inositol is implicated in diabetic complications. In animals, catabolism of MI and D-chiro-inositol depends on the enzyme MI oxygenase (MIOX), which catalyzes the first committed step of the glucuronate-xylulose pathway, and is found almost exclusively in the kidneys. The crystal structure of MIOX, in complex with MI, has been determined by multiwavelength anomalous diffraction methods and refined at 2.0-A resolution (R=0.206, Rfree=0.253). The structure reveals a monomeric, single-domain protein with a mostly helical fold that is distantly related to the diverse HD domain superfamily. Five helices form the structural core and provide six ligands (four His and two Asp) for the di-iron center, in which the two iron atoms are bridged by a putative hydroxide ion and one of the Asp ligands, Asp-124. A key loop forms a lid over the MI substrate, which is coordinated in bidentate mode to one iron atom. It is proposed that this mode of iron coordination, and interaction with a key Lys residue, activate MI for bond cleavage. The structure also reveals the basis of substrate specificity and suggests routes for the development of specific MIOX inhibitors.

Crystal Structure of the V Domain of Human Nectin-like Molecule-1/Syncam3/Tsll1/Igsf4b, a Neural Tissue-specific Immunoglobulin-like Cell-Cell Adhesion Molecule

Nectins are Ca(2+)-independent immunoglobulin (Ig) superfamily proteins that participate in the organization of epithelial and endothelial junctions. Nectins have three Ig-like domains in the extracellular region, and the first one is essential in cell-cell adhesion and plays a central role in the interaction with the envelope glycoprotein D of several viruses. Five Nectin-like molecules (Necl-1 through -5) with similar domain structures to those of Nectins have been identified. Necl-1 is specifically expressed in neural tissue, has Ca(2+)-independent homophilic and heterophilic cell-cell adhesion activity, and plays an important role in the formation of synapses, axon bundles, and myelinated axons. Here we report the first crystal structure of its N-terminal Ig-like V domain at 2.4 A, providing insight into trans-cellular recognition mediated by Necl-1. The protein crystallized as a dimer, and the dimeric form was confirmed by size-exclusion chromatography and chemical cross-linking experiments, indicating this V domain is sufficient for homophilic interaction. Mutagenesis work demonstrated that Phe(82) is a key residue for the adhesion activity of Necl-1. A model for homophilic adhesion of Necl-1 at synapses is proposed based on its structure and previous studies.