Multiple Anomalous Dispersion Research Articles

The oxygenating constituent of 3,6-diketocamphane monooxygenase from the CAM plasmid of Pseudomonas putida: the first crystal structure of a type II Baeyer-Villiger monooxygenase.

The three-dimensional structures of the native enzyme and the FMN complex of the overexpressed form of the oxygenating component of the type II Baeyer-Villiger 3,6-diketocamphane monooxygenase have been determined to 1.9 Å resolution. The structure of this dimeric FMN-dependent enzyme, which is encoded on the large CAM plasmid of Pseudomonas putida, has been solved by a combination of multiple anomalous dispersion from a bromine crystal soak and molecular replacement using a bacterial luciferase model. The orientation of the isoalloxazine ring of the FMN cofactor in the active site of this TIM-barrel fold enzyme differs significantly from that previously observed in enzymes of the bacterial luciferase-like superfamily. The Ala77 residue is in a cis conformation and forms a β-bulge at the C-terminus of β-strand 3, which is a feature observed in many proteins of this superfamily.

Structural insights into WcbI, a novel polysaccharide-biosynthesis enzyme.

Capsular polysaccharides (CPSs) are protective structures on the surfaces of many Gram-negative bacteria. The principal CPS of the human pathogen and Tier 1 select agent Burkholderia pseudomallei consists of a linear repeat of --3)--2-O-acetyl-6-deoxy-β-d-manno-heptopyranose-(1-. This CPS is critical to the virulence of this emerging pathogen and represents a key target for the development of novel therapeutics. wcbI is one of several genes in the CPS biosynthetic cluster whose deletion leads to significant attenuation of the pathogen; unlike most others, it has no homologues of known function and no detectable sequence similarity to any protein with an extant structure. Here, the crystal structure of WcbI bound to its proposed product, coenzyme A, is reported at 1.38 Å resolution, solved using the halide-soak method with multiple anomalous dispersion. This structure reveals that WcbI incorporates a previously described 100-amino-acid subdomain into a novel, principally helical fold (310 amino acids). This fold adopts a cradle-like structure, with a deep binding pocket for CoA in the loop-rich cradle. Structural analysis and biophysical assays suggest that WcbI functions as an acetyltransferase enzyme, whilst biochemical tests suggest that another functional module might be required to assist its activity in forming the mature B. pseudomallei capsule.

The C-terminal α–α superhelix of Pat is required for mRNA decapping in metazoa

Pat proteins regulate the transition of mRNAs from a state that is translationally active to one that is repressed, committing targeted mRNAs to degradation. Pat proteins contain a conserved N-terminal sequence, a proline-rich region, a Mid domain and a C-terminal domain (Pat-C). We show that Pat-C is essential for the interaction with mRNA decapping factors (i.e. DCP2, EDC4 and LSm1–7), whereas the P-rich region and Mid domain have distinct functions in modulating these interactions. DCP2 and EDC4 binding is enhanced by the P-rich region and does not require LSm1–7. LSm1–7 binding is assisted by the Mid domain and is reduced by the P-rich region. Structural analysis revealed that Pat-C folds into an α–α superhelix, exposing conserved and basic residues on one side of the domain. This conserved and basic surface is required for RNA, DCP2, EDC4 and LSm1–7 binding. The multiplicity of interactions mediated by Pat-C suggests that certain of these interactions are mutually exclusive and, therefore, that Pat proteins switch decapping partners allowing transitions between sequential steps in the mRNA decapping pathway.

Hyperstability and crystal structure of cytochromec555from hyperthermophilicAquifex aeolicus

In order to elucidate the relationship between the stability and the structure of the monohaem cytochrome c(555) (AA c(555)) from the hyperthermophilic bacterium Aquifex aeolicus, chemical denaturation and crystal structure determination were carried out. AA c(555) exhibited higher stability than the thermophilic Hydrogenobacter thermophilus cytochrome c(552) (HT c(552)), which is one of the most stable cytochromes c. The three-dimensional crystal structure of AA c(555), which was determined using the multiple anomalous dispersion technique at 1.15 A resolution, included a unique 14-residue extra helix, while the side-chain interactions of several amino-acid residues responsible for the stability of HT c(552) were conserved in AA c(555). The side chain of the Met61 residue in the extra helix was aligned towards the haem, forming a coordination bond between the Met S and haem Fe atoms. In other cytochromes c the corresponding regions always form Omega loops which also include the haem-liganding Met residue and are known to be involved in the initial step in cytochrome c denaturation. The formation of the extra helix in AA c(555) results in the highest helix content, 59.8%, among the monohaem cytochromes c. The extra helix should mainly contribute to the hyperstability of AA c(555) and is presumed to be a novel strategy of cytochromes c for adaptation to a hyperthermophilic environment.

Xanthomonas campestris PqqD in the pyrroloquinoline quinone biosynthesis operon adopts a novel saddle‐like fold that possibly serves as a PQQ carrier

<i>Xanthomonas campestris</i> PqqD in the pyrroloquinoline quinone biosynthesis operon adopts a novel saddle‐like fold that possibly serves as a PQQ carrier

XC1028 from Xanthomonas campestris adopts a PilZ domain‐like structure without a c‐di‐GMP switch

The crystal structure of XC1028 from Xanthomonas campestris has been determined to a resolution of 2.15 A using the multiple anomalous dispersion approach. It bears significant sequence identity and similarity values of 64.10% and 70.09%, respectively, with PA2960, a protein indispensable for type IV pilus-mediated twitching motility, after which the PilZ motif was first named. However, both XC1028 and PA2960 lack detectable c-di-GMP binding capability. Although XC1028 adopts a structure comprising a five-stranded beta-barrel core similar to other canonical PilZ domains with robust c-di-GMP binding ability, considerable differences are observed in the N-terminal motif; XC1028 assumes a compact five-stranded beta-barrel without an extra long N-terminal motif, whereas other canonical PilZ domains contain a long N-terminal sequence embedded with an essential "c-di-GMP switch" motif. In addition, a beta-strand (beta1) in the N-terminal motif, running in exactly opposite polarity to that of XC1028, is found inserted into the parallel beta3/beta1' strands, forming a completely antiparallel beta4 downward arrow beta3 upward arrow beta1 downward arrow beta1' upward arrow sheet in the canonical PilZ domains. Such dramatic structural differences at the N-terminus may account for the diminished c-di-GMP binding capability of XC1028, and suggest that interactions with additional proteins are necessary to bind c-di-GMP for type IV fimbriae assembly.

Crystal structure of RecX: A potent regulatory protein of RecA fromXanthomonas campestris

Crystal structure of RecX: A potent regulatory protein of RecA from<i>Xanthomonas campestris</i>

Toward chaperone‐assisted crystallography: Protein engineering enhancement of crystal packing and X‐ray phasing capabilities of a camelid single‐domain antibody (VHH) scaffold

A crystallization chaperone is an auxiliary protein that binds to a target of interest, enhances and modulates crystal packing, and provides high-quality phasing information. We critically evaluated the effectiveness of a camelid single-domain antibody (V(H)H) as a crystallization chaperone. By using a yeast surface display system for V(H)H, we successfully introduced additional Met residues in the core of the V(H)H scaffold. We identified a set of SeMet-labeled V(H)H variants that collectively produced six new crystal forms as the complex with the model antigen, RNase A. The crystals exhibited monoclinic, orthorhombic, triclinic, and tetragonal symmetry and have one or two complexes in the asymmetric unit, some of which diffracted to an atomic resolution. The phasing power of the Met-enriched V(H)H chaperone allowed for auto-building the entire complex using single-anomalous dispersion technique (SAD) without the need for introducing SeMet into the target protein. We show that phases produced by combining SAD and V(H)H model-based phases are accurate enough to easily solve structures of the size reported here, eliminating the need to collect multiple wavelength multiple-anomalous dispersion (MAD) data. Together with the presence of high-throughput selection systems (e.g., phage display libraries) for V(H)H, the enhanced V(H)H domain described here will be an excellent scaffold for producing effective crystallization chaperones.

Implementing the LIM code: the structural basis for cell type-specific assembly of LIM-homeodomain complexes

LIM-homeodomain (LIM-HD) transcription factors form a combinatorial 'LIM code' that contributes to the specification of cell types. In the ventral spinal cord, the binary LIM homeobox protein 3 (Lhx3)/LIM domain-binding protein 1 (Ldb1) complex specifies the formation of V2 interneurons. The additional expression of islet-1 (Isl1) in adjacent cells instead specifies the formation of motor neurons through assembly of a ternary complex in which Isl1 contacts both Lhx3 and Ldb1, displacing Lhx3 as the binding partner of Ldb1. However, little is known about how this molecular switch occurs. Here, we have identified the 30-residue Lhx3-binding domain on Isl1 (Isl1(LBD)). Although the LIM interaction domain of Ldb1 (Ldb1(LID)) and Isl1(LBD) share low levels of sequence homology, X-ray and NMR structures reveal that they bind Lhx3 in an identical manner, that is, Isl1(LBD) mimics Ldb1(LID). These data provide a structural basis for the formation of cell type-specific protein-protein interactions in which unstructured linear motifs with diverse sequences compete to bind protein partners. The resulting alternate protein complexes can target different genes to regulate key biological events.

Expression, crystallization and preliminary crystallographic studies of a novel bifunctional N-acetylglutamate synthase/kinase from Xanthomonas campestris homologous to vertebrate N-acetylglutamate synthase.

A novel N-acetylglutamate synthase/kinase bifunctional enzyme of arginine biosynthesis that was homologous to vertebrate N-acetylglutamate synthases was identified in Xanthomonas campestris. The protein was overexpressed, purified and crystallized. The crystals belong to the hexagonal space group P6(2)22, with unit-cell parameters a = b = 134.60, c = 192.11 A, and diffract to about 3.0 A resolution. Selenomethionine-substituted recombinant protein was produced and selenomethionine substitution was verified by mass spectroscopy. Multiple anomalous dispersion (MAD) data were collected at three wavelengths at SER-CAT, Advanced Photon Source, Argonne National Laboratory. Structure determination is under way using the MAD phasing method.

Complete automation of molecular replacement

Diamond [1] is the new third generation synchrotron source which is currently being built in the United Kingdom south of Oxford. In Phase 1 seven beamlines are funded which includes three beamlines for macromolecular crystallography (MX). Radiation from three in-vacuum undulators will be used for these beamlines. The undulators will be canted to allow for a second one to be placed in the straight to provide radiation for a side station. All three MX beamlines will be tunable over a wavelength range of 0.5-2.5 A to allow for multiple anomalous dispersion experiments. Significant emphasis is being placed on the automation of each beamline, both in terms of hardware and software. The implemented diagnostics tools will also allow for a remote monitoring and control of the beam. The experimental stations will be equipped with large area detectors, with robotic sample changers to enable automatic mounting of cryocooled and room temperature samples as well as a high quality crystal viewing system. Next to the beamlines the users will find support laboratories to prepare for their experiments. The MX beamlines are scheduled for commissioning in 2006 and will come on-line for users in 2007.

How to Avoid Premature Decay of Your Macromolecular Crystal: A Quick Soak for Long Life

Radiation damage to biological samples is currently one of the major limiting factors in macromolecular X-ray crystallography, since it severely and irreversibly affects the quality of the data that can be obtained from a diffraction experiment. However, radiation damage can effectively be reduced by utilizing the electron and radical scavenging potential of certain small-molecule compounds. We propose an approach to protect macromolecular crystals prior to data collection by quick soaking with scavengers. This, in favorable cases, can more than double crystal lifetime in the X-ray beam. The approach has the potential to yield diffraction data of superior quality and hence to increase the amount of high-quality diffraction data and of structural information attainable from a single crystal.

Old fold in a new X‐ray diffraction dataset? Low‐resolution molecular replacement using representative structural templates can provide phase information

The advent of structural genomics has led to a dramatic increase in the number of structures deposited in the Protein Data Bank. The number of new folds, however, still remains a very small fraction of the total number of deposited structures. Recent data on the progress of the structural genomics initiative reveals that more than 85% of target proteins that progress to the stage of data collection and structure determination have a known fold. Enzymes, which tend to exploit reaction space while adopting a common stable scaffold, contribute significantly to this observation. Herein, we evaluate a method to examine the "old fold in a new dataset" scenario likely to be encountered in the structural genomics pipeline. We demonstrate that a fold detection strategy based on secondary structure signatures followed by molecular replacement using a minimalist model can be effectively used to solve the phase problem in X-ray crystallography without further recourse to heavy atom derivatives or multiple anomalous dispersion techniques. Three common folds-the triosephosphate isomerase (TIM), adenine nucleotide alpha hydrolase-like (HUP), and RNA recognition motif (RRM)-were examined using this approach. The results presented herein also provide an estimate of the extent of phase information that can be derived from a single domain in a large multidomain structure.

Redox-Dependent Structural Changes in the Azotobacter vinelandii Bacterioferritin: New Insights into the Ferroxidase and Iron Transport Mechanism,

In this work, we report the X-ray crystal structure of the aerobically isolated (oxidized) and the anaerobic dithionite-reduced (at pH 8.0) forms of the native Azotobacter vinelandii bacterioferritin to 2.7 and 2.0 A resolution, respectively. Iron K-edge multiple anomalous dispersion (MAD) experiments unequivocally identified the presence of three independent iron-containing sites within the protein structure. Specifically, a dinuclear (ferroxidase) site, a b-type heme site, and the binding of a single iron atom at the four-fold molecular axis of the protein shell were observed. In addition to the novel observation of iron at the four-fold pore, these data also reveal that the oxidized form of the protein has a symmetrical ferroxidase site containing two five-coordinate iron atoms. Each iron atom is ligated by four carboxylate oxygen atoms and a single histidyl nitrogen atom. A single water molecule is found within hydrogen bonding distance of the ferroxidase site that bridges the two iron atoms on the side opposite the histidine ligands. Chemical reduction of the protein under anaerobic conditions results in an increase in the average Fe-Fe distance in the ferroxidase site from approximately 3.5 to approximately 4.0 A and the loss of one of the ligands, H130. In addition, there is significant movement of the bridging water molecule and several other amino acid side chains in the vicinity of the ferroxidase site and along the D helix to the three-fold symmetry axis. In contrast to previous work, the higher-resolution data for the dithionite-reduced structure suggest that the heme may be bound in multiple conformations. Taken together, these data allow a molecular movie of the ferroxidase gating mechanism to be developed and provide further insight into the iron uptake and/or release and mineralization mechanism of bacterioferritins in general.

The Crystal Structure of (S)-3-O-Geranylgeranylglyceryl Phosphate Synthase Reveals an Ancient Fold for an Ancient Enzyme

We report crystal structures of the citrate and sn-glycerol-1-phosphate (G1P) complexes of (S)-3-O-geranylgeranylglyceryl phosphate synthase from Archaeoglobus fulgidus (AfGGGPS) at 1.55 and 2.0 A resolution, respectively. AfGGGPS is an enzyme that performs the committed step in archaeal lipid biosynthesis, and it presents the first triose phosphate isomerase (TIM)-barrel structure with a prenyltransferase function. Our studies provide insight into the catalytic mechanism of AfGGGPS and demonstrate how it selects for the sn-G1P isomer. The replacement of "Helix 3" by a "strand" in AfGGGPS, a novel modification to the canonical TIM-barrel fold, suggests a model of enzyme adaptation that involves a "greasy slide" and a "swinging door." We propose functions for the homologous PcrB proteins, which are conserved in a subset of pathogenic bacteria, as either prenyltransferases or being involved in lipoteichoic acid biosynthesis. Sequence and structural comparisons lead us to postulate an early evolutionary history for AfGGGPS, which may highlight its role in the emergence of Archaea.

Crystallization and initial crystal characterization of the C-terminal phosphoglycerate mutase homology domain of Sts-1

Sts-1 is a multidomain protein that plays an important role in T-cell signaling. Sts-1 contains a ubiquitin-association (UBA) domain at the N-terminus, followed by an Src homology-3 (SH3) domain and a C-terminal domain that shares sequence homology to phosphoglycerate mutases (PGMs). The C-terminal domain of Sts-1, Sts-1(PGM), crystallizes in space group C2 with two different crystal forms. The first crystal form contains two or three Sts-1PGM molecules in the asymmetric unit and diffracts to 1.82 A resolution, with unit-cell parameters a = 116.2, b = 74.3, c = 100.1 A, alpha = gamma = 90, beta = 101.5 degrees. The second crystal form contains four or six Sts-1(PGM) molecules in the asymmetric unit, with unit-cell parameters a = 214.9, b = 75.1, c = 116.4 A, alpha = gamma = 90, beta = 111.6 degrees. Greater than 95% complete native and SeMet data sets have been collected and structure determination using the multiple anomalous dispersion (MAD) technique is ongoing.

Structures of a putative RNA 5-methyluridine methyltransferase,Thermus thermophilusTTHA1280, and its complex withS-adenosyl-L-homocysteine

The Thermus thermophilus hypothetical protein TTHA1280 belongs to a family of predicted S-adenosyl-L-methionine (AdoMet) dependent RNA methyltransferases (MTases) present in many bacterial and archaeal species. Inspection of amino-acid sequence motifs common to class I Rossmann-fold-like MTases suggested a specific role as an RNA 5-methyluridine MTase. Selenomethionine (SeMet) labelled and native versions of the protein were expressed, purified and crystallized. Two crystal forms of the SeMet-labelled apoprotein were obtained: SeMet-ApoI and SeMet-ApoII. Cocrystallization of the native protein with S-adenosyl-L-homocysteine (AdoHcy) yielded a third crystal form, Native-AdoHcy. The SeMet-ApoI structure was solved by the multiple anomalous dispersion method and refined at 2.55 A resolution. The SeMet-ApoII and Native-AdoHcy structures were solved by molecular replacement and refined at 1.80 and 2.60 A, respectively. TTHA1280 formed a homodimer in the crystals and in solution. Each subunit folds into a three-domain structure composed of a small N-terminal PUA domain, a central alpha/beta-domain and a C-terminal Rossmann-fold-like MTase domain. The three domains form an overall clamp-like shape, with the putative active site facing a deep cleft. The architecture of the active site is consistent with specific recognition of uridine and catalysis of methyl transfer to the 5-carbon position. The cleft is suitable in size and charge distribution for binding single-stranded RNA.

Crystal Structure of the Yersinia enterocolitica Type III Secretion Chaperone SycT

Several Gram-negative pathogens deploy type III secretion systems (TTSSs) as molecular syringes to inject effector proteins into host cells. Prior to secretion, some of these effectors are accompanied by specific type III secretion chaperones. The Yersinia enterocolitica TTSS chaperone SycT escorts the effector YopT, a cysteine protease that inactivates the small GTPase RhoA of targeted host cells. We solved the crystal structure of SycT at 2.5 angstroms resolution. Despite limited sequence similarity among TTSS chaperones, the SycT structure revealed a global fold similar to that exhibited by other structurally solved TTSS chaperones. The dimerization domain of SycT, however, differed from that of all other known TTSS chaperone structures. Thus, the dimerization domain of TTSS chaperones does not likely serve as a general recognition pattern for downstream processing of effector/chaperone complexes. Yersinia Yop effectors are bound to their specific Syc chaperones close to the Yop N termini, distinct from their catalytic domains. Here, we showed that the catalytically inactive YopT(C139S) is reduced in its ability to bind SycT, suggesting an ancillary interaction between YopT and SycT. This interaction could maintain the protease inactive prior to secretion or could influence the secretion competence and folding of YopT.

Crystal Structure of a Ribonuclease P Protein Ph1601p from Pyrococcus horikoshii OT3: An Archaeal Homologue of Human Nuclear Ribonuclease P Protein Rpp21,

Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the removal of 5' leader sequences from tRNA precursors (pre-tRNA). The human protein Rpp21 is essential for human RNase P activity in tRNA processing in vitro. The crystal structure of Ph1601p from the hyperthermophilic archaeon Pyrococcus horikoshii OT3, the archaeal homologue of Rpp21, was determined using the multiple anomalous dispersion (MAD) method with the aid of anomalous scattering in zinc and selenium at 1.6 A resolution. Ph1601p comprises an N-terminal domain (residues 1-55), a central linker domain (residues 56-79), and a C-terminal domain (residues 80-120), forming an L-shaped structure. The N-terminal domain consists of two long alpha-helices, while the central and C-terminal domains fold in a zinc ribbon domain. The electrostatic potential representation indicates the presence of positively charged clusters along the L arms, suggesting a possible role in RNA binding. A single zinc ion binds the well-ordered binding site that consists of four Cys residues (Cys68, Cys71, Cys97, and Cys100) and appears to stabilize the relative positions of the N- and C-domains. Mutations of Cys68 and Cys71 or Cys97 and Cys100 to Ser destabilize the protein structure, which results in inactivation of the RNase P activity. In addition, site-directed mutagenesis suggests that Lys69 at the central loop and Arg86 and Arg105 at the zinc ribbon domain are strongly involved in the functional activity, while Arg22, Tyr44, Arg65, and Arg84 play a modest role in the activity.

Crystal Structure of a Putative Type I Restriction–Modification S Subunit from Mycoplasma genitalium

The crystal structure of the eubacteria Mycoplasma genitalium ORF MG438 polypeptide, determined by multiple anomalous dispersion and refined at 2.3 A resolution, reveals the organization of S subunits from the Type I restriction and modification system. The structure consists of two globular domains, with about 150 residues each, separated by a pair of 40 residue long antiparallel alpha-helices. The globular domains correspond to the variable target recognition domains (TRDs), as previously defined for S subunits on sequence analysis, while the two helices correspond to the central (CR1) and C-terminal (CR2) conserved regions, respectively. The structure of the MG438 subunit presents an overall cyclic topology with an intramolecular 2-fold axis that superimposes the N and the C-half parts, each half containing a globular domain and a conserved helix. TRDs are found to be structurally related with the small domain of the Type II N6-adenine DNA MTase TaqI. These relationships together with the structural peculiarities of MG438, in particular the presence of the intramolecular quasi-symmetry, allow the proposal of a model for S subunits recognition of their DNA targets in agreement with previous experimental results. In the crystal, two subunits of MG438 related by a crystallographic 2-fold axis present a large contact area mainly involving the symmetric interactions of a cluster of exposed hydrophobic residues. Comparison with the recently reported structure of an S subunit from the archaea Methanococcus jannaschii highlights the structural features preserved despite a sequence identity below 20%, but also reveals important differences in the globular domains and in their disposition with respect to the conserved regions.