Abstract
The three-dimensional structure of recombinant human lymphotoxin (residues 24-171 of the mature protein) has been determined by x-ray crystallography at 1.9-A resolution (Rcryst = 0.215 for I greater than 3 sigma (I)). Phases were derived by molecular replacement using tumor necrosis factor (TNF-alpha) as a search model. Like TNF-alpha, lymphotoxin (LT) folds to form a "jellyroll" beta-sheet sandwich. Three-fold related LT subunits form a trimer stabilized primarily by hydrophobic interactions. A cluster of 6 basic residues around the 3-fold axis may account for the acid lability of the trimer. Although the structural cores of TNF-alpha and LT are similar, insertions and deletions relative to TNF-alpha occur in loops at the "top" of the LT trimer and significantly alter the local structure and the overall shape trimer is highly conserved. The sites of two mutations (Asp-50 and Tyr-108) that abolish the cytotoxicity of LT are contained within poorly ordered loops of polypeptide chain that flank the cleft between neighboring subunits at the base of the molecule, suggesting that the receptor recognizes an intersubunit binding site.
Highlights
Because the three-dimensional structures of LT and TNFa are key to understanding the differences in theirbiological and physical properties, we have undertaken crystallographic analysis of both molecules
Three-fold related LT sub- were grown by vapor diffusion at 4 “Cfrom hanging drops containing units form a trimer stabilized primarily by hydropho- 0.5 M NaCl, 20 mM Tris, pH 8.0, and 4 mg/ml protein over well bic interactions
A cluster of 6 basic residues around the %fold axis may account for the acid labilityof the trimer
Summary
Necrosis factor (TNF-a) as a search model. Like TNF-a, lymphotoxin (LT) folds to form a “jel- in Escherichia coli and purified as described previously (3). Atomic coordinates for monomer “A” from data set lTNF in the Brookhaven Protein Databank (10, 14) were used as a search model for phase determination by molecular replacement (15).Rotation and translation functions, and crystallographicrefinementcalculations were carried out using the program package X-PLOR (16). LT exhibits pleiotropic activities very similar to those of TNF-a Both molecules are cytotoxic to a variety of tumor cell lines in vitro and can induce hemorrhagic necrosis of tumors in vivo (3). Residues [24-32, 34-41, 39-46, 46-54, 84-91], The abbreviations used are: LT, lymphotoxin; TNFa,tumor [89-96, 99-103, 104-110, 110-117, 122-129, 158-165], and [168-171] necrosis factor-a
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