Acta Crystallographica Section D Biological Crystallography

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ISSN / EISSN : 0907-4449 / 1399-0047
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Neelanjana Janardan, Rajesh K. Harijan, Tiila-Riikka Kiema, Rikkert K. Wierenga,
Acta Crystallographica Section D Biological Crystallography, Volume 71, pp 2479-2493; https://doi.org/10.1107/s1399004715019331

Abstract:
Thiolases catalyze the degradation and synthesis of 3-ketoacyl-CoA molecules. Here, the crystal structures of a T1-like thiolase (MSM-13 thiolase) fromMycobacterium smegmatisin apo and liganded forms are described. Systematic comparisons of six crystallographically independent unliganded MSM-13 thiolase tetramers (dimers of tight dimers) from three different crystal forms revealed that the two tight dimers are connected to a rigid tetramerization domainviaflexible hinge regions, generating an asymmetric tetramer. In the liganded structure, CoA is bound to those subunits that are rotated towards the tip of the tetramerization loop of the opposing dimer, suggesting that this loop is important for substrate binding. The hinge regions responsible for this rotation occur near Val123 and Arg149. The Lα1–covering loop–Lα2 region, together with the Nβ2–Nα2 loop of the adjacent subunit, defines a specificity pocket that is larger and more polar than those of other tetrameric thiolases, suggesting that MSM-13 thiolase has a distinct substrate specificity. Consistent with this finding, only residual activity was detected with acetoacetyl-CoA as the substrate in the degradative direction. No activity was observed with acetyl-CoA in the synthetic direction. Structural comparisons with other well characterized thiolases suggest that MSM-13 thiolase is probably a degradative thiolase that is specific for 3-ketoacyl-CoA molecules with polar, bulky acyl chains.
Tao Zhang, Deqiang Yao, Jiawei Wang, ,
Acta Crystallographica Section D Biological Crystallography, Volume 71, pp 2513-2518; https://doi.org/10.1107/s139900471501603x

Abstract:
A post-experimental identification/purification procedure similar to that described in Zhanget al.[(2015),IUCrJ,2, 322–326] has been proposed for use in the treatment of multiphase protein serial crystallography (SX) diffraction snapshots. As a proof of concept, the procedure was tested using theoretical serial femtosecond crystallography (SFX) data from a mixture containing native and derivatized crystals of a protein. Two known proteins were taken as examples. Multiphase diffraction snapshots were subjected to two rounds of indexing using the programCrystFEL[Whiteet al.(2012).J. Appl. Cryst.45, 335–341]. In the first round, anab initioindexing was performed to derive a set of approximate primitive unit-cell parameters, which are roughly the average of those from the native protein and the derivative. These parameters were then used in a second round of indexing as input toCrystFEL. The results were then used to separate the diffraction snapshots into two subsets corresponding to the native and the derivative. For each test sample, integration of the two subsets of snapshots separately led to two sets of three-dimensional diffraction intensities, one belonging to the native and the other to the derivative. Based on these two sets of intensities, a conventional single isomorphous replacement (SIR) procedure solved the structure easily.
Acta Crystallographica Section D Biological Crystallography, Volume 71, pp 2494-2504; https://doi.org/10.1107/s1399004715019392

Abstract:
The virulence of theCandidapathogens is enhanced by the production of secreted aspartic proteases, which therefore represent possible targets for drug design. Here, the crystal structure of the secreted aspartic protease Sapp2p fromCandida parapsilosiswas determined. Sapp2p was isolated from its natural source and crystallized in complex with pepstatin A, a classical aspartic protease inhibitor. The atomic resolution of 0.83 Å allowed the protonation states of the active-site residues to be inferred. A detailed comparison of the structure of Sapp2p with the structure of Sapp1p, the most abundantC. parapsilosissecreted aspartic protease, was performed. The analysis, which included advanced quantum-chemical interaction-energy calculations, uncovered molecular details that allowed the experimentally observed equipotent inhibition of both isoenzymes by pepstatin A to be rationalized.
, Angelika A. M. Montada, ,
Acta Crystallographica Section D Biological Crystallography, Volume 71, pp 2505-2512; https://doi.org/10.1107/s1399004715019549

Abstract:
The U32 family is a collection of over 2500 annotated peptidases in the MEROPS database with unknown catalytic mechanism. They mainly occur in bacteria and archaea, but a few representatives have also been identified in eukarya. Many of the U32 members have been linked to pathogenicity, such as proteins fromHelicobacterandSalmonella. The first crystal structure analysis of a U32 catalytic domain fromMethanopyrus kandleri(genemk0906) reveals a modified (βα)8TIM-barrel fold with some unique features. The connecting segment between strands β7 and β8 is extended and helix α7 is located on top of the C-terminal end of the barrel body. The protein exhibits a dimeric quaternary structure in which a zinc ion is symmetrically bound by histidine and cysteine side chains from both monomers. These residues reside in conserved sequence motifs. No typical proteolytic motifs are discernible in the three-dimensional structure, and biochemical assays failed to demonstrate proteolytic activity. A tunnel in which an acetate ion is bound is located in the C-terminal part of the β-barrel. Two hydrophobic grooves lead to a tunnel at the C-terminal end of the barrel in which an acetate ion is bound. One of the grooves binds to aStrep-Tag II of another dimer in the crystal lattice. Thus, these grooves may be binding sites for hydrophobic peptides or other ligands.
Acta Crystallographica Section D Biological Crystallography, Volume 71, pp 2526-2542; https://doi.org/10.1107/s1399004715019641

Abstract:
Riboflavin kinases (RFKs) catalyse the phosphorylation of riboflavin to produce FMN. In most bacteria this activity is catalysed by the C-terminal module of a bifunctional enzyme, FAD synthetase (FADS), which also catalyses the transformation of FMN into FAD through its N-terminal FMN adenylyltransferase (FMNAT) module. The RFK module of FADS is a homologue of eukaryotic monofunctional RFKs, while the FMNAT module lacks homologyto eukaryotic enzymes involved in FAD production. Previously, the crystal structure ofCorynebacterium ammoniagenesFADS (CaFADS) was determined in its apo form. This structure predicted a dimer-of-trimers organization with the catalytic sites of two modules of neighbouring protomers approaching each other, leading to a hypothesis about the possibility of FMN channelling in the oligomeric protein. Here, two crystal structures of the individually expressed RFK module ofCaFADS in complex with the products of the reaction, FMN and ADP, are presented. Structures are complemented with computational simulations, binding studies and kinetic characterization. Binding of ligands triggers dramatic structural changes in the RFK module, which affect large portions of the protein. Substrate inhibition and molecular-dynamics simulations allowed the conformational changes that take place along the RFK catalytic cycle to be established. The influence of these conformational changes in the FMNAT module is also discussed in the context of the full-lengthCaFADS protomer and the quaternary organization.
Takanori Nakane, Changyong Song, , Eriko Nango, Jun Kobayashi, , Shigeyuki Inoue, Eiichi Mizohata, , Tomoyuki Tanaka, et al.
Acta Crystallographica Section D Biological Crystallography, Volume 71, pp 2519-25; https://doi.org/10.1107/S139900471501857X

Abstract:
Serial femtosecond crystallography (SFX) allows structures to be determined with minimal radiation damage. However, phasing native crystals in SFX is not very common. Here, the structure determination of native lysozyme from single-wavelength anomalous diffraction (SAD) by utilizing the anomalous signal of sulfur and chlorine at a wavelength of 1.77 Å is successfully demonstrated. This sulfur SAD method can be applied to a wide range of proteins, which will improve the determination of native crystal structures.
Keren Green, Nasrin Qasim, Garik Gdaelvsky, Anna Kogan, Yehuda Goldgur, , Ofra Lotan,
Acta Crystallographica Section D Biological Crystallography, Volume 71, pp 2364-2371; https://doi.org/10.1107/s139900471501799x

Abstract:
Tryptophanase (Trpase) is a pyridoxal 5′-phosphate (PLP)-dependent homotetrameric enzyme which catalyzes the degradation of L-tryptophan. Trpase is also known for its cold lability, which is a reversible loss of activity at low temperature (2°C) that is associated with the dissociation of the tetramer.Escherichia coliTrpase dissociates into dimers, whileProteus vulgarisTrpase dissociates into monomers. As such, this enzyme is an appropriate model to study the protein–protein interactions and quaternary structure of proteins. The aim of the present study was to understand the differences in the mode of dissociation between theE. coliandP. vulgarisTrpases. In particular, the effect of mutations along the molecular axes of homotetrameric Trpase on its dissociation was studied. To answer this question, two groups of mutants of theE. colienzyme were created to resemble the amino-acid sequence ofP. vulgarisTrpase. In one group, residues 15 and 59 that are located along the molecular axisR(also termed the noncatalytic axis) were mutated. The second group included a mutation at position 298, located along the molecular axisQ(also termed the catalytic axis). Replacing amino-acid residues along theRaxis resulted in dissociation of the tetramers into monomers, similar to theP. vulgarisTrpase, while replacing amino-acid residues along theQaxis resulted in dissociation into dimers only. The crystal structure of the V59M mutant ofE. coliTrpase was also determined in its apo form and was found to be similar to that of the wild type. This study suggests that inE. coliTrpase hydrophobic interactions along theRaxis hold the two monomers together more strongly, preventing the dissociation of the dimers into monomers. Mutation of position 298 along theQaxis to a charged residue resulted in tetramers that are less susceptible to dissociation. Thus, the results indicate that dissociation ofE. coliTrpase into dimers occurs along the molecularQaxis.
, , , Xin Ye, Ivan Lenov, Stephen G. Sligar,
Acta Crystallographica Section D Biological Crystallography, Volume 71, pp 2412-2421; https://doi.org/10.1107/s1399004715018702

Abstract:
Membrane proteins reconstituted into phospholipid nanodiscs comprise a soluble entity accessible to solution small-angle X-ray scattering (SAXS) studies. It is demonstrated that using SAXS data it is possible to determine both the shape and localization of the membrane protein cytochrome P450 3A4 (CYP3A4) while it is embedded in the phospholipid bilayer of a nanodisc. In order to accomplish this, a hybrid approach to analysis of small-angle scattering data was developed which combines an analytical approach to describe the multi-contrast nanodisc with a free-form bead-model description of the embedded protein. The protein shape is then reconstructedab initioto optimally fit the data. The result of using this approach is compared with the result obtained using a rigid-body description of the CYP3A4-in-nanodisc system. Here, the CYP3A4 structure relies on detailed information from crystallographic and molecular-dynamics studies of CYP3A4. Both modelling approaches arrive at very similar solutions in which the α-helical anchor of the CYP3A4 systematically stays close to the edge of the nanodisc and with the large catalytic domain leaning over the outer edge of the nanodisc. The obtained distance between the globular domains of CYP3A4 is consistent with previously published theoretical calculations.
Tomasz Manszewski, Kriti Singh, Barbara Imiolczyk,
Acta Crystallographica Section D Biological Crystallography, Volume 71, pp 2422-2432; https://doi.org/10.1107/s1399004715018659

Abstract:
S-Adenosyl-L-homocysteine hydrolase (SAHase) is involved in the enzymatic regulation ofS-adenosyl-L-methionine (SAM)-dependent methylation reactions. After methyl-group transfer from SAM,S-adenosyl-L-homocysteine (SAH) is formed as a byproduct, which in turn is hydrolyzed to adenosine (Ado) and homocysteine (Hcy) by SAHase. The crystal structure of BeSAHase, an SAHase fromBradyrhizobium elkanii, which is a nitrogen-fixing bacterial symbiont of legume plants, was determined at 1.7 Å resolution, showing the domain organization (substrate-binding domain, NAD+cofactor-binding domain and dimerization domain) of the subunits. The protein crystallized in its biologically relevant tetrameric form, with three subunits in a closed conformation enforced by complex formation with the Ado product of the enzymatic reaction. The fourth subunit is ligand-free and has an open conformation. The BeSAHase structure therefore provides a unique snapshot of the domain movement of the enzyme induced by the binding of its natural ligands.
Acta Crystallographica Section D Biological Crystallography, Volume 71, pp 2449-2456; https://doi.org/10.1107/s1399004715018635

Abstract:
Carbonic anhydrase (CA) is a zinc enzyme that catalyzes the reversible conversion of carbon dioxide to bicarbonate (hydrogen carbonate) and a proton. CAs have been extensively investigated owing to their involvement in numerous physiological and pathological processes. Currently, CA inhibitors are widely used as antiglaucoma, anticancer and anti-obesity drugs and for the treatment of neurological disorders. Recently, the potential use of CA inhibitors to fight infections caused by protozoa, fungi and bacteria has emerged as a new research direction. In this article, the cloning and kinetic characterization of the β-CA fromVibrio cholerae(VchCAβ) are reported. The X-ray crystal structure of this new enzyme was solved at 1.9 Å resolution from a crystal that was perfectly merohedrally twinned, revealing a tetrameric type II β-CA with a closed active site in which the zinc is tetrahedrally coordinated to Cys42, Asp44, His98 and Cys101. The substrate bicarbonate was found bound in a noncatalytic binding pocket close to the zinc ion, as reported for a few other β-CAs, such as those fromEscherichia coliandHaemophilus influenzae. At pH 8.3, the enzyme showed a significant catalytic activity for the physiological reaction of the hydration of CO2to bicarbonate and protons, with the following kinetic parameters: akcatof 3.34 × 105 s−1and akcat/Kmof 4.1 × 107M−1 s−1. The new enzyme, on the other hand, was poorly inhibited by acetazolamide (Kiof 4.5 µM). As this bacterial pathogen encodes at least three CAs, an α-CA, a β-CA and a γ-CA, these enzymes probably play an important role in the life cycle and pathogenicity ofVibrio, and it cannot be excluded that interference with their activity may be exploited therapeutically to obtain antibiotics with a different mechanism of action.
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