The crystal structure at 1.0 Å resolution of the cellulose-binding CBM3b from the major scaffoldin subunit ScaA of A. cellulolyticus is described.
The carbohydrate-binding module (CBM) of the major scaffoldin subunit ScaA of the cellulosome of Acetivibrio cellulolyticus is classified as a family 3b CBM and binds strongly to cellulose. The CBM3b was overexpressed, purified and crystallized, and its three-dimensional structure was determined. The structure contained a nickel-binding site located at the N-terminal region in addition to a ‘classical’ CBM3b calcium-binding site. The structure was also determined independently by the SAD method using data collected at the Ni-absorption wavelength of 1.48395 Å and even at a wavelength of 0.97625 Å in a favourable case. The new scaffoldin-borne CBM3 structure reported here provides clear evidence for the proposition that a family 3b CBM may be accommodated in scaffoldin subunits and functions as the major substrate-binding entity of the cellulosome assembly.
CBM3b; cellulose-binding module; major scaffoldin subunit; ScaA; cellulosome; Acetivibrio cellulolyticus
The equine MHC class I molecule was crystallized in complex with β2-microglobulin and a CTL epitope and X-ray diffraction data were collected to 2.3 Å resolution.
In order to clarify the structure and the peptide-presentation characteristics of the equine major histocompatibility complex (MHC) class I molecule, a complex of equine MHC class I molecule (ELA-A1 haplotype, 7-6 allele) with mouse β2-microglobulin and the cytotoxic T lymphocyte (CTL) epitope Env-RW12 (RVEDVTNTAEYW) derived from equine infectious anaemia virus (EIAV) envelope protein (residues 195–206) was refolded and crystallized. The crystal, which belonged to space group P21, diffracted to 2.3 Å resolution and had unit-cell parameters a = 82.5, b = 71.4, c = 99.8 Å, β = 102.9°. The crystal structure contained two molecules in the asymmetric unit. These results should help to determine the first equine MHC class I molecule structure presenting an EIAV CTL epitope.
equine MHC class I molecule
The enzymatic activation of human legumain requires both proteolytic cleavage and conformational reordering and is modulated by its substrate as well as cofactors. These biochemical findings are aided by the crystallization and initial crystallographic analysis of legumain.
Localized mainly to endo/lysosomes, legumain plays an important role in exogenous antigen processing and presentation. The cysteine protease legumain, also known as asparaginyl endopepetidase AEP, is synthesized as a zymogen and is known to undergo pH-dependent autoproteolytic activation whereby N-terminal and C-terminal propeptides are released. However, important mechanistic details of this pH-dependent activation as well as the characteristic pH activity profile remain unclear. Here, it is shown that all but one of the autocatalytic cleavage events occur in trans, with only the release of the C-terminal propeptide being relevant to enzymatic activity. An intriguing super-activation event that appears to be exclusively conformational in nature and enhances the enzymatic activity of proteolytically fully processed legumain by about twofold was also found. Accepting asparagines and, to lesser extent, aspartic acid in P1, super-activated legumain exhibits a marked pH dependence that is governed by the P1 residue of its substrate and conformationally stabilizing factors such as temperature or ligands. The crystallization and preliminary diffraction data analysis of active legumain are presented, which form an important basis for further studies that should clarify fundamental aspects of activation, activity and inactivation of legumain, which is a key target in (auto-)immunity and cancer.
cysteine proteases; pH regulation; conformational activation; substrate-dependent activity; autoimmune diseases; cancer
HisC2 from M. tuberculosis was cloned, overexpressed, purified and crystallized and the crystals were characterized.
HisC2 from Mycobacterium tuberculosis was overexpressed in M. smegmatis and purified to homogeneity using nickel–nitrilotriacetic acid metal-affinity and gel-filtration chromatography. Diffraction-quality crystals were grown using the hanging-drop vapour-diffusion technique from a condition consisting of 7 mg ml−1 HisC2 (in 20 mM Tris pH 8.8, 50 mM NaCl and 5% glycerol), 1 M succinic acid pH 7.0, 0.1 M HEPES pH 7.0 and 1%(w/v) polyethylene glycol monomethyl ether 2000. The crystals belonged to the orthorhombic space group P21212, with unit-cell parameters a = 255.98, b = 77.09, c = 117.97 Å. X-ray diffraction data were recorded to 2.45 Å resolution from a single crystal using the in-house X-ray facility.
HisC2; Mycobacterium tuberculosis; Mycobacterium smegmatis
The crystallization of recombinant nitrophorin 7 originating from the bloodsucking insect R. prolixus is described. The crystals diffracted to 1.8 Å resolution.
Nitrophorins (nitric oxide transport proteins) are haemproteins originating from the blood-feeding insect Rhodnius prolixus. They consist of an eight-stranded β-barrel, which classifies them into the lipocalin family. Nitrophorin 7 (NP7) and the E27V mutant protein NP7(E27V) were crystallized at 277 K using the vapour-diffusion method with PEG as the precipitating agent. Data sets for wild-type NP7 and NP7(E27V) were collected to 1.80 Å resolution from single crystals at 100 K using synchrotron radiation. The crystals belonged to space group P21, with unit-cell parameters a = 38, b = 67, c = 39 Å, β = 117°. The crystal contained one molecule per asymmetric unit, with a Matthews coefficient (V
M) of 2.11 Å3 Da−1; the solvent content was estimated to be 41.8%.
nitrophorins; Rhodnius prolixus; β-barrel proteins; haem; membrane
Chicken interferon-γ receptor α chain was crystallized and diffraction data were collected to 2.0 Å resolution.
The activity of interferon-γ (IFN-γ) relies on signal transduction, which is triggered by combination with the receptors interferon-γ receptor α chain (IFNGR1) and β chain (IFNGR2). Native recombinant chicken IFNGR1 (chIFNGR1; residues 25–237) was overexpressed in Escherichia coli, purified by refolding and crystallized using the vapour-diffusion technique. The crystals belonged to space group P6522, with unit-cell parameters a = b = 64.1, c = 216.3 Å, α = β = 90, γ = 120°. The Matthews coefficient and solvent content were calculated as 2.67 Å3 Da−1 and 53.97%, respectively. X-ray diffraction data for chIFNGR1 were collected to 2.0 Å resolution at a synchrotron source.
chicken interferon-γ receptor α chain
Human Cockayne syndrome protein A has been cocrystallized with human DNA damage-binding protein 1 and data have been collected to 2.9 Å resolution.
Cockayne syndrome protein A is one of the main components in mammalian transcription coupled repair. Here, the overproduction, purification and crystallization of human Cockayne syndrome protein A in complex with its interacting partner DNA damage binding protein 1 are reported. The complex was coproduced in insect cells, copurified and crystallized using sitting drops with PEG 3350 and sodium citrate as crystallizing agents. The crystals had unit-cell parameters a = b = 142.03, c = 250.19 Å and diffracted to 2.9 Å resolution on beamline ID14-1 at the European Synchrotron Radiation Facility.
Cockayne syndrome protein A; DNA damage-binding protein 1
ADP-ribose pyrophosphatase-I, a Nudix enzyme, from T. thermophilus was crystallized for neutron diffraction. Neutron and X-ray diffraction data sets were collected to 2.1 and 1.5 Å resolution, respectively.
ADP-ribose pyrophosphatase-I from Thermus thermophilus HB8 (TtADPRase-I) prevents the intracellular accumulation of ADP-ribose by hydrolyzing it to AMP and ribose 5′-phosphate. To understand the catalytic mechanism of TtADPRase-I, it is necessary to investigate the role of glutamates and metal ions as well as the coordination of water molecules located at the active site. A macroseeding method was developed in order to obtain a large TtADPRase-I crystal which was suitable for a neutron diffraction study to provide structural information. Neutron and X-ray diffraction experiments were performed at room temperature using the same crystal. The crystal diffracted to 2.1 and 1.5 Å resolution in the neutron and X-ray diffraction experiments, respectively. The crystal belonged to the primitive space group P3221, with unit-cell parameters a = b = 50.7, c = 119 Å.
ADP-ribose pyrophosphatase; Ndx4; neutron diffraction; Thermus thermophilus
CbsA from T. neapolitana has been crystallized. Native data were collected to 2.0 Å resolution.
The β-N-acetylglucosaminidase CbsA was cloned from the thermophilic Gram-negative bacterium Thermotoga neapolitana. Although CbsA contains a family 3 glycoside hydrolase-type (GH3-type) catalytic domain, it can be distinguished from other GH3-type β-N-acetylglucosaminidases by its high activity towards chitobiose. The homodimeric CbsA contains a unique domain at the C-terminus for which the three-dimensional structure is not yet known. In this study, CbsA was overexpressed and the recombinant protein was purified using Ni–NTA affinity and gel-filtration chromatography. The purified CbsA protein was crystallized using the vapour-diffusion method. A diffraction data set was collected to a resolution of 2.0 Å at 100 K. The crystal belonged to space group R32. To obtain initial phases, the crystallization of selenomethionyl-substituted protein and the production of heavy-atom derivative crystals are in progress.
CbsA; Thermotoga neapolitana; thermostable enzymes; β-N-acetylglucosaminidases
A human enzyme, DHDPSL, which is homologous to bacterial pyruvate-dependent aldolases but of unknown function, has been expressed, purified and crystallized with the use of in situ proteolysis. The crystals diffracted to 2.0 Å resolution and were suitable for structure determination.
Human dihydrodipicolinate synthase-like protein (DHDPSL) is a gene product of unknown function. It is homologous to bacterial pyruvate-dependent aldolases such as dihydrodipicolinate synthase (DHDPS), which functions in lysine biosynthesis. However, it cannot have this function and instead is implicated in a genetic disorder that leads to excessive production of oxalate and kidney-stone formation. In order to better understand its function, DHDPSL was expressed as an MBP-fusion protein and crystallized using an in situ proteolysis protocol. Two crystal forms were obtained, both of which diffracted X-rays to approximately 2.0 Å resolution. One of these, belonging to space group P6222 or P6422 with unit-cell parameters a = b = 142.9, c = 109.8 Å, α = β = 90, γ = 120°, was highly reproducible and suitable for structure determination by X-ray crystallography.
primary oxaluria; DHDPS; aldolase
Recombinant wild-type l-lactate dehydrogenase from B. subtilis (BsLDH) was cocrystallized with fructose 1,6-bisphosphate and NAD+ and the crystal diffracted to 2.38 Å resolution. The H171C mutant of BsLDH was also crystallized as the apoenzyme and in complex with NAD+ and the crystals diffracted to 2.20 and 2.49 Å, respectively. All crystals belonged to space group P3.
l-Lactate dehydrogenase (LDH) is an important enzyme involved in the last step of glycolysis that catalyzes the reversible conversion of pyruvate to l-lactate with the simultaneous oxidation of NADH to NAD+. In this study, wild-type LDH from Bacillus subtilis (BsLDH-WT) and the H171C mutant (BsLDH-H171C) were expressed in Escherichia coli and purified to near-homogeneity. BsLDH-WT was crystallized in the presence of fructose 1,6-bisphosphate (FBP) and NAD+ and the crystal diffracted to 2.38 Å resolution. The crystal belonged to space group P3, with unit-cell parameters a = b = 171.04, c = 96.27 Å. BsLDH-H171C was also crystallized as the apoenzyme and in complex with NAD+, and data sets were collected to 2.20 and 2.49 Å resolution, respectively. Both BsLDH-H171C crystals belonged to space group P3, with unit-cell parameters a = b = 133.41, c = 99.34 Å and a = b = 133.43, c = 99.09 Å, respectively. Tetramers were observed in the asymmetric units of all three crystals.
l-lactate dehydrogenase; fructose 1,6-bisphosphate; NADH/NAD+ binding
Recombinant pyridoxine 4-oxidase from M. loti MAFF303099 was crystallized and diffraction data were collected to 2.2 Å resolution.
Vitamin B6-degradation pathway I has recently been identified in Mesorhizobium loti MAFF303099. Pyridoxine 4-oxidase, an FAD-dependent enzyme, is the first enzyme in this pathway and catalyzes the irreversible oxidation of pyridoxine to pyridoxal. The enzyme was overexpressed in Escherichia coli with a His6 tag and purified. The recombinant enzyme was crystallized at 277 K by the sitting-drop vapour-diffusion method using PEG 4000 as the precipitant. The crystal, which belonged to space group P212121 with unit-cell parameters a = 62.38, b = 79.44, c = 136.43 Å, diffracted to 2.2 Å resolution. The calculated V
M value (3.19 Å3 Da–1) suggested that the asymmetric unit contained one molecule.
pyridoxal; vitamin B6; Mesorhizobium loti; FAD
A. thaliana dynamin-related protein 1A GTPase domain fused with its GTPase effector domain was overexpressed, purified and crystallized in a hexagonal crystal form that diffracted to 3.6 Å resolution.
Plant-specific dynamin-related proteins play crucial roles in cell-plate formation, endocytosis or exocytosis, protein sorting to the vacuole and plasma membrane and the division of mitochondria and chloroplasts. In order to determine the crystal structure and thus to obtain a better understanding of the biological functions and mechanisms of dynamin-related proteins in plant cells, the GTPase domain of Arabidopsis thaliana dynamin-related protein 1A (AtDRP1A) fused to its GTPase effector domain (GED) was crystallized in a nucleotide-associated form using polyethylene glycol 3350 as precipitant. The hexagonal crystals (space group P61) had unit-cell parameters a = b = 146.2, c = 204.3 Å, and diffraction data were collected to 3.6 Å resolution using synchrotron radiation. Four molecules, comprising two functional dimers, are assumed per asymmetric unit, corresponding to a Matthews coefficient of 3.9 Å3 Da−1 according to the molecular weight of 39 kDa.
Arabidopsis thaliana; dynamin-like protein 1A; GTPases
This study reports the expression, purification, crystallization and preliminary X-ray crystallographic analysis of human lysosomal β-d-galactosidase.
β-d-Galactosidase (β-Gal) is an exoglycosidase that cleaves β-galactosides from glycoproteins, sphingolipids and keratan sulfate. This study reports the expression, purification, crystallization and preliminary X-ray crystallographic analysis of human lysosomal β-Gal. The sitting-drop vapour-diffusion method was used to crystallize β-Gal in complexes with its product galactose and with the inhibitor 1-deoxygalactonojirimycin. The resulting crystals were isomorphous and belonged to space group P21. The crystals of the β-Gal–galactose and the β-Gal–inhibitor complexes had unit-cell parameters a = 94.8, b = 116.1, c = 140.3 Å, β = 92.2° and a = 94.8, b = 116.0, c = 140.3 Å, β = 92.2°, respectively. Diffraction data were collected to 1.8 Å resolution for both crystals.
β-galactosidase; galactose; 1-deoxygalactonojirimycin
An orthorhombic crystal of an enoyl-(acyl-carrier protein) reductase from V. fischeri was obtained and diffraction data were collected to 2.7 Å resolution.
Enoyl-(acyl-carrier protein) reductase (ENR) catalyzes the last step of the fatty-acid elongation cycle of the bacterial fatty-acid biosynthesis (FAS II) pathway. Recently, a new class of ENR has been identified from Vibrio cholerae and was named FabV. In order to understand the molecular mechanism of the new class of ENR at the structural level, FabV from V. fischeri was overexpressed, purified and crystallized. Diffraction data were collected to 2.7 Å resolution from a native crystal. The crystal belonged to the orthorhombic space group P21212, with unit-cell parameters a = 123.53, b = 164.14, c = 97.07 Å. The presence of four molecules of FabV in the asymmetric unit gave a V
M value of 2.81 Å3 Da−1, with a corresponding solvent content of 54.5%.
FAS II pathway; enoyl-(acyl-carrier protein) reductase; Vibrio fischeri; FabV
The head domain of the DNA-repair protein RecN from D. radiodurans, composed of the amino- and carboxy-terminal domains, was crystallized. X-ray diffraction data were collected to 3.0 Å resolution and the crystals belonged to space group P21.
Deinococcus radiodurans is well known for its extreme tolerance to harsh conditions and for its extraordinary ability to repair DNA. Double-strand breaks (DSBs) are the most hazardous lesions that can be induced by ionizing radiation, and homologous recombination (HR) is the principal mechanism by which the integrity of the DNA is restored. In D. radiodurans the RecFOR complex is the main actor in HR and the RecN protein is believed to play an important role in DSB recognition. Here, SAXS and preliminary X-ray diffraction studies are presented of the head domain, which is the globular region formed upon interaction of the N- and C-terminal domains of RecN. The crystal structure of this domain was solved using the single-wavelength anomalous dispersion method. Model building and refinement are in progress.
DNA repair; homologous recombination; RecFOR; RecN; SMC-like proteins
NaD1 is a potent antifungal plant defensin. Here, the crystallization and preliminary X-ray crystallographic analysis of NaD1 are reported in order to obtain insight into the structural basis of its antifungal activity.
Plant defensins are small (∼5 kDa) basic cysteine-rich proteins that are being explored in important agricultural crops for their ability to confer enhanced disease resistance against fungal pathogens. NaD1, isolated from the flowers of the ornamental tobacco (Nicotiana alata), is a particularly well characterized antifungal defensin. Here, the crystallization and preliminary X-ray crystallographic analysis of NaD1 is reported. Crystals of NaD1 were crystallized using the sitting-drop vapour-diffusion method at 291 K. Data were collected from two crystal forms to 1.4 and 1.6 Å resolution, respectively. The crystals of form A belonged to the monoclinic space group P21, with unit-cell parameters a = 32.697, b = 32.685, c = 41.977 Å, α = 90, β = 100.828, γ = 90°, whereas crystals of form B belonged to the trigonal space group P3221, with unit-cell parameters a = b = 33.091, c = 128.77 Å, α = β = 90, γ = 120°.
NaD1; plant defensins
NahF is a salicylaldehyde dehydrogenase that is involved in the naphthalene-degradation pathway, converting salicylaldehyde into salicylate. The subcloning, expression, purification and preliminary X-ray diffraction studies at 2.42 Å resolution of P. putida G7 NahF are reported.
Pseudomonas putida G7 is one of the most studied naphthalene-degrading species. The nah operon in P. putida, which is present on the 83 kb metabolic plasmid NAH7, codes for enzymes involved in the conversion of naphthalene to salicylate. The enzyme NahF (salicylaldehyde dehydrogenase) catalyzes the last reaction in this pathway. The nahF gene was subcloned into the pET28a(TEV) vector and the recombinant protein was overexpressed in Escherichia coli Arctic Express at 285 K. The soluble protein was purified by affinity chromatography followed by gel filtration. Crystals of recombinant NahF (6×His-NahF) were obtained at 291 K and diffracted to 2.42 Å resolution. They belonged to the hexagonal space group P6422, with unit-cell parameters a = b = 169.47, c = 157.94 Å. The asymmetric unit contained a monomer and a crystallographic twofold axis generated the dimeric biological unit.
Pseudomonas putida G7; naphthalene degradation; salicylaldehyde dehydrogenase; NahF
The RNA polymerase domain of primase from S. mutans strain UA159 was cloned, overexpressed, purified and crystallized. X-ray diffraction data were collected to a resolution of 1.60 Å.
Primase is the enzyme that synthesizes RNA primers on single-stranded DNA during normal DNA replication. In this study, the catalytic core domain of primase from Streptococcus mutans UA159 was overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to 1.60 Å resolution using a synchrotron-radiation source. The crystal belonged to space group P41 or P43, with unit-cell parameters a = b = 52.63, c = 110.31 Å. The asymmetric unit is likely to contain one molecule, with a corresponding V
M of 1.77 Å3 Da−1 and a solvent content of 30.7%.
Streptococcus mutans; primases
A DJ-1 homologue protein from A. thaliana (AtDJ-1D) has been crystallized. Diffraction data were collected to 2.05 Å resolution for structure determination by the multiple-wavelength anomalous dispersion (MAD) method.
A DJ-1 homologue protein from Arabidopsis thaliana (AtDJ-1D) belongs to the DJ-1/ThiJ/Pfpl superfamily and contains two tandem arrays of DJ-1-like sequences, but no structural information is available to date for this protein. AtDJ-1D was expressed in Escherichia coli, purified and crystallized for structural analysis. A crystal of AtDJ-1D was obtained by the hanging-drop vapour-diffusion method using 0.22 M NaCl, 0.1 M bis-tris pH 6.5, 21% polyethylene glycol 3350. AtDJ-1D crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 56.78, b = 75.21, c = 141.68 Å, β = 96.87°, and contained a trimer in the asymmetric unit. Diffraction data were collected to 2.05 Å resolution. The structure of AtDJ-1D has been determined using the multiple-wavelength anomalous dispersion (MAD) method.
DJ-1; Arabidopsis thaliana; AtDJ-1D; multiple-wavelength anomalous dispersion
Apolipoprotein A-IV crystals consisted of a long unit-cell edge (540 Å) with a high mosaic spread, making them intractable for X-ray diffraction analysis. Extreme dehydration in 60% PEG 3350 was utilized as a post-crystallization treatment as well a screening method to significantly sharpen the mosaic spread and increase the overall resolution of diffraction.
Apolipoproteins are the protein component of high-density lipoproteins (HDL), which are necessary for mobilizing lipid-like molecules throughout the body. Apolipoproteins undergo self-association, especially at higher concentrations, making them difficult to crystallize. Here, the crystallization and diffraction of the core fragment of apolipoprotein A-IV (apoA-IV), consisting of residues 64–335, is presented. ApoA-IV64–335 crystallized readily in a variety of hexagonal (P6) morphologies with similar unit-cell parameters, all containing a long axis of nearly 550 Å in length. Preliminary diffraction experiments with the different crystal morphologies all resulted in limited streaky diffraction to 3.5 Å resolution. Crystal dehydration was applied to the different morphologies with variable success and was also used as a quality indicator of crystal-growth conditions. The results show that the morphologies that withstood the most extreme dehydration conditions showed the greatest improvement in diffraction. One morphology in particular was able to withstand dehydration in 60% PEG 3350 for over 12 h, which resulted in well defined intensities to 2.7 Å resolution. These results suggest that the approach of integrating dehydration with variation in crystal-growth conditions might be a general technique to optimize diffraction.
apolipoproteins; dehydration; long unit cell; reflection elongation
The equilibrium relative humidity values for a number of the most commonly used precipitants in macromolecular crystallization have been measured using a humidity-control device and compared with independent values where available. The results will simplify experiments using the device.
The dehydration of crystals of macromolecules has long been known to have the potential to increase their diffraction quality. A number of methods exist to change the relative humidity that surrounds crystals, but for reproducible results, with complete characterization of the changes induced, a precise humidity-control device coupled with an X-ray source is required. The first step in these experiments is to define the relative humidity in equilibrium with the mother liquor of the system under study; this can often be quite time-consuming. In order to reduce the time spent on this stage of the experiment, the equilibrium relative humidity for a range of concentrations of the most commonly used precipitants has been measured. The relationship between the precipitant solution and equilibrium relative humidity is explained by Raoult’s law for the equilibrium vapour pressure of water above a solution. The results also have implications for the choice of cryoprotectant and solutions used to dehydrate crystals. For the most commonly used precipitants (10–30% PEG 2000–8000), the starting point will be a relative humidity of 99.5%.
controlled dehydration; macromolecular crystallography; humidity control; automation
The crystal structures of engineered C. botulinum neurotoxin–SNARE derivatives have been and exhibit strong stability of the LHn fragment.
Targeted secretion inhibitors (TSIs) are a new class of engineered biopharmaceutical molecules derived from the botulinum neurotoxins (BoNTs). They consist of the metalloprotease light chain (LC) and translocation domain (Hn) of BoNT; they thus lack the native toxicity towards motor neurons but are able to target soluble N-ethylmaleimide-sensitive fusion protein attachment receptor (SNARE) proteins. These functional fragment (LHn) derivatives are expressed as single-chain proteins and require post-translational activation into di-chain molecules for function. A range of BoNT derivatives have been produced to demonstrate the successful use of engineered SNARE substrate peptides at the LC–Hn interface that gives these molecules self-activating capabilities. Alternatively, recognition sites for specific exoproteases can be engineered to allow controlled activation. Here, the crystal structures of three LHn derivatives are reported between 2.7 and 3.0 Å resolution. Two of these molecules are derivatives of serotype A that contain a SNARE peptide. Additionally, a third structure corresponds to LHn serotype B that includes peptide linkers at the exoprotease activation site. In all three cases the added engineered segments could not be modelled owing to disorder. However, these structures highlight the strong interactions holding the LHn fold together despite the inclusion of significant polypeptide sequences at the LC–Hn interface.
BoNT; SNARE; protein engineering
The crystal structure of At2g44920, a pentapeptide repeat protein (PRP) from Arabidopsis thaliana, has been determined at 1.7 Å resolution. The structure represents the first PRP protein whose subcellular localization has been experimentally confirmed to be the thylakoid lumen of a plant species.
At2g44920 belongs to a diverse family (Pfam PF00805) of pentapeptide-repeat proteins (PRPs) that are present in all known organisms except yeast. PRPs contain at least eight tandem-repeating sequences of five amino acids with an approximate consensus sequence (STAV)(D/N)(L/F)(S/T/R)(X). Recent crystal structures show that PRPs adopt a highly regular four-sided right-handed β-helical structure consisting mainly of type II and type IV β-turns, sometimes referred to as a repeated five-residue (or Rfr) fold. Among sequenced genomes, PRP genes are most abundant in cyanobacteria, leading to speculation that PRPs play an important role in the unique lifestyle of photosynthetic cyanobacteria. Despite the recent structural characterization of several cyanobacterial PRPs, most of their functions remain unknown. Plants, whose chloroplasts are of cyanobacterial origin, have only four PRP genes in their genomes. At2g44920 is one of three PRPs located in the thylakoid lumen. Here, the crystal structure of a double methionine mutant of residues 81–224 of At2g44920, the naturally processed fragment of one of its full-length isoforms, is reported at 1.7 Å resolution. The structure of At2g44920 consists of the characteristic Rfr fold with five uninterrupted coils made up of 25 pentapeptide repeats and α-helical elements capping both termini. A disulfide bridge links the two α-helices with a conserved loop between the helical elements at its C-terminus. This structure represents the first structure of a PRP protein whose subcellular location has been experimentally confirmed to be the thylakoid lumen in a plant species.
At2g44920; pentapeptide-repeat proteins; Arabidopsis thaliana
The crystallographic structures of the subunit B mutants F427W and F508W of the Pyrococcus horikoshii OT3 of the A1AO ATP synthase reveal that the exact volume of the adenine ribose binding pocket is essential for ATP-/ADP-binding.
A reporter tryptophan residue was individually introduced by site-directed mutagenesis into the adenine-binding pocket of the catalytic subunit A (F427W and F508W mutants) of the motor protein A1AO ATP synthase from Pyrococcus horikoshii OT3. The crystal structures of the F427W and F508W mutant proteins were determined to 2.5 and 2.6 Å resolution, respectively. The tryptophan substitution caused the fluorescence signal to increase by 28% (F427W) and 33% (F508W), with a shift from 333 nm in the wild-type protein to 339 nm in the mutant proteins. Tryptophan emission spectra showed binding of Mg-ATP to the F427W mutant with a K
d of 8.5 µM. In contrast, no significant binding of nucleotide could be observed for the F508W mutant. A closer inspection of the crystal structure of the F427W mutant showed that the adenine-binding pocket had widened by 0.7 Å (to 8.70 Å) in comparison to the wild-type subunit A (8.07 Å) owing to tryptophan substitution, as a result of which it was able to bind ATP. In contrast, the adenine-binding pocket had narrowed in the F508W mutant. The two mutants presented demonstrate that the exact volume of the adenine ribose binding pocket is essential for nucleotide binding and even minor narrowing makes it unfit for nucleotide binding. In addition, structural and fluorescence data confirmed the viability of the fluorescently active mutant F427W, which had ideal tryptophan spectra for future structure-based time-resolved dynamic measurements of the catalytic subunit A of the ATP-synthesizing enzyme A-ATP synthase.
A1AO ATP synthase; tryptophan; Pyrococcus horikoshii OT3