Crystallization and preliminary X-ray analysis of the beta-isoform of glutamate decarboxylase from Escherichia coli.

Glutamate decarboxylase (GAD) is a vitamin B6 enzyme which catalyzes the alpha-decarboxylation of L-glutamate to gamma-aminobutyric acid (GABA). Escherichia coli cells coexpress two highly homologous enzyme isoforms, GADalpha and GADbeta. Well diffracting crystals of GADbeta were obtained by taking advantage of the possibility of expressing each isoform separately. They belong to space group P31 or P32 with the unit-cell dimensions a = b = 115.6 and c = 206.6 A and contain one GAD hexamer in the asymmetric unit. High-resolution synchrotron data were collected at 100 K for the native protein and a potential heavy-atom derivative.

The crystal structure of a 3D domain-swapped dimer of RNase A at a 2.1-A resolution.

The dimer of bovine pancreatic ribonuclease A (RNase A) discovered by Crestfield, Stein, and Moore in 1962 has been crystallized and its structure determined and refined to a 2.1-A resolution. The dimer is 3D domain-swapped. The N-terminal helix (residues 1-15) of each subunit is swapped into the major domain (residues 23-124) of the other subunit. The dimer of bull seminal ribonuclease (BS-RNase) is also known to be domain-swapped, but the relationship of the subunits within the two dimers is strikingly different. In the RNase A dimer, the 3-stranded beta sheets of the two subunits are hydrogen-bonded at their edges to form a continuous 6-stranded sheet across the dimer interface; in the BS-RNase dimer, it is instead the two helices that abut. Whereas the BS-RNase dimer has 2-fold molecular symmetry, the two subunits of the RNase A dimer are related by a rotation of approximately 160 degrees. Taken together, these structures show that intersubunit adhesion comes mainly from the swapped helical domain binding to the other subunit in the "closed interface" but that the overall architecture of the domain-swapped oligomer depends on the interactions in the second type of interface, the "open interface." The RNase A dimer crystals take up the dye Congo Red, but the structure of a Congo Red-stained crystal reveals no bound dye molecule. Dimer formation is inhibited by excess amounts of the swapped helical domain. The possible implications for amyloid formation are discussed.

3D domain swapping: a mechanism for oligomer assembly.

3D domain swapping is a mechanism for forming oligomeric proteins from their monomers. In 3D domain swapping, one domain of a monomeric protein is replaced by the same domain from an identical protein chain. The result is an intertwined dimer or higher oligomer, with one domain of each subunit replaced by the identical domain from another subunit. The swapped "domain" can be as large as an entire tertiary globular domain, or as small as an alpha-helix or a strand of a beta-sheet. Examples of 3D domain swapping are reviewed that suggest domain swapping can serve as a mechanism for functional interconversion between monomers and oligomers, and that domain swapping may serve as a mechanism for evolution of some oligomeric proteins. Domain-swapped proteins present examples of a single protein chain folding into two distinct structures.

Comparison of two crystal structures of TGF-beta2: the accuracy of refined protein structures.

Transforming growth factor-beta is a multifunctional cell-growth regulator and is a member of the TGF-beta superfamily of cytokines. Each monomer is 112 amino acids long and the mature active form is a 25 kDa homodimer. Recently, the crystal structure of TGF-beta2 has been determined independently in two laboratories [Daopin, Piez, Ogawa & Davies (1992). Science, 257, 369-373; Schlunegger & Grütter (1992). Nature (London), 358, 430-434] and subsequently refined to higher resolutions [Daopin, Li & Davies (1993). Proteins Struct. Funct. Genet. In the press; Schlunegger & Grütter (1993). J. Mol. Biol. In the press]. A detailed structural comparison shows that the two structures are nearly identical with the differences mostly located on the mobile regions of the molecule. The r.m.s. differences between the two structures are 0.10 A for 104 pairs of C(alpha) atoms, 0.15 A for 434 pairs of main-chain atoms, 0.33 A for 860 out of 890 pairs of protein atoms and a correlation of 90% between the temperature B factors of all protein atoms. Based on a comparison of the water molecules, a B value of 60.0 A(2) is recommended as the cut off for modeling new waters. The structural identity is striking because in one case the material was expressed in vivo in CHO cells whereas in the other case it was expressed in E. coli and had to be refolded in vitro. The overall coordinate errors are estimated to be 0.21 A from the Luzzati plot, 0.18 A from the sigma(A) plot, 0.24 A with Cruickshank's equations and 0.25 A using the empirical method of Perry & Stroud. These estimates are comparable to the r.m.s. structure superposition. The r.m.s. differences correlate very well with the crystallographic B values and the relation is best described with the Cruickshank formula. In addition to the estimation of an overall error, a new application of the Cruickshank formula is presented here to estimate the local errors.

Crystallization and preliminary crystallographic investigations of the soluble glucose dehydrogenase from Acinetobacter calcoaceticus.

Single crystals of the soluble glucose dehydrogenase (GDH) from Acinetobacter calcoaceticus have been grown by the vapour diffusion method. These crystals diffract to beyond 2.1 A and are suitable for X-ray crystallography. The space group was determined to be P2(1) with unit cell parameters a = 55.5 A, b = 104.5 A, c = 86.5 A and beta = 99.8 degrees. One asymmetric unit contains a dimer of the GDH molecule.

Refined crystal structure of human transforming growth factor beta 2 at 1.95 A resolution.

Transforming growth factor beta 2 (TGF-beta 2), a homodimeric protein, is a member of a family of structurally related polypeptides that regulate various growth and differentiation processes in many cell types. The crystal structure of recombinant human TGF-beta 2 has been determined using a single heavy-atom derivative, anomalous scattering and by applying solvent flattening. The molecular model has been refined by a combination of simulated annealing and restrained least-squares refinement to a crystallographic R-factor of 0.194 including all data from 1.95 A to 8.0 A resolution. In the final structure, the root-mean-square deviation for bond lengths is 0.007 A and for bond angles 1.97 degrees. The final model includes 890 protein atoms (all 112 amino acid residues) as well as 84 water molecules. The new monomer fold consists of a separate alpha-helix and two pairs of antiparallel beta-sheet segments, which can be subdivided into nine individual beta-strands. The extended monomer lacks the typical hydrophobic core. A cluster of disulfide bridges, including the TGF-beta knot, connects the beta-strands with each other as well as the alpha-helix. Two monomers are covalently linked by a single disulfide bridge. In the dimer the alpha-helix of one subunit interacts with the beta-sheet of the other subunit forming two symmetrically related hydrophobic cores. The center of the dimer interaction is stabilized by a network of hydrogen bonds including several well-defined water molecules, which surround the central intersubunit disulfide bridge. The refined structure reveals the details of hydrogen bonding, electrostatic and hydrophobic interactions between intra- and intersubunit residues and allows the identification of possible receptor binding segments.

An unusual feature revealed by the crystal structure at 2.2 A resolution of human transforming growth factor-beta 2.

Transforming growth factor type beta 2 (TGF-beta 2) is a member of an expanding family of growth factors that regulate proliferation and differentiation of many different cell types. TGF-beta 2 binds to various receptors, one of which was shown to be a serine/threonine kinase. TGF-beta 2 is involved in wound healing, bone formation and modulation of immune functions. We report here the crystal structure of TGF-beta 2 at 2.2 A resolution, which reveals a novel monomer fold and dimer association. The monomer consists of two antiparallel pairs of beta-strands forming a flat curved surface and a separate, long alpha-helix. The disulphide-rich core has one disulphide bone pointing through a ring formed by the sequence motifs Cys-Ala-Gly-Ala-Cys and Cys-Lys-Cys, which are themselves connected through the cysteines. Two monomers are connected through a single disulphide bridge and associate such that the helix of one subunit interacts with the concave beta-sheet surface of the other. Four exposed loop regions might determine receptor specificity. The structure provides a suitable model for the TGF-beta s and other members of the super-family and is the basis for the analysis of the TGF-beta 2 interactions with the receptor.

Crystallization and preliminary X-ray analysis of recombinant human transforming growth factor beta 2.

Recombinant human transforming growth factor beta 2 (TGF-beta 2) was cloned and expressed in E. coli. The protein was isolated from inclusion bodies, renatured and purified to a single component as judged by reversed-phase HPLC. The recombinant TGF-beta 2 was shown to have a biological activity equal to that of native TGF-beta 2 in a fibroblast migration assay. Pure, active recombinant TGF-beta 2 has been crystallized from polyethylene glycol 400. The trigonal crystals of spacegroup P3(1)21 or P3(2)21 have unit cell dimensions of a=b=60.6 A, c=75.2 A and diffract beyond 2.0 A.