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Preview: Acta Crystallographica Section F

Acta Crystallographica Section F

Acta Crystallographica Section F: Structural Biology Communications is a rapid all-electronic journal, which provides a home for short communications on the crystallization and structure of biological macromolecules. Structures determined through structur

Published: 2017-06-01


Crystal structures of human Fabs targeting the Bexsero meningococcal vaccine antigen NHBA


Neisserial heparin-binding antigen (NHBA) is a surface-exposed lipoprotein from Neisseria meningitidis and is a component of the meningococcus B vaccine Bexsero. As part of a study to characterize the three-dimensional structure of NHBA and the molecular basis of the human immune response to Bexsero, the crystal structures of two fragment antigen-binding domains (Fabs) isolated from human monoclonal antibodies targeting NHBA were determined. Through a high-resolution analysis of the organization and the amino-acid composition of the CDRs, these structures provide broad insights into the NHBA epitopes recognized by the human immune system. As expected, these Fabs also show remarkable structural conservation, as shown by a structural comparison of 15 structures of apo Fab 10C3 which were obtained from crystals grown in different crystallization conditions and were solved while searching for a complex with a bound NHBA fragment or epitope peptide. This study also provides indirect evidence for the intrinsically disordered nature of two N-terminal regions of NHBA.

Crystal structure of Rv1220c, a SAM-dependent O-methyltransferase from Mycobacterium tuberculosis


Rv1220c from Mycobacterium tuberculosis is annotated as an O-methyltransferase (MtbOMT). Currently, no structural information is available for this protein. Here, the crystal structure of MtbOMT refined to 2.0 Å resolution is described. The structure reveals the presence of a methyltransferase fold and shows clear electron density for one molecule of S-adenosylmethionine (SAM), which was apparently bound by the protein during its production in Escherichia coli. Although the overall structure of MtbOMT resembles the structures of O-methyltransferases from Cornybacterium glutamicum, Coxiella burnetti and Alfa alfa, differences are observed in the residues that make up the active site. Notably, substitution of Asp by His164 seems to abrogate metal binding by MtbOMT. A putative catalytic His–Asp pair located in the vicinity of SAM is absolutely conserved in MtbOMT homologues from all species of Mycobacterium, suggesting a conserved function for this protein.

Crystallization and X-ray diffraction analysis of SpaE, a basal pilus protein from the gut-adapted Lactobacillus rhamnosus GG


SpaE is the predicted basal pilin subunit in the sortase-dependent SpaFED pilus from the gut-adapted and commensal Lactobacillus rhamnosus GG. Thus far, structural characterization of the cell-wall-anchoring basal pilins has remained difficult and has been limited to only a few examples from pathogenic genera and species. To gain a further structural understanding of the molecular mechanisms that are involved in the anchoring and assembly of sortase-dependent pili in less harmful bacteria, L. rhamnosus GG SpaE for crystallization was produced by recombinant expression in Escherichia coli. Although several attempts to crystallize the SpaE protein were unsuccessful, trigonal crystals that diffracted to a resolution of 3.1 Å were eventually produced using PEG 3350 as a precipitant and high protein concentrations. Further optimization with a combination of additives led to the generation of SpaE crystals in an orthorhombic form that diffracted to a higher resolution of 1.5 Å. To expedite structure determination by SAD phasing, selenium-substituted (orthorhombic) SpaE crystals were grown and X-ray diffraction data were collected to 1.8 Å resolution.

Crystal structure of the Thermoplasma acidophilum protein Ta1207


The crystal structure of the Ta1207 protein from Thermoplasma acidophilum is reported. Ta1207 was identified in a screen for high-molecular-weight protein complexes in T. acidophilum. In solution, Ta1207 forms homopentamers of 188 kDa. The crystal structure of recombinant Ta1207 solved by Se-MAD at 2.4 Å resolution revealed a complex with fivefold symmetry. In the crystal lattice, calcium ions induce the formation of a nanocage from two pentamers. The Ta1207 protomers comprise two domains with the same novel α/β topology. A deep pocket with a binding site for a negatively charged group suggests that Ta1207 functions as an intracellular receptor for an unknown ligand. Homologues of Ta1207 occur only in Thermoplasmatales and its function might be related to the extreme lifestyle of these archaea. The thermostable Ta1207 complex might provide a useful fivefold-symmetric scaffold for future nanotechnological applications.

Crystal structure of the active form of native human thymidylate synthase in the absence of bound substrates


Human thymidylate synthase (hTS) provides the sole de novo intracellular source of thymidine 5′-monophosphate (dTMP). hTS is required for DNA replication prior to cell division, making it an attractive target for anticancer chemotherapy and drug discovery. hTS binds 2′-deoxyuridine 5′-monophosphate (dUMP) and the folate co-substrate N5,N10-methylenetetrahydrofolate (meTHF) in a pocket near the catalytic residue Cys195. The catalytic loop, which is composed of amino-acid residues 181–197, can adopt two distinct conformations related by a 180° rotation. In the active conformation Cys195 is close to the active site, while in the inactive conformation it is rotated and Cys195 is too distant from the active site for catalysis. Several hTS structures, either native or engineered, have been solved in the active conformation in complex with ligands or inhibitors and at different salt concentrations. However, apo hTS structures have been solved in an inactive conformation in high-salt and low-salt conditions (PDB entries 1ypv, 4h1i, 4gyh, 3egy and 3ehi). Here, the structure of apo hTS crystallized in the active form with sulfate ions coordinated by the arginine residue that binds dUMP is reported.

Phosphate-binding protein from Polaromonas JS666: purification, characterization, crystallization and sulfur SAD phasing


Phosphate-binding proteins (PBPs) are key proteins that belong to the bacterial ABC-type phosphate transporters. PBPs are periplasmic (or membrane-anchored) proteins that capture phosphate anions from the environment and release them to the transmembrane transporter. Recent work has suggested that PBPs have evolved for high affinity as well as high selectivity. In particular, a short, unique hydrogen bond between the phosphate anion and an aspartate residue has been shown to be critical for selectivity, yet is not strictly conserved in PBPs. Here, the PBP from Polaromonas JS666 is focused on. Interestingly, this PBP is predicted to harbor different phosphate-binding residues to currently known PBPs. Here, it is shown that the PBP from Polaromonas JS666 is capable of binding phosphate, with a maximal binding activity at pH 8. Its structure is expected to reveal its binding-cleft configuration as well as its phosphate-binding mode. Here, the expression, purification, characterization, crystallization and X-ray diffraction data collection to 1.35 Å resolution of the PBP from Polaromonas JS666 are reported.

The crystal structure of human DEAH-box RNA helicase 15 reveals a domain organization of the mammalian DEAH/RHA family


DEAH-box RNA helicase 15 (DHX15) plays important roles in RNA metabolism, including in splicing and in ribosome biogenesis. In addition, mammalian DHX15 also mediates the innate immune sensing of viral RNA. However, structural information on this protein is not available, although the structure of the fungal orthologue of this protein, Prp43, has been elucidated. Here, the crystal structure of the ADP-bound form of human DHX15 is reported at a resolution of 2.0 Å. This is the first structure to be revealed of a member of the mammalian DEAH-box RNA helicase (DEAH/RHA) family in a nearly complete form, including the catalytic core consisting of the two N-terminal RecA domains and the C-terminal regulatory domains (CTD). The ADP-bound form of DHX15 displayed a compact structure, in which the RecA domains made extensive contacts with the CTD. Notably, a potential RNA-binding site was found on the surface of a RecA domain with positive electrostatic potential. Almost all structural features were conserved between the fungal Prp43 and the human DHX15, suggesting that they share a fundamentally common mechanism of action and providing a better understanding of the specific mammalian functions of DHX15.

Crystal structure of N-acetylmannosamine kinase from Fusobacterium nucleatum


Sialic acids comprise a varied group of nine-carbon amino sugars that are widely distributed among mammals and higher metazoans. Some human commensals and bacterial pathogens can scavenge sialic acids from their environment and degrade them for use as a carbon and nitrogen source. The enzyme N-acetylmannosamine kinase (NanK; EC belongs to the transcriptional repressors, uncharacterized open reading frames and sugar kinases (ROK) superfamily. NanK catalyzes the second step of the sialic acid catabolic pathway, transferring a phosphate group from adenosine 5′-triphosphate to the C6 position of N-acetylmannosamine to generate N-acetylmannosamine 6-phosphate. The structure of NanK from Fusobacterium nucleatum was determined to 2.23 Å resolution by X-ray crystallography. Unlike other NanK enzymes and ROK family members, F. nucleatum NanK does not have a conserved zinc-binding site. In spite of the absence of the zinc-binding site, all of the major structural features of enzymatic activity are conserved.

Crystal structure of the multiple antibiotic resistance regulator MarR from Clostridium difficile


Regulators of multiple antibiotic resistance (MarRs) are key players against toxins in prokaryotes. MarR homologues have been identified in many bacterial and archaeal species which pose daunting antibiotic resistance issues that threaten public health. The continuous prevalence of Clostridium difficile infection (CDI) throughout the world is associated with the abuse of antibiotics, and antibiotic treatments of CDI have limited effect. In the genome of C. difficile strain 630, the marR gene (ID 4913953) encodes a MarR protein. Here, MarR from C. difficile (MarRC.difficile) was subcloned and crystallized for the first time. MarRC.difficile was successfully expressed in Escherichia coli in a soluble form and was purified to near-homogeneity (>95%) by a two-step purification protocol. The structure of MarRC.difficile has been solved at 2.3 Å resolution. The crystal belonged to the monoclinic space group P43212, with unit-cell parameters a = b = 66.569, c = 83.654 Å. The structure reported reveals MarRC.difficile to be a dimer, with each subunit consisting of six α-helices and three antiparallel β-hairpins. MarRC.difficile shows high structural similarity to the MarR proteins from E. coli and Staphylococcus aureus, indicating that MarRC.difficile might be a DNA-binding protein.

Crystal structure of recombinant phosphoribosylpyrophosphate synthetase 2 from Thermus thermophilus HB27 complexed with ADP and sulfate ions


Phosphoribosylpyrophosphate synthetase (PRPPS) from the thermophilic bacterial strain Thermus thermophilus HB27 catalyzes the synthesis of phosphoribosylpyrophosphate from ribose 5-phosphate and ATP, and belongs to the class I PRPPSs. The three-dimensional structure of the recombinant enzyme was solved at 2.2 Å resolution using crystals grown in microgravity from protein solution containing ATP, magnesium and sulfate ions. An ADP molecule was located in the active site of each subunit of the hexameric enzyme molecule and sulfate ions were located in both the active and allosteric sites. It was found that the catalytic loop that restricts the active-site area and is usually missing from the electron-density map of class I PRPPSs adopts different conformations in three independent subunits in T. thermophilus PRPPS. A closed conformation of the active site was found in one of subunits where the highly ordered catalytic β-hairpin delivers the Lys and Arg residues that are essential for activity directly to the ADP molecule, which occupies the ATP-binding site. A comparison of the conformations of the catalytic loop in the three independent subunits reveals a possible mode of transition from the open to the closed state of the active site during the course of the catalyzed reaction.