A phosphomimetic mutation in subunit ∊ dramatically increases reproducibility for crystallization of Escherichia coli ATP synthase catalytic complex (F1) (subunit composition α3β3γ∊). Diffraction data were collected to ∼3.15 Å resolution using synchrotron radiation.
The bacterial ATP synthase (FOF1) of Escherichia coli has been the prominent model system for genetics, biochemical and more recently single-molecule studies on F-type ATP synthases. With 22 total polypeptide chains (total mass of ∼529 kDa), E. coli FOF1 represents nature’s smallest rotary motor, composed of a membrane-embedded proton transporter (FO) and a peripheral catalytic complex (F1). The ATPase activity of isolated F1 is fully expressed by the α3β3γ ‘core’, whereas single δ and ∊ subunits are required for structural and functional coupling of E. coli F1 to FO. In contrast to mitochondrial F1-ATPases that have been determined to atomic resolution, the bacterial homologues have proven very difficult to crystallize. In this paper, we describe a biochemical strategy that led us to improve the crystallogenesis of the E. coli F1-ATPase catalytic core. Destabilizing the compact conformation of ∊’s C-terminal domain with a phosphomimetic mutation (∊S65D) dramatically increased crystallization success and reproducibility, yielding crystals of E. coli F1 that diffract to ∼3.15 Å resolution.
Crystallization; FOF1-ATP synthase; phosphomimetic mutation; ∊ subunit
The crystallization and initial diffraction analysis of human Drp1 GTPase-GED fusion protein are reported.
The mechano-enzyme dynamin-related protein 1 plays an important role in mitochondrial fission and is implicated in cell physiology. Dysregulation of Drp1 is associated with abnormal mitochondrial dynamics and neuronal damage. Drp1 shares structural and functional similarities with dynamin 1 with respect to domain organization, ability to self-assemble into spiral-like oligomers and GTP-cycle-dependent membrane scission. Structural studies of human dynamin-1 have greatly improved the understanding of this prototypical member of the dynamin superfamily. However, high-resolution structural information for full-length human Drp1 covering the GTPase domain, the middle domain and the GTPase effector domain (GED) is still lacking. In order to obtain mechanistic insights into the catalytic activity, a nucleotide-free GTPase-GED fusion protein of human Drp1 was expressed, purified and crystallized. Initial X-ray diffraction experiments yielded data to 2.67 Å resolution. The hexagonal-shaped crystals belonged to space group P21212, with unit-cell parameters a = 53.59, b = 151.65, c = 43.53 Å, one molecule per asymmetric unit and a solvent content of 42%. Expression of selenomethionine-labelled protein is currently in progress. Here, the expression, purification, crystallization and X-ray diffraction analysis of the Drp1 GTPase-GED fusion protein are presented, which form a basis for more detailed structural and biophysical analysis.
dynamin-related protein 1; GTPase domain; GTPase effector domain
The pH 6 antigen Psa displayed on the surface of Yersinia pestis, the bacterium that causes plague in humans, consists of polymers of a single protein subunit termed PsaA. Donor-strand complemented PsaA was purified and crystallized.
Yersinia pestis has been responsible for a number of high-mortality epidemics throughout human history. Like all other bacterial infections, the pathogenesis of Y. pestis begins with the attachment of bacteria to the surface of host cells. At least five surface proteins from Y. pestis have been shown to interact with host cells. Psa, the pH 6 antigen, is one of them and is deployed on the surface of bacteria as thin flexible fibrils that are the result of the polymerization of a single PsaA pilin subunit. Here, the crystallization of recombinant donor-strand complemented PsaA by the hanging-drop vapor-diffusion method is reported. X-ray diffraction data sets were collected to 1.9 Å resolution from a native crystal and to 1.5 Å resolution from a bromide-derivatized crystal. These crystals displayed the symmetry of the orthorhombic space group P2221, with unit-cell parameters a = 26.3, b = 54.6, c = 102.1 Å. Initial phases were derived from single isomorphous replacement with anomalous scattering experiments, resulting in an electron-density map that showed a single molecule in the crystallographic asymmetric unit. Sequence assignment was aided by residues binding to bromide ions of the heavy-atom derivative.
Psa; pilins; Yersinia pestis; PsaA
A proteolytically stable fragment of a plasmid replication initiation protein from the thermophile G. stearothermophilus has been biochemically characterized, crystallized and diffraction data collected to a resolution of 2.5 Å.
Antibiotic resistance in bacterial pathogens poses an ever-increasing risk to human health. In antibiotic-resistant strains of Staphylococcus aureus this resistance often resides in extra-chromosomal plasmids, such as those of the pT181 family, which replicate via a rolling-circle mechanism mediated by a plasmid-encoded replication initiation protein. Currently, there is no structural information available for the pT181-family Rep proteins. Here, the crystallization of a catalytically active fragment of a homologous replication initiation protein from the thermophile Geobacillus stearothermophilus responsible for the replication of plasmid pSTK1 is reported. Crystals of the RepSTK1 fragment diffracted to a resolution of 2.5 Å and belonged to space group P212121.
rolling-circle replication; plasmid pSTK1; plasmid pT181; replication initiation proteins; Staphylococcus aureus; Geobacillus stearothermophilus
The crystal structure of C. jejuni anabolic ornithine transcarbamoylase has been determined at a resolution of 2.7 Å in an unliganded state.
Anabolic ornithine transcarbamoylase (aOTC) catalyzes the reaction between carbamoyl phosphate (CP) and l-ornithine (ORN) to form l-citrulline and phosphate in the urea cycle and l-arginine biosynthesis. The crystal structure of unliganded aOTC from Campylobacter jejuni (Cje aOTC) was determined at 2.7 Å resolution and refined to an R
work of 20.3% and an R
free of 24.0%. Cje aOTC is a trimer that forms a head-to-head pseudohexamer in the asymmetric unit. Each monomer is composed of an N-terminal CP-binding domain and a C-terminal ORN-binding domain joined by two interdomain helices. The Cje aOTC structure presents an open conformation of the enzyme with a relatively flexible orientation of the ORN-binding domain respective to the CP-binding domain. The conformation of the B2–H3 loop (residues 68–78), which is involved in binding CP in an adjacent subunit of the trimer, differs from that seen in homologous proteins with CP bound. The loop containing the ORN-binding motif (DxxxSMG, residues 223–230) has a conformation that is different from those observed in unliganded OTC structures from other species, but is similar to those in structures with bound ORN analogs. The major differences in tertiary structure between Cje aOTC and human aOTC are described.
Campylobacter jejuni; anabolic ornithine transcarbamoylases; carbamoyl phosphate; l-ornithine; l-citrulline; arginine biosynthesis; biocatalysis
AIM2 is an innate immune sensor of microbial double-stranded DNA. The HIN-200 domain of mouse AIM2 bound to a 15 bp and an 18 bp dsDNA were crystallized and diffract to about 4.0 Å.
AIM2 (absent in melanoma 2) is an innate immune receptor for cytosolic double-stranded DNA (dsDNA). The engagement of dsDNA by AIM2 activates the AIM2 inflammasome, resulting in the cleavage of pro-interleukin-1β by caspase-1. The DNA-binding HIN-200 domain of mouse AIM2 bound to a 15 bp dsDNA and to an 18 bp dsDNA was purified and crystallized. The AIM2 HIN-200 domain in complex with the 15 bp DNA crystallized in the cubic space group I23 or I213, with unit-cell parameter a = 235.60 Å. The complex of the AIM2 HIN-200 domain and the 18 bp DNA crystallized in a similar unit cell. Diffraction data for the two complexes were collected to about 4.0 Å resolution. Mutagenesis and DNA-binding studies suggest that mouse AIM2 uses a similar surface to human AIM2 to recognize DNA.
mouse AIM2; HIN-200 domain; DNA binding
Comparison of bound and unbound DNA in protein–DNA co-crystal complexes reveals insights into controller-protein binding and DNA distortion in transcriptional regulation.
The controller protein of the type II restriction–modification (RM) system Esp1396I binds to three distinct DNA operator sequences upstream of the methyltransferase and endonuclease genes in order to regulate their expression. Previous biophysical and crystallographic studies have shown molecular details of how the controller protein binds to the operator sites with very different affinities. Here, two protein–DNA co-crystal structures containing portions of unbound DNA from native operator sites are reported. The DNA in both complexes shows significant distortion in the region between the conserved symmetric sequences, similar to that of a DNA duplex when bound by the controller protein (C-protein), indicating that the naked DNA has an intrinsic tendency to bend when not bound to the C-protein. Moreover, the width of the major groove of the DNA adjacent to a bound C-protein dimer is observed to be significantly increased, supporting the idea that this DNA distortion contributes to the substantial cooperativity found when a second C-protein dimer binds to the operator to form the tetrameric repression complex.
transcriptional regulation; DNA-binding proteins; helix–turn–helix motif; DNA distortion
The 23S rRNA methyltransferase RlmJ from E. coli has been cloned, expressed, purified and crystallized. X-ray diffraction data to 1.85 Å resolution have been collected from the apo RlmJ crystals.
Methyltransferase RlmJ uses the cofactor S-adenosylmethionine to methylate the exocyclic nitrogen N6 of nucleotide A2030 in 23S rRNA during ribosome assembly in Escherichia coli. RlmJ with a C-terminal hexahistidine tag was overexpressed in E. coli and purified as a monomer using Ni2+-affinity and size-exclusion chromatography. The recombinant RlmJ was crystallized using the sitting-drop vapour-diffusion method and a full data set was collected to 1.85 Å resolution from a single apo crystal. The crystals belonged to space group P21, with unit-cell parameters a = 46.9, b = 77.8, c = 82.5 Å, β = 104°. Data analysis suggested two molecules per asymmetric unit and a Matthews coefficient of 2.20 Å3 Da−1.
RlmJ; S-adenosylmethionine; methyltransferases; 23S rRNA; m6A2030; ribosome assembly; Escherichia coli
Serial femtosecond X-ray (SFX) diffraction extending beyond 6 Å resolution using T. thermophilus 30S ribosomal subunit crystals is reported.
High-resolution ribosome structures determined by X-ray crystallography have provided important insights into the mechanism of translation. Such studies have thus far relied on large ribosome crystals kept at cryogenic temperatures to reduce radiation damage. Here, the application of serial femtosecond X-ray crystallography (SFX) using an X-ray free-electron laser (XFEL) to obtain diffraction data from ribosome microcrystals in liquid suspension at ambient temperature is described. 30S ribosomal subunit microcrystals diffracted to beyond 6 Å resolution, demonstrating the feasibility of using SFX for ribosome structural studies. The ability to collect diffraction data at near-physiological temperatures promises to provide fundamental insights into the structural dynamics of the ribosome and its functional complexes.
30S ribosomal subunit; serial femtosecond X-ray crystallography; X-ray free-electron laser; ribosome
A correction is made to the article by Harris et al. [(2010) Acta Cryst. F66, 938–940].
The article by Harris et al. [(2010) Acta Cryst. F66, 938–940] is corrected.
enolase; Lactobacillus jensenii; Lactobacillus gasseri; corrigendum
The crystal structure of A. pernix fibrillarin includes a tightly bound S-adenosyl-l-methionine molecule.
Fibrillarin is the key methyltransferase associated with the C/D class of small nuclear ribonucleoproteins (snRNPs) and participates in the preliminary step of pre-ribosomal rRNA processing. This molecule is found in the fibrillar regions of the eukaryotic nucleolus and is involved in methylation of the 2′-O atom of ribose in rRNA. Human fibrillarin contains an N-terminal GAR domain, a central RNA-binding domain comprising an RNP-2-like superfamily consensus sequence and a catalytic C-terminal helical domain. Here, Aeropyrum pernix fibrillarin is described, which is homologous to the C-terminal domain of human fibrillarin. The protein was crystallized with an S-adenosyl-l-methionine (SAM) ligand bound in the active site. The molecular structure of this complex was solved using X-ray crystallography at a resolution of 1.7 Å using molecular replacement with fibrillarin structural homologs. The structure shows the atomic details of SAM and its active-site interactions; there are a number of conserved residues that interact directly with the cofactor. Notably, the adenine ring of SAM is stabilized by π–π interactions with the conserved residue Phe110 and by electrostatic interactions with the Asp134, Ala135 and Gln157 residues. The π–π interaction appears to play a critical role in stabilizing the association of SAM with fibrillarin. Furthermore, comparison of A. pernix fibrillarin with homologous structures revealed different orientations of Phe110 and changes in α-helix 6 of fibrillarin and suggests key differences in its interactions with the adenine ring of SAM in the active site and with the C/D RNA. These differences may play a key role in orienting the SAM ligand for catalysis as well as in the assembly of other ribonucleoproteins and in the interactions with C/D RNA.
fibrillarin; S-adenosyl-l-methionine; methyltransferases; Aeropyrum pernix
Crystal structures of Mn2
2+- and Co2
2+-substituted human arginase I complexed with an unreactive analog of substrate l-arginine are described at 1.82 and 1.90 Å resolution, respectively.
Human arginase I (HAI) is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of l-arginine to form l-ornithine and urea through a metal-activated hydroxide mechanism. Since HAI regulates l-Arg bioavailability for NO biosynthesis, it is a potential drug target for the treatment of cardiovascular diseases such as atherosclerosis. X-ray crystal structures are now reported of the complexes of Mn2
2+-HAI and Co2
2+-HAI with l-2-amino-3-guanidinopropionic acid (AGPA; also known as dinor-l-arginine), an amino acid bearing a guanidinium side chain two methylene groups shorter than that of l-arginine. Hydrogen bonds to the α-carboxylate and α-amino groups of AGPA dominate enzyme–inhibitor recognition; the guanidinium group does not interact directly with the metal ions.
human arginase I; l-2-amino-3-guanidinopropionic acid; dinor-l-arginine
The therapeutic antibody MORAb-009 disrupts the interaction of mesothelin and the ovarian cancer antigen CA-125. Crystals have been grown of the Fab fragment derived from MORAb-009 and of its complex with an N-terminal fragment of mesothelin.
The mesothelin-specific monoclonal antibody MORAb-009 is capable of blocking the binding of mesothelin to CA-125 and displays promising anticancer potential. It is currently undergoing clinical trials. In order to understand the basis of the interaction between MORAb-009 and mesothelin at atomic resolution, both the Fab fragment of MORAb-009 and the complex between the Fab and an N-terminal fragment of mesothelin (residues 7–64) were crystallized. The crystals of the Fab diffracted X-rays to 1.75 Å resolution and had the symmetry of space group P41212, with unit-cell parameters a = b = 140.6, c = 282.0 Å. The crystals of the mesothelin–Fab complex diffracted to 2.6 Å resolution and belonged to the hexagonal space group P64, with unit-cell parameters a = b = 146.2, c = 80.9 Å. Structural analyses of these molecules are in progress.
mesothelin; MORAb-009; monoclonal antibodies
title change; editorial
The putative small terminase protein from the thermostable bacteriophage G20C has been produced, purified and crystallized.
The assembly of double-stranded DNA bacteriophages is dependent on a small terminase protein that normally plays two important roles. Firstly, the small terminase protein specifically recognizes viral DNA and recruits the large terminase protein, which makes the initial cut in the dsDNA. Secondly, once the complex of the small terminase, the large terminase and the DNA has docked to the portal protein, and DNA translocation into a preformed empty procapsid has begun, the small terminase modulates the ATPase activity of the large terminase. Here, the putative small terminase protein from the thermostable bacteriophage G20C, which infects the Gram-negative eubacterium Thermus thermophilus, has been produced, purified and crystallized. Size-exclusion chromatography–multi-angle laser light scattering data indicate that the protein forms oligomers containing nine subunits. Crystals diffracting to 2.8 Å resolution have been obtained. These belonged to space group P212121, with unit-cell parameters a = 94.31, b = 125.6, c = 162.8 Å. The self-rotation function and Matthews coefficient calculations are consistent with the presence of a nine-subunit oligomer in the asymmetric unit.
putative small terminase; Thermus thermophilus; bacteriophage G20C
The crystallization and preliminary X-ray diffraction analysis at 1.25 Å resolution of free-ligand arginine kinase from the Pacific whiteleg shrimp Litopenaeus vannamei are reported. Crystals belong to space group P212121, phases were determined by molecular replacement and refinement was performed with Phenix.
Crystals of an unligated monomeric arginine kinase from the Pacific whiteleg shrimp Litopenaeus vannamei (LvAK) were successfully obtained using the microbatch method. Crystallization conditions and preliminary X-ray diffraction analysis to 1.25 Å resolution are reported. Data were collected at 100 K on NSLS beamline X6A. The crystals belonged to space group P212121, with unit-cell parameters a = 56.5, b = 70.2, c = 81.7 Å. One monomer per asymmetric unit was found, with a Matthews coefficient (V
M) of 2.05 Å3 Da−1 and 40% solvent content. Initial phases were determined by molecular replacement using a homology model of LvAK as the search model. Refinement was performed with PHENIX, with final R
work and R
free values of 0.15 and 0.19, respectively. Biological analysis of the structure is currently in progress.
arginine kinases; Litopenaeus vannamei
Construct engineering and crystallization of E. coli PgaB using in situ proteolysis and mass spectrometry is reported.
The periplasmic poly-β-1,6-N-acetyl-d-glucosamine (PNAG) de-N-acetylase PgaB from Escherichia coli was overexpressed and purified, but was recalcitrant to crystallization. Use of the in situ proteolysis technique produced crystals of PgaB, but these crystals could not be optimized for diffraction studies. By analyzing the initial crystal hits using SDS–PAGE and mass spectrometry, the boundaries of the protein species that crystallized were determined. The re-engineered protein target crystallized reproducibly without the addition of protease and with significantly increased crystal quality. Crystals of the selenomethionine-incorporated protein exhibited the symmetry of space group P212121 and diffracted to 2.1 Å resolution.
in situ proteolysis; protein modification and truncation; mass spectrometry; PgaB; poly-β-1,6-N-acetyl-d-glucosamine de-N-acetylase
crystallization communications; editorial
A truncated mutant missing the first 62 residues of the N-terminal, cytoplasmic domain of the sodium-bicarbonate NBCe1-A cotransporter crystallizes in space group P31 with pseudo-P3121 symmetry and a hemihedral twin fraction of 33.0%. Twinned fractions and twin-pair statistics over binned resolutions confirm that the calculated twin fraction is associated with hemihedral twinning and not to non-crystallographic symmetry.
NBCe1-A membrane-embedded macromolecules that cotransport sodium and bicarbonate ions across the bilayer serve to maintain acid–base homeostasis throughout the body. Defects result in a number of renal and eye disorders, including type-II renal tubular acidosis and cataracts. Here, crystals of a human truncated mutant of the cytoplasmic N-terminal domain of NBCe1 (Δ1–62NtNBCe1-A) are reported that diffract X-rays to 2.4 Å resolution. The crystal symmetry of Δ1–62NtNBCe1-A is of space group P31 with pseudo-P3121 symmetry and it has a hemihedral twin fraction of 33.0%. The crystals may provide insight into the pathogenic processes observed in a subset of patients with truncating and point mutations in the gene encoding NBCe1.
NBCe1; bicarbonate transport
T. maritima CheA P3-P4-P5 domains were crystallized in complex with CheW. Low-resolution diffraction data were collected to ∼8 Å using synchrotron X-ray radiation.
The CheA–CheW complex plays a key role in bacterial chemotaxis signal transduction by initiating phosphotransfer to response regulators via coupling to the chemoreceptors. CheA (P3-P4-P5 domains) and CheW from Thermotoga maritima were overexpressed in Escherichia coli and crystallized as a complex at 298 K using ammonium dihydrogen phosphate as a precipitant. X-ray diffraction data were collected to ∼8 Å resolution at 100 K using synchrotron radiation. The crystal belonged to space group I222 or I212121, with unit-cell parameters a = 184.2, b = 286.4, c = 327.7 Å. The asymmetric unit may contain six to ten CheA–CheW molecules.
CheA; histidine kinases; CheW; coupling proteins; bacterial chemotaxis; signal transduction; Thermotoga maritima
The catalytic domain of human ADAM-8 was expressed, purified and crystallized in complex with a hydroxamic acid inhibitor, batimastat. The crystal structure of the enzyme–inhibitor complex was refined to 2.1 Å resolution.
The role of ADAM-8 in cancer and inflammatory diseases such as allergy, arthritis and asthma makes it an attractive target for drug development. Therefore, the catalytic domain of human ADAM-8 was expressed, purified and crystallized in complex with a hydroxamic acid inhibitor, batimastat. The crystal structure of the enzyme–inhibitor complex was refined to 2.1 Å resolution. ADAM-8 has an overall fold similar to those of other ADAM members, including a central five-stranded β-sheet and a catalytic Zn2+ ion. However, unique differences within the S1′ binding loop of ADAM-8 are observed which might be exploited to confer specificity and selectivity to ADAM-8 competitive inhibitors for the treatment of diseases involving this enzyme.
ADAM-8; metalloproteases; inhibitors; batimastat; inflammation
The structure of the L166K variant of G protein-coupled receptor kinase 1 has been determined at 2.5 Å resolution in order to determine how a dimer interface observed in prior crystal structures influences the conformation of the enzyme and how the C-terminal amino acids are configured while in a monomeric state.
G protein-coupled receptor kinase 1 (GRK1 or rhodopsin kinase) phosphorylates activated rhodopsin and initiates a cascade of events that results in the termination of phototransduction by the receptor. Although GRK1 seems to be a monomer in solution, seven prior crystal structures of GRK1 revealed a similar domain-swapped dimer interface involving the C-terminus of the enzyme. The influence of this interface on the overall conformation of GRK1 is not known. To address this question, the crystalline dimer interface was disrupted with a L166K mutation and the structure of GRK1-L166K was determined in complex with Mg2+·ATP to 2.5 Å resolution. GRK1-L166K crystallized in a novel space group as a monomer and exhibited little overall conformational difference from prior structures of GRK1, although the C-terminal domain-swapped region had reorganized owing to loss of the dimer interface.
rhodopsin kinase; GRK1; RGS homology domain; dimerization
The crystal structure of a short-chain dehydrogenase/reductase from B. anthracis strain ‘Ames Ancestor’ is reported.
The crystal structure of a short-chain dehydrogenase/reductase from Bacillus anthracis strain ‘Ames Ancestor’ complexed with NADP has been determined and refined to 1.87 Å resolution. The structure of the enzyme consists of a Rossmann fold composed of seven parallel β-strands sandwiched by three α-helices on each side. An NADP molecule from an endogenous source is bound in the conserved binding pocket in the syn conformation. The loop region responsible for binding another substrate forms two perpendicular short helices connected by a sharp turn.
short-chain dehydrogenases/reductases; NADP; Bacillus anthracis
The amino-terminal domain of the Hsp70 co-chaperone Bag2 from M. musculus has been crystallized in native and selenomethionyl forms diffracting to 2.27 and 3.1 Å resolution, respectively.
Bag2, an atypical member of the Bag family of Hsp70 co-chaperones, acts as both an Hsp70 nucleotide-exchange factor and an inhibitor of the Hsp70-binding E3 ubiquitin ligase CHIP (carboxyl-terminus of Hsp70-interacting protein). The amino-terminal domain of Bag2 (Bag2-NTD), which is required for inhibition of CHIP, has no sequence homologs in the PDB. Native and selenomethionyl (SeMet) forms of Bag2-NTD were crystallized by hanging-drop vapor diffusion. Native Bag2-NTD crystals diffracted to 2.27 Å resolution and belonged to space group P212121, with unit-cell parameters a = 75.5, b = 84.7, c = 114.1 Å. SeMet Bag2-NTD crystals diffracted to 3.10 Å resolution and belonged to space group P212121, with unit-cell parameters a = 37.2, b = 53.3, c = 86.7 Å. Phases for the SeMet Bag2-NTD crystal were solved by single-wavelength anomalous diffraction. Initial phasing and model building using the 3.10 Å resolution SeMet Bag2-NTD data set suggested that Bag2-NTD forms a dimer and adopts a fold distinct from those of any domains annotated in the Pfam or SMART domain databases.
Bag2; selenomethionine; SAD phasing
A corrigendum to the article by Inoguchi et al. [(2013). Acta Cryst. F69, 393–398].
The affiliation of two authors in the article by Inoguchi et al. [(2013). Acta Cryst. F69, 393–398] is corrected.
hemoglobin; deer mouse; oxygen affinity; Peromyscus maniculatus; corrigendum