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.

Crystal structure of TGF-beta 2 refined at 1.8 A resolution.

The crystal structure of TGF-beta 2 has been refined using data collected with synchrotron radiation (CHESS) to 1.8 A resolution with a residual R (= sigma magnitude of Fo-magnitude of Fc/sigma magnitude of Fo) factor of 17.3%. The model consists of 890 protein atoms from all 112 residues and 59 water molecules. The monomer of TGF-beta 2 assumes a rather extended conformation and lacks a well-defined hydrophobic core. Surface accessibility calculations show only 44% of the nonpolar surface is buried in the monomer. In contrast, 55.8% of the nonpolar surface area is buried when the two monomers form a dimer, a typical value for globular proteins. This includes a 1300 A2 buried interface area that is largely hydrophobic. Sequence comparisons using a profile derived from the refined TGF-beta 2 structure suggest that the cluster of four disulfides (three intramonomeric disulfide bonds 15-78, 44-109, 48-111 forming a disulfide knot, and one intermonomeric disulfide 77-77) together with the extended beta strand region constitutes the conserved structural motif for the TGF-beta superfamily. This structural motif, without the 77-77 disulfide bond, defines also the common fold for a general family of growth factors, including the nerve growth factor and platelet-derived growth factor families. The fold is conserved only at the monomer level, while the active forms are dimers, suggesting that dimerization plays an important role in regulating the binding of these cytokines to their receptors and in modulating the biological responses.

Crystal structure of transforming growth factor-beta 2: an unusual fold for the superfamily.

The transforming growth factors-beta (TGF-beta 1 through -beta 5) are a family of homodimeric cytokines that regulate proliferation and function in many cell types. Family members have 66 to 80% sequence identity and nine strictly conserved cysteines. A crystal structure of a member of this family, TGF-beta 2, has been determined at 2.1 angstrom (A) resolution and refined to an R factor of 0.172. The monomer lacks a well-defined hydrophobic core and displays an unusual elongated nonglobular fold with dimensions of approximately 60 A by 20 A by 15 A. Eight cysteines form four intrachain disulfide bonds, which are clustered in a core region forming a network complementary to the network of hydrogen bonds. The dimer is stabilized by the ninth cysteine, which forms an interchain disulfide bond, and by two identical hydrophobic interfaces. Sequence profile analysis of other members of the TGF-beta superfamily, including the activins, inhibins, and several developmental factors, imply that they also adopt the TGF-beta fold.

Structural and thermodynamic analysis of the packing of two alpha-helices in bacteriophage T4 lysozyme.

Packing interactions in bacteriophage T4 lysozyme were explored by determining the structural and thermodynamic effects of substitutions for Ala98 and neighboring residues. Ala98 is buried in the core of T4 lysozyme in the interface between two alpha-helices. The Ala98 to Val (A98V) replacement is a temperature-sensitive lesion that lowers the denaturation temperature of the protein by 15 degrees C (pH 3.0, delta delta G = -4.9 kcal/mol) and causes atoms within the two helices to move apart by up to 0.7 A. Additional structural shifts also occur throughout the C-terminal domain. In an attempt to compensate for the A98V replacement, substitutions were made for Val149 and Thr152, which make contact with residue 98. Site-directed mutagenesis was used to construct the multiple mutants A98V/T152S, A98V/V149C/T152S and the control mutants T152S, V149C and A98V/V149I/T152S. These proteins were crystallized, and their high-resolution X-ray crystal structures were determined. None of the second-site substitutions completely alleviates the destabilization or the structural changes caused by A98V. The changes in stability caused by the different mutations are not additive, reflecting both direct interactions between the sites and structural differences among the mutants. As an example, when Thr152 in wild-type lysozyme is replaced with serine, the protein is destabilized by 2.6 kcal/mol. Except for a small movement of Val94 toward the cavity created by removal of the methyl group, the structure of the T152S mutant is very similar to wild-type T4 lysozyme. In contrast, the same Thr152 to Ser replacement in the A98V background causes almost no change in stability. Although the structure of A98V/T152S remains similar to A98V, the combination of T152S with A98V allows relaxation of some of the strain introduced by the Ala98 to Val replacement. These studies show that removal of methyl groups by mutation can be stabilizing (Val98----Ala), neutral (Thr152----Ser in A98V) or destabilizing (Val149----Cys, Thr152----Ser). Such diverse thermodynamic effects are not accounted for by changes in buried surface area or free energies of transfer of wild-type and mutant side-chains. In general, the changes in protein stability caused by a mutation depend not only on changes in the free energy of transfer associated with the substitution, but also on the structural context within which the mutation occurs and on the ability of the surrounding structure to relax in response to the substitution.(ABSTRACT TRUNCATED AT 400 WORDS)