Carmen Berthet-Colominas

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 Å 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 Å 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.

Structural study of the calcifying collagen in turkey leg tendons

Abstract The calcified turkey leg tendon represents a simple bone-like tissue that is ideally suited to analysis by diffraction methods. In this paper we report some structural studies of the tendon collagen in the uncalcified, fully calcified and partially calcified states. The low-angle meridional X-ray pattern from the uncalcified tendon is very similar to that of the rat tail tendon, and the resulting one-dimensional structure of the collagen fibril exhibits no feature that could be related to its eventual calcification. The structure of the fully calcified tendon, as determined by a combination of X-ray and neutron diffraction analyses, shows that the mineral is associated with the collagen at the level of the hole or gap region. In the calcifying tendon, increases in the amplitudes of the first and second X-ray meridional reflections are correlated with an increase in the mineral content of the collagen. On the basis of simple models, it is shown that this change in the pattern can be explained by a nucleation mechanism of calcification. It is concluded that when collagen becomes calcified the mineral penetrates throughout the fibril and is crystalline in the hole region but amorphous between the collagen molecules. The mechanism of calcification and the mechanical implications of the fully calcified structure are also discussed.

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 Å 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 Å 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.

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.

Crystallization of the seryl-tRNA synthetase: tRNAser complex of Escherichia coli

Crystals of the complex between seryl‐tRNA synthetase and tRNA2 ser 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,, have cell dimensions of a = 128.9 Å, b = 164.9 Å, c = 127.3 Å and diffract anisotropically from 3.5 to 4.5 Å. An X‐ray diffraction data set to 4 Å 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 Å 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 TΨC loop and the long extra arm of the tRNA.

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 Å. 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 Å. Only minor differences were observed (notably in regions of crystal contacts) with the known tetragonal lysozyme structure (Brookhaven Protein Data Bank entry 1HEL).

Structural studies of adenovirus type 2 by neutron and X-ray scattering.

Small-angle neutron and X-ray scattering have been used to investigate various aspects of the structural organization of adenovirus type 2. Neutron scattering allows the determination of the radial distribution of DNA and protein, which because of the highly icosahedral form of the virus allows it to be described in terms of three icosahedral shells. X-ray scattering shows that the distance between the major coat proteins (hexons) in the capsid is 100 +/- A. Evidence was also observed for an organization in the nucleoprotein core that gives rise to a maximum in the X-ray scattering at 1/29 A-1.

Structural studies of collagen fibres from intervertebral disc.

A combined biochemical, X-ray and electron microscopic study is presented on the molecular structure of bovine and human intervertebral disc. Positively stained electron micrographs of Type I and Type II collagen are shown to have slightly but significantly different banding patterns. New low-angle meridional patterns from different anatomical parts of the intervertebral disc are reported from native and trypsin-extracted discs. Qualitative measurements of these intensities lead to the conclusion that in the nucleus pulposus of the disc, another molecule, probably proteoglycan, is ordered on the collagen fibrils at the axial level of the gap region.

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

Two distinct complexes between seryl‐tRNA synthetase and tRNA Ser from Thermus themophilus 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 Å, b = 126.8 Å, c = 197.1 Å, β = 132.4° and diffract to about 3.5 Å. 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 P212121 with unit cell dimensions a = 124.5 Å, b = 128.9 Å, c = 121.2 Å and diffract to 2.8 Å resolution. Preliminary crystallographic results show that these crystals contain only one tRNA molecule bound to a synthetase dimer.

Crystal packing and stoichiometry of the fibre protein of adenovirus type 2

Crystals of the fibre protein of adenovirus type 2 have been grown and studied by electron microscopy and X-ray powder diffraction. The molecular packing and density of the crystals suggest that the fibre is dimeric.