The structure of human vascular adhesion protein 1 in space group C2 with seven molecules in the asymmetric unit has been refined at a resolution of 2.9 Å.
Human vascular adhesion protein 1 (VAP-1) is involved in lymphocyte–endothelial cell adhesion and has been implicated in many human inflammatory diseases. VAP-1 is a member of the copper amine oxidase family of enzymes with a trihydroxyphenylalanine quinone (TPQ) cofactor. Previously characterized crystals of VAP-1 suffered from anisotropy and contained disordered regions; in addition, one form was consistently twinned. In an effort to grow crystals that diffracted to higher resolution for inhibitor-binding studies, a construct with an N-terminal deletion was made and expressed in the Chinese hamster ovary (CHO) glycosylation mutant cell line Lec8. Screening produced crystals that displayed some anisotropy and contained seven molecules per asymmetric unit. These crystals belonged to space group C2, with unit-cell parameters a = 394.5, b = 115.8, c = 179.3 Å, β = 112.3°. The structure was refined to a resolution of 2.9 Å, with R
cryst and R
free values of 0.250 and 0.286, respectively.
vascular adhesion protein 1; VAP-1; SSAO; copper amine oxidases; topaquinone; TPQ
The polytopic membrane protein FeoB is a ferrous iron transporter in prokaryotes. The protein contains a potassium-activated GTPase domain that is essential in regulating the import of iron and conferring virulence to many disease-causing bacteria. However, the mechanism by which the G-domain of FeoB hydrolyzes GTP is not well understood. In particular, it is not yet known how the pivotal step in GTP hydrolysis is achieved: alignment of a catalytic water molecule. In the current study, the crystal structure of the soluble domains from Streptococcus thermophilus FeoB (NFeoBSt) in complex with the activating potassium ion and a transition-state analogue, GDP⋅AlF4−, reveals a novel mode of water alignment involving contacts with the protein backbone only. In parallel to the structural studies, a series of seven mutant proteins were constructed that targeted conserved residues at the active site of NFeoBSt, and the nucleotide binding and hydrolysis properties of these were measured and compared to the wild-type protein. The results show that mutations in Thr35 abolish GTPase activity of the protein, while other conserved residues (Tyr58, Ser64, Glu66 and Glu67) are not required for water alignment by NFeoBSt. Together with the crystal structure, the findings suggest a new mechanism for hydrolysis initiation in small G-proteins, in which the attacking water molecule is aligned by contacts with the protein backbone only.
The MA protein from HIV-1 is a small, multifunctional protein responsible for regulating various stages of the viral replication cycle. To achieve its diverse tasks MA interacts with host cell proteins and it has been reported that one of these is the ubiquitous calcium -sensing calmodulin (CaM) which is up-regulated upon HIV-1 infection. The nature of the CaM-MA interaction has been the subject of structural studies using peptides based on the MA sequence that have led to conflicting conclusions. The results presented here show that CaM binds intact MA with 1:1 stoichiometry in a Ca2+-dependent manner and that the complex adopts a highly extended conformation in solution as revealed by small-angle X-ray scattering. Alterations in tryptophan fluorescence suggest that the two tryptophans at the N-terminus of MA mediate the CaM interaction. Major chemical shift changes occur in the NMR spectrum of MA upon complex formation, while chemical shift changes in the CaM spectrum are quite modest and are assigned to residues within the target-protein binding hydrophobic clefts of CaM. The NMR data indicate that CaM binds MA via its N-and C-terminal lobes and induces a dramatic conformational change involving a significant loss of secondary and tertiary structure within MA. Circular dichroism experiments suggest that MA looses ~20% of its α-helical content upon CaM binding. Thus CaM binding is expected to impact upon the accessibility of interaction sites within MA that are involved in its various functions.
A complex comprising the LIM domains of the LIM-homeodomain protein Lhx4 tethered to a peptide region of Isl2 has been engineered, purified and crystallized. Crystals of this intramolecular complex diffracted to 2.16 Å resolution.
A stable intramolecular complex comprising the LIM domains of the LIM-homeodomain protein Lhx4 tethered to a peptide region of Isl2 has been engineered, purified and crystallized. The monoclinic crystals belonged to space group P21, with unit-cell parameters a = 46.8, b = 88.7, c = 49.9 Å, β = 111.9°, and diffracted to 2.16 Å resolution.
Lhx4; Isl2; Lim domains; Lim-homeodomain transcription factors
The structure of human diamine oxidase has been refined to 2.1 Å resolution in space group C2221.
Copper amine oxidases (CAOs) are ubiquitous in nature and catalyse the oxidative deamination of primary amines to the corresponding aldehydes. Humans have three viable CAO genes (AOC1–3). AOC1 encodes human diamine oxidase (hDAO), which is the frontline enzyme for histamine metabolism. hDAO is unique among CAOs in that it has a distinct substrate preference for diamines. The structure of hDAO in space group P212121 with two molecules in the asymmetric unit has recently been reported. Here, the structure of hDAO refined to 2.1 Å resolution in space group C2221 with one molecule in the asymmetric unit is reported.
diamine oxidases; copper amine oxidases; topaquinone; histaminase; kidney amine oxidases
The second RanBP2-type zinc finger from ZRANB2 has been crystallized in complex with a single-stranded RNA target sequence.
ZRANB2 is a zinc-finger protein that has been shown to influence alternative splice-site selection. The protein comprises a C-terminal arginine/serine-rich domain that interacts with spliceosomal proteins and two N-terminal RanBP2-type zinc fingers that have been implicated in RNA recognition. The second zinc finger bound to a six-nucleotide single-stranded RNA target sequence crystallized in the hexagonal space group P6522 or P6122, with unit-cell parameters a = 54.52, b = 54.52, c = 48.07 Å; the crystal contains one monomeric complex per asymmetric unit. This crystal form has a solvent content of 39% and diffracted to 1.4 Å resolution using synchrotron radiation.
RanBP2-type zinc fingers; RNA-binding proteins; splicing factors
The crystal structure of Sda, a DNA-replication/damage checkpoint inhibitor of sporulation in B. subtilis, has been solved via the MAD method. The subunit arrangement in the crystal has enabled a reappraisal of previous biophysical data, resulting in a new model for the behaviour of the protein in solution.
The crystal structure of the DNA-damage checkpoint inhibitor of sporulation, Sda, from Bacillus subtilis, has been solved by the MAD technique using selenomethionine-substituted protein. The structure closely resembles that previously solved by NMR, as well as the structure of a homologue from Geobacillus stearothermophilus solved in complex with the histidine kinase KinB. The structure contains three molecules in the asymmetric unit. The unusual trimeric arrangement, which lacks simple internal symmetry, appears to be preserved in solution based on an essentially ideal fit to previously acquired scattering data for Sda in solution. This interpretation contradicts previous findings that Sda was monomeric or dimeric in solution. This study demonstrates the difficulties that can be associated with the characterization of small proteins and the value of combining multiple biophysical techniques. It also emphasizes the importance of understanding the physical principles behind these techniques and therefore their limitations.
Sda; sporulation histidine kinase inhibitors
An intramolecular complex comprising the LIM domains of Lhx3 and the interacting domain of Isl1 tethered by a flexible linker was engineered, overexpressed in E. coli, purified and crystallized.
A stable intramolecular complex comprising the LIM domains of the LIM-homeodomain protein Lhx3 tethered to a peptide region of Isl1 has been engineered, purified and crystallized. The monoclinic crystals belong to space group C2, with unit-cell parameters a = 119, b = 62.2, c = 51.9 Å, β = 91.6°, and diffract to 2.05 Å resolution.
Lhx3; Isl1; LIM-homeodomain proteins; protein complex; motor-neuron development
Humans have three functioning genes that code for copper-containing amine oxidases. The product of the AOC1 gene is a so-called diamine oxidase (hDAO), named for its substrate preference for diamines, particularly histamine. hDAO has been cloned and expressed in insect cells and the structure of the native enzyme determined by X-ray crystallography to a resolution of 1.8 Å. The homodimeric structure has the archetypal amine oxidase fold. Two active sites, one in each subunit, are characterized by the presence of a copper ion and a topaquinone residue formed by the post-translational modification of a tyrosine. Although hDAO shares 37.9 % sequence identity with another human copper amine oxidase, semicarbazide sensitive amine oxidase or vascular adhesion protein-1, its substrate binding pocket and entry channel are distinctly different in accord with the different substrate specificities. The structures of two inhibitor complexes of hDAO, berenil and pentamidine, have been refined to resolutions of 2.1 Å and 2.2 Å, respectively. They bind non-covalently in the active site channel. The inhibitor binding suggests that an aspartic acid residue, conserved in all diamine oxidases but absent from other amine oxidases, is responsible for the diamine specificity by interacting with the second amino group of preferred diamine substrates.
The structures of complexes of the copper-containing amine oxidase from A. globiformis with benzylhydrazine and tranylcypromine have been refined at 1.86 and 1.65 Å resolution, respectively.
Complexes of Arthrobacter globiformis amine oxidase (AGAO) with the inhibitors benzylhydrazine and tranylcypromine (an antidepressant drug) have been refined at 1.86 and 1.65 Å resolution, respectively. Both inhibitors form covalent adducts with the TPQ cofactor. A tyrosine residue, proposed to act as a gate to the AGAO active site, is in its open conformation.
amine oxidases; AGAO; benzylhydrazine; tranylcypromine; topaquinone; copper
In a new crystal form of PSAO, the homodimeric molecules lack twofold symmetry and the C-termini of the two protomers are linked by a previously unrevealed disulfide bond.
The structure of a newly crystallized form of the copper-dependent amine oxidase from pea seedlings has been refined at a resolution of 2.2 Å to a final R factor of 0.181. The structure (form II) was originally discovered during a study of xenon binding to copper-dependent amine oxidases as a probe for dioxygen-binding sites [Duff et al. (2004 ▶), J. Mol. Biol.
344, 599–607]. The form II crystals belong to space group P21, with two dimers in the asymmetric unit. The overall structure is very similar to the crystals of form I in space group P212121 with a dimer in the asymmetric unit [Kumar et al. (1996 ▶), Structure, 4, 943–955]. In form I the last three residues (644–647) observable in the two subunits were apparently splayed apart. It was noted that the absence of a disulfide bond between the Cys647 residues of the two subunits was inconsistent with chemical evidence for the absence of free sulfhydryl groups. In both of the crystallographically independent dimers of form II the two subunits are clearly joined by a disulfide bridge between the C-terminal cysteine residues. This is only possible if the two polypeptide chains in the dimer adopt different conformations near the C-terminus so that the twofold symmetry is lost. A proline residue (645) two residues before the cysteine has a cis conformation in one chain and a trans conformation in the other. As a result, the disulfide bond lies more than 5 Å from the twofold axis. The loss of local twofold symmetry in form II can be explained by intermolecular contacts, which provide an asymmetric environment.
copper amine oxidase; PSAO
The previously reported structure of the copper-containing amine oxidase from A. globiformis has been refined in two different crystal forms at resolutions of 1.55 and 2.20 Å.
Copper-containing amine oxidases are found in all the major kingdoms of life. They catalyse the oxidation of organic amines in the presence of molecular dioxygen to aldehydes and hydrogen peroxide. The catalytic centres contain a Cu atom and a topaquinone cofactor formed autocatalytically from a tyrosine residue in the presence of Cu and molecular oxygen. The structure of the Cu-containing amine oxidase from Arthrobacter globiformis, which was previously refined at 1.8 Å resolution in space group C2 with unit-cell parameters a = 157.84, b = 63.24, c = 91.98 Å, β = 112.0° [Wilce et al. (1997 ▶), Biochemistry, 36, 16116–16133], has been re-refined with newly recorded data at 1.55 Å resolution. The structure has also been solved and refined at 2.2 Å resolution in a new crystal form, space group C2, with unit-cell parameters a = 158.04, b = 64.06, c = 69.69 Å, β = 111.7°.
copper amine oxidase; topaquinone; Arthrobacter globiformis
The copper amine oxidase from Arthrobacter globiformis (AGAO) is reversibly inhibited by molecular wires comprising a Ru(II)-complex head group and an aromatic tail group joined by an alkane linker. The crystal structures of a series of Ru(II)-wire-AGAO complexes differing with respect to the length of the alkane linker have been determined. All wires lie in the AGAO active-site channel, with their aromatic tail group in contact with the trihydroxyphenylalanine quinone (TPQ) cofactor of the enzyme. The TPQ cofactor is consistently in its active (‘off-Cu’) conformation, and the side chain of the so-called ‘gate’ residue Tyr296 is consistently in the ‘gate-open’ conformation. Among the wires tested, the most stable complex is produced when the wire has a –(CH2)4– linker. In this complex, the Ru(II)(phen)(bpy)2 head group is level with the protein molecular surface. Crystal structures of AGAO in complex with optically pure forms of the ‘C4’ wire show that the linker and head group in the two enantiomers occupy slightly different positions in the active-site channel. Both the Λ and Δ isomers are effective competitive inhibitors of amine oxidation. Remarkably, inhibition by the ‘C4’ wire shows a high degree of selectivity for AGAO in comparison with other copper-containing amine oxidases.
A single-point mutant (T109S) of E. coli dihydroorotase initially crystallizes so that the two monomers of the dimer are related by a crystallographic twofold axis. In the presence of substrate, conversion to the previously observed asymmetric dimer with substrate bound in one subunit and product in the other is observed.
Crystals of a single-point mutant (T109S) of Escherichia coli dihydroorotase (DHOase) with diminished activity grown in the presence of l-dihydroorotate (l-DHO) are tetragonal, with a monomer in the asymmetric unit. These crystals are extremely unstable and disintegrate shortly after formation, which is followed by the growth of orthorhombic crystals from the remnants of the tetragonal crystals or at new nucleation sites. Orthorhombic crystals, for which a structure has previously been reported [Thoden et al. (2001 ▶), Biochemistry, 40, 6989–6997; Lee et al. (2005 ▶), J. Mol. Biol.
348, 523–533], contain a dimer of DHOase in the asymmetric unit; the active site of one monomer contains the substrate N-carbamyl-l-aspartate (l-CA-asp) and the active site of the other monomer contains the product of the reaction, l-DHO. In the subunit with l-DHO in the active site, a surface loop (residues 105–115) is ‘open’. In the other subunit, with l-CA-asp in the active site, the loop folds inwards, forming specific hydrogen bonds from the loop to the l-CA-asp. The tetragonal crystal form can be stabilized by crystallization in the presence of the inhibitor 5-fluoroorotate (FOA), a product (l-DHO) mimic. Crystals of the complex of T109S DHOase with FOA are tetragonal, space group P41212, with unit-cell parameters a = b = 72.6, c = 176.1 Å. The structure has been refined to R and R
free values of 0.218 and 0.257, despite severe anisotropy of the diffraction. In this structure, the flexible loops are both in the ‘open’ conformation, which is consistent with FOA, like l-DHO, binding at both sites. The behaviour of the T109S mutant crystals of DHOase in the presence of l-DHO is explained by initial binding of l-DHO to both subunits, followed by slow conversion to l-CA-asp, with consequent movement of the flexible loop and dissolution of the crystals. Orthorhombic crystals are then able to grow in the presence of l-DHO and l-CA-asp.
dihydroorotase; conformational change; loop movement; catalytic state; crystal contacts; crystal instability
The structure of the complex formed between d(CGTACG)2 and 9-amino-N-[2-(4-morpholinyl)ethyl]-4-acridinecarboxamide, an inactive derivative of the antitumour agents N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (DACA) and 9-amino-DACA, has been solved to a resolution of 1.8 Å using X-ray crystallography. The complex crystallises in the space group P64 and the final structure has an overall R factor of 21.9%. A drug molecule intercalates between each of the CpG dinucleotide steps with its side chain lying in the major groove, and its protonated morpholino nitrogen partially occupying positions close to the N7 and O6 atoms of guanine G2. The morpholino group is disordered, the major conformer adopting a twisted boat conformation that makes van der Waals contact with the O4 oxygen of thymine T3. A water molecule forms bridging hydrogen bonds between the 4-carboxamide NH and the phosphate group of guanine G2. Sugar rings are found in alternating C3′-exo/C2′-endo conformations except for cytosine C1 which is C3′-endo. Intercalation perturbs helix winding throughout the hexanucleotide compared with B-DNA, steps 1 and 2 being unwound by 10 and 8°, respectively, while the central TpA step is overwound by 11°. An additional drug molecule lies at the end of each DNA helix linking it to the next duplex to form a continuously stacked structure. The protonated morpholino nitrogen of this ‘end-stacked’ drug hydrogen bonds to the N7 atom of guanine G6, and its conformationally disordered morpholino ring forms a C–H···O hydrogen bond with the guanine O6 oxygen. In both drug molecules the 4-carboxamide group is internally hydrogen bonded to the protonated N10 atom of the acridine ring. We discuss our findings with respect to the potential role played by the interaction of the drug side chain and the topoisomerase II protein in the poisoning of topoisomerase activity by the acridinecarboxamides.
The structures of the complexes formed between 9-amino-[N-(2-dimethyl-amino)butyl]acridine-4-carboxamide and d(CG5BrUACG)2 and d(CGTACG)2 have been solved by X-ray crystallography using MAD phasing methodology and refined to a resolution of 1.6 Å. The complexes crystallised in space group C222. An asymmetric unit in the brominated complex comprises two strands of DNA, one disordered drug molecule, two cobalt (II) ions and 19 water molecules (31 in the native complex). Asymmetric units in the native complex also contain a sodium ion. The structures exhibit novel features not previously observed in crystals of DNA/drug complexes. The DNA helices stack in continuous columns with their central 4 bp adopting a B-like motif. However, despite being a palindromic sequence, the terminal GC base pairs engage in quite different interactions. At one end of the duplex there is a CpG dinucleotide overlap modified by ligand intercalation and terminal cytosine exchange between symmetry-related duplexes. A novel intercalation complex is formed involving four DNA duplexes, four ligand molecules and two pairs of base tetrads. The other end of the DNA is frayed with the terminal guanine lying in the minor groove of the next duplex in the column. The structure is stabilised by guanine N7/cobalt (II) coordination. We discuss our findings with respect to the effects of packing forces on DNA crystal structure, and the potential effects of intercalating agents on biochemical processes involving DNA quadruplexes and strand exchanges. NDB accession numbers: DD0032 (brominated) and DD0033 (native).