HIGH-RESOLUTION CRYSTAL-STRUCTURE OF A HUMAN TUMOR-NECROSIS-FACTOR-ALPHA MUTANT WITH LOW SYSTEMIC TOXICITY

A human tumor necrosis factor-alpha (TNF-alpha) mutant (M3S) with low systemic toxicity in vivo was designed, and its structures in two diff erent crystal packings were determined crystallographically at 1.8 and 2.15-Angstrom resolution, respectively, to explain altered biological activities of the mutant, M3S contains four changes: a hydrophilic su bstitution of L29S,two hydrophobic substitutions of S52I and Y56F, and a deletion of the N-terminal seven amino acids that is disordered in the structure of wild type TNF-alpha. Compared with wild-type TNF-alph a, it exhibits 11- and 71-fold lower binding affinities for the human TNF-R55 and TNF-R75 receptors, respectively, and in vitro cytotoxic ef fect and in vivo systemic toxicity of M3S are 20 and 10 times lower, r espectively, However, in a transplanted solid tumor mouse model, M3S s uppresses tumor growth more efficiently than wild-type TNF-alpha, M3S is highly resistant to proteolysis by trypsin, and it exhibits increas ed thermal stability and a prolonged half-life in vivo, The L29S mutat ion causes substantial restructuring of the loop containing residues 2 9-36 into a rigid segment as a consequence of induced formation of int ra-and intersubunit interactions, explaining the altered receptor bind ing affinity and thermal stability, A mass spectrometric analysis iden tified major proteolytic cleavage sites located on this loop, and thus the increased resistance of M3S to the proteolysis is consistent with the increased rigidity of the loop, The S52I and Y56F mutations do no t induce a noticeable conformational change, The side chain of Phe(56) projects into a hydrophobic cavity, while Ile(52) is exposed to the b ulk solvent, Ile(52) should be involved in hydrophobic interactions wi th the receptors, since a mutant containing the same mutations as in M 3S except for the L29S mutation exhibits an increased receptor binding affinity. The low systemic toxicity of M3S appears to be the effect o f the reduced and selective binding affinities for the TNF receptors, and the superior tumor-suppression of M3S appears to be the effect of its weak but longer antitumoral activity in vivo compared with wild-ty pe TNF-alpha, It is also expected that the 1.8-Angstrom resolution str ucture will serve as an accurate model for explaining the structure-fu nction relationship of wild-type TNF-alpha and many TNF-alpha mutants reported previously and for the design of new TNF-alpha mutants.