The structure of a bifunctional deaminase/reductase involved in riboflavin biosynthesis in the pathogen A. baumannii has been determined in two crystal forms.
The bifunctional diaminohydroxyphosphoribosylaminopyrimidine deaminase/5-amino-6-(5-phosphoribosylamino)uracil reductase (RibD) represents a potential antibacterial drug target. The structure of recombinant Acinetobacter baumannii RibD is reported in orthorhombic and tetragonal crystal forms at 2.2 and 2.0 Å resolution, respectively. Comparisons with orthologous structures in the Protein Data Bank indicated close similarities. The tetragonal crystal form was obtained in the presence of guanosine monophosphate, which surprisingly was observed to occupy the adenine-binding site of the reductase domain.
bifunctional deaminase/reductase; Acinetobacter baumannii; RibD; riboflavin biosynthesis
The X-ray crystal structure of PatF from P. didemni was solved by the single-wavelength anomalous diffraction method to a resolution of 2.13 Å.
Patellamides are macrocyclic peptides with potent biological effects and are a subset of the cyanobactins. Cyanobactins are natural products that are produced by a series of enzymatic transformations and a common modification is the addition of a prenyl group. Puzzlingly, the pathway for patellamides in Prochloron didemni contains a gene, patF, with homology to prenylases, but patellamides are not themselves prenylated. The structure of the protein PatF was cloned, expressed, purified and determined. Prenylase activity could not be demonstrated for the protein, and examination of the structure revealed changes in side-chain identity at the active site. It is suggested that these changes have inactivated the protein. Attempts to mutate these residues led to unfolded protein.
patellamide; cyanobactins; natural products; prenyltransferases
The structure of ErpC, a member of the complement regulator-acquiring surface protein family from B. burgdorferi, has been solved, providing insights into the strategies of complement evasion by this zoonotic bacterium and suggesting a common architecture for other members of this protein family.
Borrelia burgdorferi is a spirochete responsible for Lyme disease, the most commonly occurring vector-borne disease in Europe and North America. The bacterium utilizes a set of proteins, termed complement regulator-acquiring surface proteins (CRASPs), to aid evasion of the human complement system by recruiting and presenting complement regulator factor H on its surface in a manner that mimics host cells. Presented here is the atomic resolution structure of a member of this protein family, ErpC. The structure provides new insights into the mechanism of recruitment of factor H and other factor H-related proteins by acting as a molecular mimic of host glycosaminoglycans. It also describes the architecture of other CRASP proteins belonging to the OspE/F-related paralogous protein family and suggests that they have evolved to bind specific complement proteins, aiding survival of the bacterium in different hosts.
BbCRASP-4; Borrelia burgdorferi; ErpC; factor H; complement
B. burgdorferi binds complement factor H using a dimeric surface protein, CspA (BbCRASP-1). Presented here is a new structure of CspA that suggests that there is a degree of flexibility between subunits which may have implications for complement regulator binding.
Borrelia burgdorferi has evolved many mechanisms of evading the different immune systems across its range of reservoir hosts, including the capture and presentation of host complement regulators factor H and factor H-like protein-1 (FHL-1). Acquisition is mediated by a family of complement regulator-acquiring surface proteins (CRASPs), of which the atomic structure of CspA (BbCRASP-1) is known and shows the formation of a homodimeric species which is required for binding. Mutagenesis studies have mapped a putative factor H binding site to a cleft between the two subunits. Presented here is a new atomic structure of CspA which shows a degree of flexibility between the subunits which may be critical for factor H scavenging by increasing access to the binding interface and allows the possibility that the assembly can clamp around the bound complement regulators.
complement; factor H; BbCRASP-1; CspA; Borrelia burgdorferi
The structure of copper amine oxidase 1 from H. polymorpha in its metal-free precursor (apo) form is reported along with structures of the apo protein in complex with CuI and CoII.
Copper amine oxidases (CAOs) catalyze the oxidative deamination of primary amines to their corresponding aldehydes, with the concomitant reduction of O2 to H2O2. Catalysis requires two cofactors: a mononuclear copper center and the cofactor 2,4,5-trihydroxyphenylalanine quinone (TPQ). TPQ is synthesized through the post-translational modification of an endogenous tyrosine residue and requires only oxygen and copper to proceed. TPQ biogenesis in CAO can be supported by alternate metals, albeit at decreased rates. A variety of factors are thought to contribute to the degree to which a metal can support TPQ biogenesis, including Lewis acidity, redox potential and electrostatic stabilization capability. The crystal structure has been solved of one of two characterized CAOs from the yeast Hansenula polymorpha (HPAO-1) in its metal-free (apo) form, which contains an unmodified precursor tyrosine residue instead of fully processed TPQ (HPAO-1 was denoted HPAO in the literature prior to 2010). Structures of apoHPAO-1 in complex with CuI and CoII have also been solved, providing structural insight into metal binding prior to biogenesis.
amine oxidases; biogenesis; TPQ; copper; cofactor
The structure of the catalytic chain of Methanococcus jannaschii aspartate transcarbamoylase has been determined in a hexagonal crystal form and gives insight into the possible paths that the substrate carbamoyl phosphate may follow to reach the active site during catalysis.
Crystals of the catalytic chain of Methanococcus jannaschii aspartate transcarbamoylase (ATCase) grew in the presence of the regulatory chain in the hexagonal space group P6322, with one monomer per asymmetric unit. This is the first time that crystals with only one monomer in the asymmetric unit have been obtained; all known structures of the catalytic subunit contain several crystallographically independent monomers. The symmetry-related chains form the staggered dimer of trimers observed in the other known structures of the catalytic subunit. The central channel of the catalytic subunit contains a sulfate ion and a K+ ion as well as a glycerol molecule at its entrance. It is possible that it is involved in channeling carbamoyl phosphate (CP) to the active site of the enzyme. A second sulfate ion near Arg164 is near the second CP position in the wild-type Escherichia coli ATCase structure complexed with CP. It is suggested that this position may also be in the path that CP takes when binding to the active site in a partial diffusion process at 310 K. Additional biochemical studies of carbamoylation and the molecular organization of this enzyme in M. jannaschii will provide further insight into these points.
aspartate transcarbamoylase; Methanococcus jannaschii; catalytic chain; enzyme mechanisms; protein structure-function
A novel hyperactive antifreeze protein from R. inquisitor (RiAFP) has been overexpressed, purified and crystallized. A complete native X-ray diffraction data set was recorded to 1.3 Å resolution.
Antifreeze proteins (AFPs) are a specialized evolutionary adaptation of a variety of bacteria, fish, arthropods and other organisms to inhibit ice-crystal growth for survival in harsh subzero environments. The recently reported novel hyperactive AFP from Rhagium inquisitor (RiAFP) is the second distinct type of AFP in beetles and its structure could reveal important molecular insights into the evolution of AFPs. For this purpose, RiAFP was overexpressed in Escherichia coli, purified and crystallized at 293 K using a combination of 23% PEG 3350 and 0.2 M ammonium sulfate as a precipitant. X-ray diffraction data were collected to 1.3 Å resolution using a synchrotron-radiation source. The crystals belonged to the trigonal space group P3121 (or P3221), with unit-cell parameters a = b = 46.46, c = 193.21 Å.
antifreeze proteins; Rhagium inquisitor
The expression, purification and crystallization of the alginate lyase AlgL from P. aeruginosa is described. The crystals diffracted to a resolution of 1.64 Å.
The periplasmic alginate lyase AlgL is essential for the synthesis and export of the exopolysaccharide alginate in Pseudomonas sp. and also plays a role in its depolymerization. P. aeruginosa PAO1 AlgL has been overexpressed and purified and diffraction-quality crystals were grown using the hanging-drop vapour-diffusion method. The crystals grew as thin plates, with unit-cell parameters a = 56.4, b = 59.6, c = 102.1 Å, α = β = γ = 90°. The AlgL crystals exhibited the symmetry of space group P212121 and diffracted to a minimum d-spacing of 1.64 Å. Based on the Matthews coefficient (V
M = 2.20 Å3 Da−1), one molecule is estimated to be present in the asymmetric unit.
AlgL; Pseudomonas aeruginosa; alginate biosynthetic complex; biofilms; exopolysaccharides; alginate lyases
The utility of differential scanning fluorimetry for homogeneity assessment and crystallization improvement of PLP-dependent enzymes is demonstrated using the potential drug target BioA from M. tuberculosis.
Differential scanning fluorimetry (DSF) is a practical and accessible technique that allows the assessment of multiphasic unfolding behavior resulting from subsaturating binding of ligands. Multiphasic unfolding is indicative of a heterogenous protein solution, which frequently interferes with crystallization and complicates functional characterization of proteins of interest. Along with UV–Vis spectroscopy, DSF was used to guide purification and crystallization improvements for the pyridoxal 5′-phosphate (PLP) dependent transaminase BioA from Mycobacterium tuberculosis. The incompatibility of the primary amine-containing buffer 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris) and PLP was identified as a significant contributor to heterogeneity. It is likely that the utility of DSF for ligand-binding assessment is not limited to the cofactor PLP but will be applicable to a variety of ligand-dependent enzymes.
differential scanning fluorimetry; ThermoFluor; crystallization optimization; pyridoxal 5′-phosphate; UV–Vis spectroscopy
The structure of the third catalytic domain of the human protein disulfide isomerase ERp46 has been determined to 2.0 Å resolution.
Protein disulfide isomerases are responsible for catalyzing the proper oxidation and isomerization of disulfide bonds of newly synthesized proteins in the endoplasmic reticulum. Here, the crystal structure of the third catalytic domain of protein disulfide isomerase ERp46 (also known as protein disulfide isomerase A5 and TXNDC5) was determined to 2.0 Å resolution. The structure shows a typical thioredoxin-like fold, but also identifies regions of high structural variability. In particular, the loop between helix α2 and strand β3 adopts strikingly different conformations among the five chains of the asymmetric unit. Cys381 and Cys388 form a structural disulfide and its absence in one of the molecules leads to dramatic conformational changes. The tryptophan residue Trp349 of this molecule inserts into the cavity formed by helices α1 and α3 of a neighbouring molecule, potentially mimicking the interactions of ERp46 with misfolded substrates.
thioredoxin-like domains; endoplasmic reticulum; ERp46; protein disulfide isomerases
Structural data for the ternary complex of P. carinii DHFR with NADPH and the potent inhibitor PY1014 reveal conformational changes that help to explain the weaker potency and selectivity of the pyrido[2,3-d]pyrimidine scaffold compared with the homologous pyrimidine series of antifolates.
Structural data are reported for 2,4-diamino-6-[2-(5-carboxypent-1-yn-1-yl)-5-methoxybenzyl]-5-methylpyrido[2,3-d]pyrimidine (PY1014) complexed with Pneumocystis carinii dihydrofolate reductase (pcDHFR) refined to 1.8 Å resolution. These data reveal that the carboxylate of the ω-carboxyalkynyl side chain of PY1014, the most pcDHFR-selective analog in this series, forms ionic interactions with the conserved Arg75 in the substrate-binding pocket of pcDHFR. The reversal of the 2′,5′-substitution pattern of this analog compared with the highly selective diaminopyrimidine analog PY1011 (i.e. the 5′-pentynylcarboxy-5′-methoxy pattern of PY1014 versus the 3′,4′-dimethoxy-5′-pentynylcarboxy pattern of PY1011) is necessary to achieve optimal interaction with Arg75 as observed in other structures. The larger diaminopyrido[2,3-d]pyrimidine ring of PY1014 places the 5′-methoxy group closer to Leu25 and Ser64 than does the diaminopyrimidine ring of PY1011. The 5′-methoxy O atom forms a hydrogen bond to the amide of Leu25 (O⋯N, 2.7 Å) and the 5′-methoxy methyl group makes a hydrophobic contact of 3.1 Å with Cβ of Ser64. Although the IC50 values of PY1014 and PY1011 are similar, inhibition data show that the selectivity of PY1011 for pcDHFR is significantly greater. The greater selectivity for pcDHFR compared with mammalian DHFR of these inhibitors is also influenced by the enhanced hydrophobic interactions of the side-chain methylene atoms with Phe69 of pcDHFR compared with Asn64 of mammalian DHFR.
dihydrofolate reductase; inhibitors; Pneumocystis carinii
Crystals of the SCAN domain of Zfp206 are tetragonal, belonging to space group I422 with unit-cell parameters a = 67.57, c = 87.54 Å and one molecule in the asymmetric unit, and diffract to 1.85 Å resolution.
Zfp206 (also named Zscan10) is a transcription factor that plays an important role in maintaining the pluripotent state of embryonic stem cells. Zfp206 is a member of the SCAN-domain family of C2H2 zinc-finger transcription factors. The SCAN domain is a highly conserved motif of 84 residues which mediates the self-association of and heterodimerization between SCAN-domain family transcription factors. The SCAN domain may therefore be the key to the selective oligomerization of and may combinatorially enhance the regulatory versatility of C2H2 zinc fingers. This paper describes crystallization attempts with the SCAN domain of Zfp206 (Zfp206SCAN) and optimization strategies to obtain diffraction-quality crystals. The best diffracting crystal was grown in a solution consisting of 0.3 M ammonium sulfate, 0.1 M Tris–HCl pH 8.6, 25% PEG 3350, 0.1 M ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA) using the hanging-drop vapour-diffusion technique. Optimized crystals diffracted to 1.85 Å resolution and belonged to space group I422, with unit-cell parameters a = 67.57, c = 87.54 Å. A Matthews analysis indicated the presence of one Zfp206SCAN molecule per asymmetric unit.
Zfp206; SCAN domain; transcription factors
AAC(6′)-Im is an N-acetyltransferase enzyme responsible for aminoglycoside resistance in E. faecium and E. coli isolates. Crystals of the kanamycin complex of this enzyme have been prepared and preliminary X-ray diffraction experiments have been undertaken.
Bacterial resistance to the aminoglycoside antibiotics is primarily the result of enzymatic deactivation of the drugs. The aminoglycoside N-acetyltransferases (AACs) are a large family of bacterial enzymes that are responsible for coenzyme-A-facilitated acetylation of aminoglycosides. The gene encoding one of these enzymes, AAC(6′)-Im, has been cloned and the protein (comprising 178 amino-acid residues) was expressed in Escherichia coli, purified and crystallized as the kanamycin complex. Synchrotron diffraction data to approximately 2.0 Å resolution were collected from a crystal of this complex on beamline BL12-2 at SSRL (Stanford, California, USA). The crystals belonged to the hexagonal space group P65, with approximate unit-cell parameters a = 107.75, c = 37.33 Å, and contained one molecule in the asymmetric unit. Structure determination is under way using molecular replacement.
N-acetyltransferases; AAC(6′)-Im; aminoglycoside resistance
The gene encoding the hypothetical DUF1811-family protein GK0453 from G. kaustophilus was cloned and expressed. The crystal structure of the protein was determined by the molecular-replacement method and was refined to 2.2 Å resolution.
The crystal structure of a conserved hypothetical protein, GK0453, from Geobacillus kaustophilus has been determined to 2.2 Å resolution. The crystal belonged to space group P43212, with unit-cell parameters a = b = 75.69, c = 64.18 Å. The structure was determined by the molecular-replacement method and was refined to a final R factor of 22.6% (R
free = 26.3%). Based on structural homology, the GK0453 protein possesses two independent binding sites and hence it may simultaneously interact with two proteins or with a protein and a nucleic acid.
DUF1811; Geobacillus kaustophilus; helix–turn–helix motif; β-barrel domain
In order to characterize the type IV pili of nontypeable Haemophilus influenzae, an attempt to solve the atomic structure of the major pilin subunit PilA was initiated. A 1.73 Å resolution X-ray diffraction data set was collected from native N-terminally truncated PilA (ΔN-PilA).
The type IV pili of nontypeable Haemophilus influenzae (NTHi) are involved in twitching motility, adherence, competence and biofilm formation. They are potential virulence factors for this important human pathogen and are thus considered to be vaccine targets. To characterize these pili, an attempt to solve the atomic structure of the major pilin subunit PilA was initiated. A 1.73 Å resolution X-ray diffraction data set was collected from native N-terminally truncated PilA (ΔN-PilA). Data processing indicated a hexagonal crystal system, which was determined to belong to space group P61 or P65 based on the systematic absences and near-perfect twinning of the crystal. The unit-cell parameters were a = b = 68.08, c = 197.03 Å with four molecules in the asymmetric unit, giving a solvent content of 50%. Attempts to solve the ΔN-PilA structure by molecular replacement with existing type IV pilin and type II secretion pseudopilin structures are in progress.
nontypeable Haemophilus influenzae; type IV pili; otitis media
The crystal structure of inosine 5′-monophosphate dehydrogenase from P. aeruginosa has been determined to 2.25 Å resolution.
Inosine 5′-monophosphate dehydrogenase (IMPDH) represents a potential antimicrobial drug target. The crystal structure of recombinant Pseudomonas aeruginosa IMPDH has been determined to a resolution of 2.25 Å. The structure is a homotetramer of subunits dominated by a (β/α)8-barrel fold, consistent with other known structures of IMPDH. Also in common with previous work, the cystathionine β-synthase domains, residues 92–204, are not present in the model owing to disorder. However, unlike the majority of available structures, clearly defined electron density exists for a loop that creates part of the active site. This loop, composed of residues 297–315, links α8 and β9 and carries the catalytic Cys304. P. aeruginosa IMPDH shares a high level of sequence identity with bacterial and protozoan homologues, with residues involved in binding substrate and the NAD+ cofactor being conserved. Specific differences that have been proven to contribute to selectivity against the human enzyme in a study of Cryptosporidium parvum IMPDH are also conserved, highlighting the potential value of IMPDH as a drug target.
inosine 5′-monophosphate dehydrogenase; Pseudomonas aeruginosa; antimicrobial drug targets
This paper reports the successful purification, crystallization and preliminary structure solution of the transfer protein TraM from the Gram-positive conjugative plasmid pIP501.
The major means of horizontal gene spread (e.g. of antibiotic resistance) is conjugative plasmid transfer. It presents a serious threat especially for hospitalized and immuno-suppressed patients, as it can lead to the accelerated spread of bacteria with multiple antibiotic resistances. Detailed information about the process is available only for bacteria of Gram-negative (G−) origin and little is known about the corresponding mechanisms in Gram-positive (G+) bacteria. Here we present the purification, biophysical characterization, crystallization and preliminary structure determination of the TraM C-terminal domain (TraMΔ, comprising residues 190–322 of the full-length protein), a putative transfer protein from the G+ conjugative model plasmid pIP501. The crystals diffracted to 2.5 Å resolution and belonged to space group P1, with unit-cell parameters a = 39.21, b = 54.98, c = 93.47 Å, α = 89.91, β = 86.44, γ = 78.63° and six molecules per asymmetric unit. The preliminary structure was solved by selenomethionine single-wavelength anomalous diffraction.
Gram-positive; conjugative plasmid transfer; pIP501; TraM
The use of UV imaging as a means of locating protein crystals is a fairly new tool, however not suitable for all protein crystallization trials. Practical examples of the strengths and some of the pitfalls of the technology are presented.
High-throughput imaging of protein crystallization experiments with ultraviolet (UV) light has recently become commercially available and can enable crystallographers to differentiate between crystals of protein and those of salt, as the visualization of protein crystals is based on intrinsic tryptophan fluorescence. Unfortunately, UV imaging is not a panacea, as some protein crystals will not fluoresce under UV excitation and some salt crystals are UV-fluorescently active. As a new technology, there is little experience within the general community on how to use this technology effectively and what caveats to look out for. Here, an attempt is made to identify some of the common problems that may arise using UV-imaging technology by examining test proteins, common crystallization reagents and a range of proteins by assessing their UV–Vis absorbance spectra. Some pointers are offered as to which systems may not be appropriate for this methodology.
A focused strategy has been directed towards the structural characterization of selected proteins from the bacterial pathogen P. aeruginosa. The objective is to exploit the resulting structural data, in combination with ligand-binding studies, and to assess the potential of these proteins for early-stage antimicrobial drug discovery.
Bacterial infections are increasingly difficult to treat owing to the spread of antibiotic resistance. A major concern is Gram-negative bacteria, for which the discovery of new antimicrobial drugs has been particularly scarce. In an effort to accelerate early steps in drug discovery, the EU-funded AEROPATH project aims to identify novel targets in the opportunistic pathogen Pseudomonas aeruginosa by applying a multidisciplinary approach encompassing target validation, structural characterization, assay development and hit identification from small-molecule libraries. Here, the strategies used for target selection are described and progress in protein production and structure analysis is reported. Of the 102 selected targets, 84 could be produced in soluble form and the de novo structures of 39 proteins have been determined. The crystal structures of eight of these targets, ranging from hypothetical unknown proteins to metabolic enzymes from different functional classes (PA1645, PA1648, PA2169, PA3770, PA4098, PA4485, PA4992 and PA5259), are reported here. The structural information is expected to provide a firm basis for the improvement of hit compounds identified from fragment-based and high-throughput screening campaigns.
protein structure; Gram-negative bacteria; Pseudomonas aeruginosa; infectious diseases; structure-based inhibitor design
Three different truncation constructs of the S-layer protein SbsC containing domains crucial for self-assembly could be crystallized. Native data were collected for the three crystal forms from crystals that diffracted to 3.4, 2.8 and 1.5 Å resolution.
The Gram-positive bacterium Geobacillus stearothermophilus ATCC 12980 is completely covered with a two-dimensional crystalline monolayer composed of the S-layer protein SbsC. In order to complete the structure of the full-length protein, additional soluble constructs containing the crucial domains for self-assembly have been successfully cloned, expressed and purified. Crystals obtained from three different recombinant constructs yielded diffraction to 3.4, 2.8 and 1.5 Å resolution. Native data have been collected.
S-layer; self-assembly; Geobacillus stearothermophilus
Obituary for Dame Louise Napier Johnson.
A corrigendum to the article by Dutta et al.
[(2012) Acta Cryst. F68, 786–789].
In the article by Dutta et al. [(2012) Acta Cryst. F68, 786–789] two citations were given erroneously. These are now corrected.
FabG4; high-molecular-weight ketoacyl reductases; Mycobacterium tuberculosis; corrigendum
An addendum to the article by Do et al. [(2012) Acta Cryst. F68, 806–809].
An additional acknowledgement is published for the article by Do et al. [(2012) Acta Cryst. F68, 806–809].
antifreeze proteins; ice-binding proteins; Flavobacterium frigoris PS1; psychrophilic bacteria; addendum