Mutual conformational adaptations in antigen and antibody upon complex formation between an Fab and HIV-1 capsid protein p24.

BACKGROUND: Elucidating the structural basis of antigen-antibody recognition ideally requires a structural comparison of free and complexed components. To this end we have studied a mouse monoclonal antibody, denoted 13B5, raised against p24, the capsid protein of HIV-1. We have previously described the first crystal structure of intact p24 as visualized in the Fab13B5-p24 complex. Here we report the structure of the uncomplexed Fab13B5 at 1.8 A resolution and analyze the Fab-p24 interface and the conformational changes occurring upon complex formation. RESULTS: Fab13B5 recognizes a nearly continuous epitope comprising a helix-turn-helix motif in the C-terminal domain of p24. Only 4 complementarity-determining regions (CDRs) are in contact with p24 with most interactions being by the heavy chain. Comparison of the free and complexed Fab reveals that structural changes upon binding are localized to a few side chains of CDR-H1 and -H2 but involve a larger, concerted displacement of CDR-H3. Antigen binding is also associated with an 8 degrees relative rotation of the heavy and light chain variable regions. In p24, small conformational changes localized to the turn between the two helices comprising the epitope result from Fab binding. CONCLUSIONS: The relatively small area of contact between Fab13B5 and p24 may be related to the fact that the epitope is a continuous peptide rather than a more complex protein surface and correlates with a relatively low affinity of antigen and antibody. Despite this, a significant quaternary structural change occurs in the Fab upon complex formation, with additional smaller adaptations of both antigen and antibody.

Anomalous X-ray diffraction with soft X-ray synchrotron radiation.

Anomalous diffraction with soft X-ray synchrotron radiation opens new possibilities in protein crystallography and materials science. Low-Z elements like silicon, phosphorus, sulfur and chlorine become accessible as new labels in structural studies. Some of the heavy elements like uranium exhibit an unusually strong dispersion at their M(V) absorption edge (lambdaMV = 3.497 A, E(MV) = 3545 eV) and so does thorium. Two different test experiments are reported here showing the feasibility of anomalous X-ray diffraction at long wavelengths with a protein containing uranium and with a salt containing chlorine atoms. With 110 electrons the anomalous scattering amplitude of uranium exceeds by a factor of 4 the resonance scattering of other strong anomalous scatterers like that of the lanthanides at their L(III) edge. The resulting exceptional phasing power of uranium is most attractive in protein crystallography using the multi-wavelength anomalous diffraction (MAD) method. The anomalous dispersion of an uranium derivative of asparaginyl-tRNA synthetase (hexagonal unit cell; a = 123.4 A, c = 124.4 A) has been measured for the first time at 4 wavelengths near the M(V) edge using the beamline ID1 of ESRF (Grenoble, France). The present set up allowed to measure only 30% of the possible reflections at a resolution of 4 A, mainly because of the low sensitivity of the CCD detector. In the second experiment, the dispersion of the intensity of 5 X-ray diffraction peaks from pentakismethylammonium undecachlorodibismuthate (PMACB, orthorhombic unit cell; a = 13.003 A, b = 14.038 A, c = 15.450 A) has been measured at 30 wavelengths near the K absorption edge of chlorine (lambdaK = 4.397 A, EK= 2819.6 eV). All reflections within the resolution range from 6.4 A to 3.4 A expected in the 20 degree scan were observed. The chemical state varies between different chlorine atoms of PMACB, and so does the dispersion of different Bragg peaks near the K-edge of chlorine. The results reflect the performance of the beamline ID1 of ESRF at wavelengths beyond 3 A at the end of 1998. A gain by a factor 100 for diffraction experiments with 4.4 A photons was achieved in Autumn 1999 when two focusing mirrors had been added to the X-ray optics. Further progress is expected from area detectors more sensitive to soft X-rays. Both CCD detectors and image plates would provide a gain of two orders of measured intensity. Image plates would have the additional advantage that they can be bent cylindrically and thus cover a larger solid angle in reciprocal space. In many cases, samples need to be cooled: closed and open systems are presented. A comparison with the state of art of soft X-ray diffraction, as it had been reached at HASYLAB (Hamburg, Germany), and as it is developing at the Brookhaven National Laboratory (USA), is given.

Feasibility and review of anomalous X-ray diffraction at long wavelengths in materials research and protein crystallography.

The feasibility and a review of progress in the long-wavelengths anomalous dispersion technique is given in the context of the development of beamline ID1 of the ESRF for such studies. First experiments on this beamline and their analyses are described. The first study reports on the use of uranium which exhibits an unusually strong anomalous dispersion at its M(V) absorption edge (lambda(M(V)) = 3.5 A). The anomalous scattering amplitude of uranium with 110 anomalous electrons exceeds the resonance scattering of other strong anomalous scatterers like that of the rare earth ions by a factor of four. The resulting exceptional phasing power of uranium is most attractive in protein crystallography using the MAD method. The anomalous dispersion of a uranium derivative of asparaginyl-tRNA synthetase (hexagonal, a = 124.4 A, c = 123.4 A) has been measured at three wavelengths near the M(V) edge using beamline ID1 of the ESRF. The present set-up allowed the measurement of 10% of the possible reflections at a resolution of 8 A. This is mainly due to the low sensitivity of the CCD camera. The second study, involving DAFS experiments at wavelengths near the K-absorption edge of chlorine (lambda(K) = 4.4 A), reports the use of salt crystals which give rise to much stronger intensities of diffraction peaks than those of protein crystals. In the case of a crystal of pentamethylammonium undecachlorodibismuthate (PMACB, orthorhombic, a = 13.00 A, b = 14.038 A, c = 15.45 A), all reflections within the resolution range from 6.4 A to 3.5 A and the total scan width of 24 degrees were collected. The crystalline structure of PMACB implies two chemically distinct states of the Cl atom. Consequently, different dispersions near the K-edge of chlorine are expected. The dispersion of the intensity of five Bragg peaks of the PMACB crystal has been measured at 30 wavelengths. The relative success of these preliminary experiments with X-rays of long wavelength shows that the measurement of anomalous X-ray diffraction at wavelengths beyond 3 A is feasible. Starting from the experience gained in these experiments, an increased efficiency of the instrument ID1 by two to three orders of magnitude will be achieved in this wavelength range. A comparison with different techniques of anomalous diffraction which rely on the use of argon/ethane-filled multiwire chambers and image plates as detectors for wavelengths near the K-edge of sulfur and phosphorus is also given.

Head-to-tail dimers and interdomain flexibility revealed by the crystal structure of HIV-1 capsid protein (p24) complexed with a monoclonal antibody Fab.

The crystal structure of an intact molecule of HIV-1 capsid protein (p24) in complex with a monoclonal antibody fragment recognizing an epitope on the C-terminal domain has been determined at 3 A resolution. The helical N- and C-terminal domains of p24 are linked by an extended peptide forming a flexibly linked dumb-bell-shaped molecule 75 A in overall length. The p24 construct used is a variant with an N-terminal extension that mimics to some extent the Gag context of p24. We observed a novel head-to-tail dimer of p24 molecules which occurs through the formation of a substantial intermolecular interface between the N- and C-terminal domains. Comparison with previously observed p24 dimers shows that the same residues and secondary structural elements can partake in different interfaces revealing a remarkable stickiness and plasticity of the p24 molecule, properties which, combined with the inter-domain flexibility, are presumably important in the assembly and maturation of viral particles. Previous mutagenesis studies designed to test specific N-N and C-C homodimer interfaces do not discriminate fully against the possibility of the observed N-C interface.

The crystal structure of asparaginyl-tRNA synthetase from Thermus thermophilus and its complexes with ATP and asparaginyl-adenylate: the mechanism of discrimination between asparagine and aspartic acid.

The crystal structure of Thermus thermophilus asparaginyl-tRNA synthetase has been solved by multiple isomorphous replacement and refined at 2.6 A resolution. This is the last of the three class IIb aminoacyl-tRNA synthetase structures to be determined. As expected from primary sequence comparisons, there are remarkable similarities between the tertiary structures of asparaginyl-tRNA synthetase and aspartyl-tRNA synthetase, and most of the active site residues are identical except for three key differences. The structure at 2.65 A of asparaginyl-tRNA synthetase complexed with a non-hydrolysable analogue of asparaginyl-adenylate permits a detailed explanation of how these three differences allow each enzyme to discriminate between their respective and very similar amino acid substrates, asparagine and aspartic acid. In addition, a structure of the complex of asparaginyl-tRNA synthetase with ATP shows exactly the same configuration of three divalent cations as previously observed in the seryl-tRNA synthetase-ATP complex, showing that this a general feature of class II synthetases. The structural similarity of asparaginyl- and aspartyl-tRNA synthetases as well as that of both enzymes to the ammonia-dependent asparagine synthetase suggests that these three enzymes have evolved relatively recently from a common ancestor.

Preliminary X-ray diffraction studies on asparaginyl-tRNA synthetase from Thermus thermophilus.

The recombinant asparginyl-tRNA synthetase from the thermophilic bacterium Thermus thermophilus expressed in Escherichia coli has been crystallized from PEG 6000 solutions. Depending on the PEG concentrations the crystals were in either tetragonal or hexagonal space groups. Although generally smaller, the latter (space group P6(4)22) diffracted better, to a resolution of 2.8 A. Using the coordinates of the yeast aspartyl-tRNA synthetase structure molecular replacement methods were applied to both tetragonal and hexagonal crystals; a solution was found which gave excellent crystal packing in both space groups.

Crystallization and preliminary structural analysis of catalase A from Saccharomyces cerevisiae.

Yeast peroxisomal catalase A, obtained at high yields by over expression of the C-terminally modified gene from a 2 mu-plasmid, has been crystallized in a form suitable for high resolution X-ray diffraction studies. Brownish crystals with bipyrimidal morphology and reaching ca. 0.8 mm in size were produced by the hanging drop method using ammonium sulphate as precipitant. These crystals diffract better than 2.0 A resolution and belong to the hexagonal space group P6(1)22 with unit cell parameters a = b = 184.3 A and c = 305.5 A. An X-ray data set with 76% completeness at 3.2 A resolution was collected in a rotating anode generator using mirrors to improve the collimation of the beam. An initial solution was obtained by molecular replacement only when using a beef liver catalase tetramer model in which fragments with no sequence homology had been omitted, about 150 residues per subunit. In the structure found a single molecule of catalase A (a tetramer with accurate 222 molecular symmetry) is located in the asymmetric unit of the crystal with an estimated solvent content of about 61%. The preliminary analysis of the structure confirms the absence of a carboxy terminal domain as the one found in the catalase from Penicillium vitalae, the only other fungal catalase structure available. The NADPH binding site appears to be involved in crystal contacts, suggesting that heterogeneity in the occupancy of the nucleotide can be a major difficulty during crystallization.

Interconversion of crystals of the Escherichia coli EF-Tu.EF-Ts complex between high- and low-diffraction forms.

Crystals of the complex formed between the two bacterial polypeptide elongation factors, EF-Tu and EF-Ts, produced from solutions of PEG 6000 can be of two morphologically similar forms both of space group P2(1)2(1)2(1). One form diffracts to only about 3 A resolution, the other to better than 2.4 A resolution. These forms can be interconverted and the transformation of one into the other has been shown to be solely a result of dehydration/hydration processes. By designing a suitable soaking protocol and careful control of the experimental parameters for data collection at cryotemperatures, complete data sets for the high-resolution form could be obtained.

The structure of the Escherichia coli EF-Tu.EF-Ts complex at 2.5 A resolution.

The crystal structure of the EF-Tu.EF-Ts complex from Escherichia coli has been determined to a resolution of 2.5 A. The complex contains two subunits of each of the elongation factors. The two EF-Ts molecules form a tight dimer, but there is little contact between the two EF-Tu molecules. The interaction of EF-Ts with EF-Tu results principally in the disruption of the Mg2+ ion binding site, thereby reducing the affinity of EF-Tu for guanine nucleotides.

The structural basis for seryl-adenylate and Ap4A synthesis by seryl-tRNA synthetase.

BACKGROUND: Seryl-tRNA synthetase is a homodimeric class II aminoacyl-tRNA synthetase that specifically charges cognate tRNAs with serine. In the first step of this two-step reaction, Mg.ATP and serine react to form the activated intermediate, seryl-adenylate. The serine is subsequently transferred to the 3'-end of the tRNA. In common with most other aminoacyl-tRNA synthetases, seryl-tRNA synthetase is capable of synthesizing diadenosine tetraphosphate (Ap4A) from the enzyme-bound adenylate intermediate and a second molecule of ATP. Understanding the structural basis for the substrate specificity and the catalytic mechanism of aminoacyl-tRNA synthetases is of considerable general interest because of the fundamental importance of these enzymes to protein biosynthesis in all living cells. RESULTS: Crystal structures of three complexes of seryl-tRNA synthetase from Thermus thermophilus are described. The first complex is of the enzyme with ATP and Mn2+. The ATP is found in an unusual bent conformation, stabilized by interactions with conserved arginines and three manganese ions. The second complex contains seryl-adenylate in the active site, enzymatically produced in the crystal after soaking with ATP, serine and Mn2+. The third complex is between the enzyme, Ap4A and Mn2+. All three structures exhibit a common Mn2+ site in which the cation is coordinated by two active-site residues in addition to the alpha-phosphate group from the bound ligands. CONCLUSIONS: Superposition of these structures allows a common reaction mechanism for seryl-adenylate and Ap4A formation to be proposed. The bent conformation of the ATP and the position of the serine are consistent with nucleophilic attack of the serine carboxyl group on the alpha-phosphate by an in-line displacement mechanism leading to the release of the inorganic pyrophosphate. A second ATP molecule can bind with its gamma-phosphate group in the same position as the beta-phosphate of the original ATP. This can attack the seryl-adenylate with the formation of Ap4A by an identical in-line mechanism in the reverse direction. The divalent cation is essential for both reactions and may be directly involved in stabilizing the transition state.

A new additive for protein crystallization.

The potential usefulness of the new zwitterionic solubilizing agent, dimethyl ethylammonium propane sulfonate (NDSB195), in protein crystallization was shown using hen egg-white lysozyme. In the presence of this agent, highly diffracting crystals were obtained using ammonium sulphate as a precipitant, whereas in its absence only amorphous precipitates were obtained. The crystals possess a triclinic unit cell not previously described and diffract to a resolution of 2 A. To ascertain that the new reagent had not produced significant changes in the protein fold the structure was determined to a resolution of 2.6 A. Only minor differences were observed (notably in regions of crystal contacts) with the known tetragonal lysozyme structure (Brookhaven Protein Data Bank entry 1HEL).

Crystal structures at 2.5 angstrom resolution of seryl-tRNA synthetase complexed with two analogs of seryl adenylate.

Crystal structures of seryl-tRNA synthetase from Thermus thermophilus complexed with two different analogs of seryl adenylate have been determined at 2.5 A resolution. The first complex is between the enzyme and seryl-hydroxamate-AMP (adenosine monophosphate), produced enzymatically in the crystal from adenosine triphosphate (ATP) and serine hydroxamate, and the second is with a synthetic analog of seryl adenylate (5'-O-[N-(L-seryl)-sulfamoyl]adenosine), which is a strong inhibitor of the enzyme. Both molecules are bound in a similar fashion by a network of hydrogen bond interactions in a deep hydrophilic cleft formed by the antiparallel beta sheet and surrounding loops of the synthetase catalytic domain. Four regions in the primary sequence are involved in the interactions, including the motif 2 and 3 regions of class 2 synthetases. Apart from the specific recognition of the serine side chain, the interactions are likely to be similar in all class 2 synthetases.

Refined crystal structure of the seryl-tRNA synthetase from Thermus thermophilus at 2.5 A resolution.

The three-dimensional structure of the seryl-tRNA synthetase from Thermus thermophilus has been determined and refined at 2.5 A resolution. The final model consists of a dimer of 421 residues each and 190 water molecules. The R-factor is 18.4% for all the data between 10 and 2.5 A resolution. The structure is very similar to that of the homologous enzyme from Escherichia coli, with an r.m.s. difference of 1.5 A for the 357 alpha-carbon atoms considered equivalent. The comparison of the two structures indicates increased hydrophobicity, reduced conformational entropy and reduced torsional strain as possible mechanisms by which thermostability is obtained in the enzyme from the thermophile.

Crystallization of the seryl-tRNA synthetase:tRNAS(ser) complex of Escherichia coli.

Crystals of the complex between seryl-tRNA synthetase and tRNA(2ser) from Escherichia coli have been obtained from ammonium sulphate solutions. The crystals are of the 1:2 enzyme:tRNA complex, belong to the space group C222(1), have cell dimensions of a = 128.9 A, b = 164.9 A, c = 127.3 A and diffract anisotropically from 3.5 to 4.5 A. An X-ray diffraction data set to 4 A has been collected. The combination of molecular replacement using the refined structure of the catalytic domain of the native enzyme, data from a heavy atom derivative and solvent flattening was used to produce a map at 4 A resolution. This shows that a tRNA molecule binds across the dimer, the anticodon stem and loop do not contact the protein and the helical arm of the enzyme contacts the T psi C loop and the long extra arm of the tRNA.

A new crystal form of the complex between seryl-tRNA synthetase and tRNA(Ser) from Thermus thermophilus that diffracts to 2.8 A resolution.

Two distinct complexes between seryl-tRNA synthetase and tRNA(Ser) from Thermus thermophilus have been crystallized using ammonium sulphate as a precipitant. Form III crystals grow from solutions containing a 1:2.5 stoichiometry of synthetase dimer to tRNA. They are of monoclinic space group C2 with unit cell dimensions a = 211.6 A, b = 126.8 A, c = 197.1 A, beta = 132.4 degrees and diffract to about 3.5 A. Preliminary crystallographic results show that the crystallographic asymmetric unit contains two synthetase dimers. Form IV crystals grow from solutions containing a 1:1.5 stoichiometry of synthetase dimer to tRNA. They are of orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 124.5 A, b = 128.9 A, c = 121.2 A and diffract to 2.8 A resolution. Preliminary crystallographic results show that these crystals contain only one tRNA molecule bound to a synthetase dimer.

Crystallization of the seryl-tRNA synthetase-tRNA(Ser) complex from Thermus thermophilus.

The complex between seryl-tRNA synthetase and its cognate tRNA from the extreme thermophile Thermus thermophilus has been crystallized from ammonium sulphate solutions. Two different tetragonal crystal forms have been characterized, both diffracting to about 6 A using synchrotron radiation. One form grows as large bipyramids and has cell dimensions a = b = 127 A, c = 467 A, and the second form occurs as long, thin square prisms with cell dimensions a = b = 101 A, c = 471 A. Analysis of washed and dissolved crystals demonstrates the presence of both protein and tRNA.

Structure of adenovirus fibre. I. Analysis of crystals of fibre from adenovirus serotypes 2 and 5 by electron microscopy and X-ray crystallography.

An analysis by electron microscopy in amorphous ice and X-ray diffraction of four types of three-dimensional crystals of adenovirus fibre is presented. Fibre from adenovirus type 2 (Ad2) crystallizes in two forms depending on whether it is native or cleaved near the N terminus at Tyr17. Fibre from Ad5 also crystallizes in two forms, both of which contained fibre cleaved at Tyr17. Analysis of the packing of the fibres in each of these crystals suggests that the overall length of the fibre may be considerably longer (about 350 to 370 A) than previously reported. Crystals of cleaved Ad2 fibre are of sufficient quality to be characterized by X-ray diffraction. They are of space group C2 and cell dimensions a = 134.4 A, b = 77.6 A, c = 539.4 A, beta = 92.7 degrees. These crystals are remarkable in that, despite being monoclinic, the ab plane forms a perfect hexagonal lattice. This is explained by a trigonal packing of the trimeric fibre heads in the crystal. A similar feature is found for one type of Ad5 crystal, although the hexagonal lattice is 12% smaller. The crystals of cleaved Ad2 show very strong meridional intensity at a Bragg spacing of 4.4 A and weaker diffuse intensity corresponding to layer-lines of spacing 26.4 A. This must reflect the quasiperiodicity of the structure of the fibre shaft, which is apparent in the primary sequence. The occurrence of these features combined with the new determination of the length of the fibre (see also the accompanying paper) require a reappraisal of the cross-beta model of the fibre shaft proposed by Green et al.

A second class of synthetase structure revealed by X-ray analysis of Escherichia coli seryl-tRNA synthetase at 2.5 A.

The three-dimensional crystal structure of seryl-transfer RNA synthetase from Escherichia coli, refined at 2.5 A resolution, is described. It has an N-terminal domain that forms an antiparallel alpha helical coiled-coil, stretching 60 A out into the solvent and stabilized by interhelical hydrophobic interactions and an active-site alpha-beta domain based around a seven-stranded antiparallel beta sheet. Unlike the three other known synthetase structures, the enzyme contains no classical nucleotide-binding fold, and is the first representative of a second class of aminoacyl-tRNA synthetase structures.

Crystals of seryl-tRNA synthetase from Thermus thermophilus. Preliminary crystallographic data.

Crystals have been obtained of seryl-tRNA synthetase from the extreme thermophile Thermus thermophilus, using mixed solutions of ammonium sulphate and methane pentane diol. The crystals are very stable and diffract to at least 2 A. The crystals are monoclinic (space group P21) with cell parameters a = 87.1 A, b = 126.9 A, c = 63.5 A and beta = 109.7 degrees.

Comparative structural studies of reconstituted and native type I and type II collagen fibrils by low-angle X-ray diffraction.

Acid-soluble and pepsin-soluble type I collagen from calf skin and pepsin-soluble type II collagen from bovine articular cartilage were precipitated in fibrillar form by various methods. Reconstituted native-like fibrils were analysed by low-angle X-ray diffraction, and the patterns were compared with those obtained with native type I (rat tail tendon) and type II fibrils (bovine articular cartilage). For both orientated and disorientated forms of these samples, we measured the ratio of the first/third-order intensities of the meridional diffraction peaks which are associated with the gap-filling. The values obtained with the reconstituted native-like fibrils (types I and II) were double and 20-times the values, respectively, measured for rat tail tendon and bovine articular cartilage. These differences reflect the extent of specific interactions of other components (proteoglycans) at the gap level along the collagen fibrils in the two tissues.