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Caged and clustered structures of endothelin inhibitor BQ123, cyclo(-D-Trp-D-Asp--Pro-D-Val-Leu-)-Na+, forming five and six coordination bonds between sodium ions and peptides.

BQ123 is a cyclic pentapeptide and a potent endothelin-1 inhibitor. The crystal structure of the BQ123 sodium salt was determined as the first example of an endothelin inhibitor. Four independent molecules and many solvent molecules were found in the asymmetric unit; the total weight was about 3000 Da. The precise structure including the solvent molecules was determined using high-resolution data collected on a synchrotron source. Sodium ions formed unique structures with five and six coordination bonds and their forms were distinguished into three classes. An ion was sandwiched by two BQ123 molecules. This peptide-sodium (2:1) complex showed a cage-like structure and octahedral coordination was observed. Sodium ions also formed a cluster composed of hydrated water molecules and peptides. Two sodium ions were contained in this cluster, making five coordination bonds. Despite having the same coordination numbers, these ions were distinguishable by differences in the polyhedra. One was trigonal bipyramidal (having six planes) and the other was square pyramidal (having five planes). Both shapes were very similar to each other, although the synchrotron data clearly revealed slight geometrical differences.

Endothelin Receptor Antagonists↗

X-ray diffraction and atomic force microscopy analysis of twinned crystals: rhombohedral canavalin.

The structure of canavalin, the vicilin-class storage protein from jack bean, was refined to 1.7 A resolution in a highly twinned rhombohedral crystal of space group R3 and unit-cell parameters a = b = c = 83.0 A, alpha = beta = gamma = 111.1 degrees. The resulting R and R(free) were 0.176 and 0.245, respectively. The orthorhombic crystal structure (space group C222(1), unit-cell parameters a = 136.5, b = 150.3, c = 133.4 A) was also refined with threefold non-crystallographic symmetry restraints. R and R(free) were 0.181 and 0.226, respectively, for 2.6 A resolution data. No significant difference in the protein structure was seen between these two crystal forms, nor between these two and the hexagonal and cubic crystal forms reported elsewhere [Ko et al. (1993), Acta Cryst. D49, 478-489; Ko et al. (1993), Plant Physiol. 101, 729-744]. A phosphate ion was identified in the lumen of the C-terminal beta-barrel. Lattice interactions showed that the trimeric molecule could be well accommodated in both 'top-up' and 'bottom-up' orientations in a rhombohedral unit cell of the R3 crystal and explained the presence of a high twin fraction. The large inter-trimer stacking interface of the C222(1) crystal may account for its relative stability. Atomic force microscopy (AFM) investigations of the growth of three crystal forms of canavalin indicate the rhombohedral form to be unique. Unlike the other two crystal forms, it contains at least an order of magnitude more screw dislocations and stacking faults than any other macromolecular crystal yet studied, and it alone grows principally by generation of steps from the screw dislocations. The unusually high occurrence of the screw dislocations and stacking faults is attributed to mechanical stress produced by the alternate molecular orientations in the rhombohedral crystals and their organization into discrete domains or blocks. At boundaries of alternate domains, lattice strain is relieved by the formation of the screw dislocations.

Crystallization↗

Crystallization and preliminary crystallographic studies of RhoGDI in complex with the radixin FERM domain.

The Rho guanine nucleotide-dissociation inhibitor (RhoGDI) is a general regulator that forms a complex with the GDP-bound form of Rho-family GTPases and suppresses their activation. The FERM domains of ERM (ezrin/radixin/moesin) proteins bind to RhoGDI and dissociate Rho from RhoGDI. The formation of a complex between RhoGDI and the FERM domain is an important step in the regulatory cycle of Rho activation. In this study, crystals of RhoGDI complexed with the FERM domain of radixin were obtained. The crystals of the binary complex belong to the space group P2(1)2(1)2, with unit-cell parameters a = 130.9 (2), b = 151.2 (2), c = 71.2 (1) A, and contain two protein complexes in the crystallographic asymmetric unit. A 2.9 A resolution data set was collected using synchrotron radiation at SPring-8.

Animals↗

Structure of a new polymorphic monoclinic form of human transthyretin at 3 A resolution reveals a mixed complex between unliganded and T4-bound tetramers of TTR.

The crystal structure of a new polymorphic form of human transthyretin (hTTR) with a lattice containing a unique assembly of apo hTTR and TTR-T(4) complex has been determined to 3 A resolution. The monoclinic form of human TTR reported here crystallizes in space group P2(1), with unit-cell parameters a = 76.7 (6), b = 96.7 (8), c = 81.7 (4) A, beta = 106.8 (4) degrees. The asymmetric unit contains two tetramers of transthyretin related by the non-crystallographic symmetry (NCS) operation of a 90.28 degrees rotation between two hTTR molecules around an axis close to crystallographic z. The r.m.s. difference between the two tetramers calculated from their C(alpha) positions is 0.48 A. The structure was refined using 15.0-3.0 A resolution data to R = 22.9% and R(free) = 28.9% for reflections F > 0.0sigma(F), and R = 19.7% and R(free) = 25.8% for reflections F > 3.0sigma(F). The intermolecular interactions involve the tips of alpha-helices and loops around Arg21, Glu61 and Ser100 of all monomers. The electron-density maps revealed residual thyroxine (T(4)) bound in only one of the two unique tetrameric TTR molecules, with an occupancy of 53%, while the second tetramer is unliganded. One thyroxine ligand is bound in a way similar to the orientations described for the orthorhombic form of the hTTR-T(4) complex. The T(4) bound in the second site is positioned similar to 3',5'-dinitro-N-acetyl-L-thyronine in its hTTR complex. Differences in the size of the central channel defined by the D, A, G and H beta-strands of two monomeric subunits are observed between the apo TTR and T(4)-bound tetramer. The averaged distances between Ala108 C(alpha) and its equivalent measured across each binding site are 12.34 A for the T(4)-bound and 10.96 A for the unliganded TTR tetramer, respectively. The observed differences might reflect the mechanics of the ligand binding in the channel and possibly explain the observed negative cooperativity effect for ligand binding.

Animals↗

Combining image-seeking functions and a subtraction strategy: a vector-space procedure to improve many-body searches in molecular replacement.

Many-body searches in molecular replacement are usually carried out sequentially and each step benefits from the structural information obtained in previous rotational and translational stages. In this context, the incorporation of known structural information has proved to enhance the discrimination of a rotation function in Patterson space when many independent molecules have to be located in the asymmetric unit of the crystal cell. This improvement is achieved by subtraction of the contributions of already positioned molecules from the observed Patterson map, which makes the determination of the correct orientation of the remaining molecules easier. The quality of the resultant difference Patterson map is greatly influenced by the application of a bulk-solvent correction to the structure-factor amplitudes of the molecules that are being subtracted. The results obtained in the rotation search benefit both from the availability of high-resolution data and from the combination of the subtraction strategy and the refinement of a great number of the peaks of the rotation function.

Bacterial Proteins↗

Crystallization and preliminary crystallographic studies of a novel antifungal protein with five disulfide bridges from Eucommia ulmoides Oliver.

Two antifungal proteins, named Eucommia antifungal peptides 1 and 2 (EAFP1 and EAFP2), have been purified from the bark of the tree E. ulmoides Oliver and show a relatively broad spectrum of antifungal activity against several agriculturally important plant pathogens. One of these small proteins (EAFP2) has been crystallized. The crystal belongs to space group P2(1), with unit-cell parameters a = 19.01, b = 23.16, c = 30.69 A, beta = 98.54 degrees. 1.0 A resolution data were collected from an EAFP2 crystal and have been used to obtain phase information directly by an ab initio method.

Antifungal Agents↗

Multiple rotation function.

A simultaneous analysis of several rotation functions allows identification of the model orientation in situations when a single rotation function fails to find the answer. Multiple rotation functions can be obtained by the usual modification of the search model or by variation of the resolution at which the function is calculated. A specially suitable case is a search with several NMR models. When the orientation of the model becomes available, its position can be found much more easily; low-resolution data can help in such a search. Many difficult cases of molecular replacement can be solved by new tools discussed in the article.

Magnetic Resonance Spectroscopy↗

Is the bond-valence method able to identify metal atoms in protein structures?

The proper assignment of metal ions in X-ray structures of proteins is not always easy, but in many cases this knowledge can be important, e.g. for an understanding of enzyme mechanism. In this publication, the bond-valence approach is assessed critically. A simplified version, the calcium bond-valence sum (CBVS), is proposed for the convenient analysis of the geometric environment of potential sites with a view to metal-ion assignment. The bond-valence approach is found to be more reliable for structures determined from high-resolution data (1.5 A or better).

Algorithms↗

Structure of a constitutively activated RhoA mutant (Q63L) at 1.55 A resolution.

Mutants of the small G protein RhoA that are deficient in GTPase activity and thereby exhibit constitutive molecular signaling activity are commonly used to discover its cellular functions. In particular, two such mutants, Gly14-->Val (G14V) and Gln63-->Leu (Q63L), are often used interchangeably for such studies. However, while their in vitro rates of GTP hydrolysis are very similar, differences are observed in their other functional properties. The structure of G14V-RhoA is known; in order to assess whether structural variations are responsible for functional differences, the crystal structure of a Q63L-RhoA bound to the GTP-analog 5'-guanylylimidodiphosphate (GMPPNP) was determined at 1.5 A resolution. Overall, the structure is very similar to that of G14V-RhoA, but the significantly higher resolution data permit an improved basis for structural analysis and comparison. The data support the notion that differences observed between the mutants in vivo are likely to arise from altered affinities for RhoGDI and not from direct structural differences.

Amino Acid Substitution↗

Structure of superoxide dismutase from Pyrobaculum aerophilum presents a challenging case in molecular replacement with multiple molecules, pseudo-symmetry and twinning.

The crystal structure of superoxide dismutase from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum was determined by molecular replacement at 1.8 A resolution. The structure determination was made especially challenging by the large number of molecules (24) in the asymmetric unit, the presence of a pseudo-crystallographic twofold operator close to a twinning operator and the inability to detect twinning by conventional means. Molecular replacement proceeded at low resolution in pseudo (apparent) space group P3(2)12 and was facilitated by examination of the self-rotation function and native Patterson map. Refinement, however, stalled at an R factor of 40% when high-resolution data were included. Expanding to the lower symmetry space group P3(2) decreased R (to 22%) and R(free) (to 26%), but not by as much as expected for the quality of data. Finally, despite the apparent lack of evidence from conventional twinning tests [i.e. plots of the second moment of I and N(Z) distributions], a twinning operator was included in the refinement, lowering R and R(free) to 16.2 and 21.7%, respectively. The early detection of twinning appears to have been masked by a deviation in the expected intensity distribution caused by the presence of non-crystallographic translational symmetry. These findings suggest the importance of testing twinning operators in cases where pseudo-translational symmetry can explain negative results from conventional twinning tests. The structure reveals a tetrameric assembly with 222 symmetry, similar to superoxide dismutase structures from other organisms. The current structural model represents the metal-free state of the enzyme.

Archaeal Proteins↗

Co-crystallization of Leptospira interrogans peptide deformylase with a potent inhibitor and molecular-replacement schemes with eight subunits in an asymmetric unit.

Translation initiation in eubacteria involves a formylmethionine at the N-terminus of newly synthesized polypeptides. This N-formyl group is removed by peptide deformylase (PDF) during the post-translation process. Such a formylation/deformylation cycle is essential for the cell survival of eubacteria, but is not utilized in eukaryotic cytosolic protein biosynthesis. In view of the absence of deformylase activity in mammalian cells, this is an attractive target for the design of novel antibiotic drugs. Co-crystallization of peptide deformylase from Leptospira interrogans (LiPDF) with its natural inhibitor actinonin produced diffraction-quality crystals that belong to space group P2(1), with unit-cell parameters a = 87.5, b = 119.1, c = 95.8 A, beta = 111.6 degrees. The 3.1 A resolution data set collected in-house was used to obtain phases by molecular replacement. Three schemes for the correction of the preliminary solutions were proposed and proved successful in determining the structure of LiPDF with eight subunits in the asymmetric unit.

Amidohydrolases↗

Structure of the hybrid cluster protein (HCP) from Desulfovibrio desulfuricans ATCC 27774 containing molecules in the oxidized and reduced states.

The hybrid cluster protein (HCP) from the sulfate-reducing bacteria Desulfovibrio desulfuricans ATCC 27774 has been isolated and crystallized anaerobically. The protein sample used in the crystallization studies was several months old, having been stored at 193 K, and initial crystal structure studies were unable to fully resolve details of the hybrid cluster despite the use of high-resolution data to 1.25 A collected at the ESRF, Grenoble, France. Full elucidation of the structure has only become possible with the complete knowledge of the as-isolated and fully reduced crystal structures. The analysis clarifies the significant movements in the position of the Fe atom linked to the persulfide moiety in the oxidized as-isolated protein and the S atom of the persulfide itself as the protein is reduced. The structures of the as-isolated and reduced states are discussed in terms of the putative function of the HCP proteins.

Bacterial Proteins↗

Purification, crystallization and X-ray diffraction analysis of a recombinant Fab that recognizes a human blood-group antigen.

The NNA7 Fab fragment recognizes the human glycopeptide N blood-group antigen and has a high affinity for N-type glycophorin A (GPA). To provide insight into how antibodies recognize glycopeptide antigens, soluble Fab fragments were expressed in Escherichia coli, purified and crystallized using the hanging-drop vapor-diffusion method at 293 K. The best crystals were obtained from solutions of PEG monomethyl ether 5000 containing 4-8 mM yttrium chloride (YCl3). This rare-earth ion, which could be substituted with various lanthanides, changed the habit of crystals from multinucleated rods with a diffraction limit of 4.25 A resolution to a diamond-shaped morphology that grew as single crystals and diffracted X-rays to 1.75 A resolution. Data were collected that indicated that the crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 57.9, b = 77.1, c = 118.1 A and one Fab fragment per asymmetric unit. A molecular-replacement solution has been obtained and 86% of the molecule was fitted by use of an automated refinement procedure (ARP).

Antigen-Antibody Complex↗

The atomic resolution structure of methanol dehydrogenase from Methylobacterium extorquens.

The crystal structure of methanol dehydrogenase (MDH) from Methylobacterium extorquens has been refined without stereochemical restraints at a resolution of 1.2 A. The high-resolution data have defined the conformation of the tricyclic pyrroloquinoline quinone (PQQ) cofactor ring as entirely planar. The detailed definition of the active-site geometry has shown many features that are similar to the quinohaemo-protein alcohol dehydrogenases from Comamonas testosteroni and Pseudomonas putida, both of which possess MDH-like and cytochrome c-like domains. Conserved features between the two types of PQQ-containing enzyme suggest a common pathway for electron transfer between MDH and its physiological electron acceptor cytochrome cL. A pathway for proton transfer from the active site to the bulk solvent is also suggested.

Alcohol Dehydrogenase↗

Structure of a yellow lupin pathogenesis-related PR-10 protein belonging to a novel subclass.

Pathogenesis-related (PR) proteins of class 10 are abundant in higher plants. Some of these proteins are induced under stress conditions as part of the plant defence mechanism. Other homologues are developmentally regulated and their expression varies in different plant organs. The PR-10 proteins are encoded by multigene families, have a weight of about 17 kDa and are found in the cytosol. In yellow lupin, nine different homologues have been identified and divided into two subclasses, LlPR-10.1 and LlPR-10.2. Within each subclass the sequence identity is about 75-91%, while across the subclasses it is only 59-60%. Here, the crystal structure of a yellow lupin PR-10 protein from the second subclass, LlPR-10.2A, is presented. The structure was solved by molecular replacement and refined to R = 0.205 using 1.9 A resolution data. The general fold of LlPR-10.2A resembles that of the other PR-10 proteins and consists of a long C-terminal alpha-helix surrounded by a seven-stranded antiparallel beta-sheet, with two shorter alpha-helices located between strands beta1 and beta2. The most variable part of the structure, the C-terminal helix, is strongly kinked towards the beta-sheet core in both LlPR-10.2A molecules present in the asymmetric unit. This unexpected feature reduces the size of the hydrophobic cavity observed in other PR-10 proteins that is reported to be the ligand-binding site. As in other PR-10 structures, a surface loop located near the entrance to the cavity shows very high structural conservation and stability despite the high glycine content in its sequence.

Binding Sites↗

High-resolution structure of recombinant Trichomonas vaginalis thioredoxin.

The structure of thioredoxin from the anaerobic organism Trichomonas vaginalis (TvTrx) has been determined at 1.9 angstroms resolution. The structure is that of a typical thioredoxin: a five-stranded beta-sheet structure with two alpha-helices on either side. The active site of the protein carries a Trp-Cys-Gly-Pro-Cys motif, residues 34-38, at the N-terminus of an alpha-helix (alpha2). The cysteine residues in this motif form a redox-active disulfide necessary for thioredoxin activity. With high-resolution data available, it was possible to model numerous amino-acid side chains in alternate conformations and this includes the redox-active disulfide cysteine residues. The sample was initially in the oxidized state and the use of X-rays from an intense third-generation synchrotron source resulted in partial photoreduction of this labile redox centre. Comparisons with previously determined thioredoxin structures indicate that TvTrx is most similar to the human homologue, although the insertion of three residues between strands beta4 and beta5 makes the corresponding turn longer and more flexible in TvTrx. In addition, three significant amino-acid differences are identified on the protein surfaces near to the active-site Cys35. These residues may contribute to the interactions that specific thioredoxins form with their cognate physiological partners.

Amino Acid Sequence↗

High-resolution structure of human D-glyceraldehyde-3-phosphate dehydrogenase.

GAPDH (D-glyceraldehyde-3-phosphate dehydrogenase) is a multifunctional protein that is a target for the design of antitrypanosomatid and anti-apoptosis drugs. Here, the first high-resolution (1.75 Angstroms) structure of a human GAPDH is reported. The structure shows that the intersubunit selectivity cleft that has been leveraged in the design of antitrypanosomatid compounds is closed in human GAPDH. Modeling of an anti-trypanosomatid GAPDH inhibitor in the human GAPDH active site provides insights into the basis for the observed selectivity of this class of inhibitor. Moreover, the high-resolution data reveal a new feature of the cleft: water-mediated intersubunit hydrogen bonds that assist closure of the cleft in the human enzyme. The structure is used in a computational ligand-docking study of the small-molecule compound CGP-3466, which inhibits apoptosis by preventing nuclear accumulation of GAPDH. Plausible binding sites are identified in the adenosine pocket of the NAD(+)-binding site and in a hydrophobic channel located in the center of the tetramer near the intersection of the three molecular twofold axes. The structure is also used to build a qualitative model of the complex between GAPDH and the E3 ubiquitin ligase Siah1. The model suggests that the convex surface near GAPDH Lys227 interacts with a large shallow groove of the Siah1 dimer. These results are discussed in the context of the recently discovered NO-S-nitrosylation-GAPDH-Siah1 apoptosis cascade.

Binding Sites↗

Combining experimental data for structure determination of flexible multimeric macromolecules by molecular replacement.

A major effort has been made by the structural biology community to develop user-friendly software for the use of biologists. However, structural projects become more and more challenging and their solution often relies on a combination of information from various sources. Here, it is described how X-ray data, normal-mode analysis (NMA) and electron-microscopy (EM) data can be successfully combined in order to obtain a molecular-replacement (MR) solution for crystal structures containing multimeric molecules. NMA is used to simulate computationally the inherent internal flexibility of the monomer and thus enhance, together with the crystal noncrystallographic symmetry (NCS), the MR capabilities. NCS is also used to obtain a reliable EM reconstruction, which is then employed as a filter to construct oligomers starting from monomers. The feasibility of the direct use of EM reconstructions as a template for MR when the X-ray and EM data resolutions overlap is also discussed.

Agrobacterium tumefaciens↗