Vol. 273, Issue 4, 2153-2160, January 23, 1998
High Resolution Crystal Structure of a Human Tumor Necrosis
Factor-
Mutant with Low Systemic Toxicity
Sun-Shin
Cha
,
Jeong-Sun
Kim,
Hyun-Soo
Cho,
Nam-Kyu
Shin§,
Woojin
Jeong§,
Hang-Cheol
Shin§,
Yeoun Jin
Kim¶,
Jong Hoon
Hahn¶, and
Byung-Ha
Oh
From the Department of Life Science and School of
Environmental Engineering and the ¶ Center for Biofunctional
Molecules and Department of Chemistry, Pohang University of Science and
Technology, Hyoja-dong, San 31, Pohang, Kyungbuk, 790-784, South Korea
and the § Protein Engineering Laboratory, Hanhyo Institute
of Technology, 461-6 Jeonmin-dong, Yusung-gu,
Taejon 305-390, South Korea
A human tumor necrosis factor-
(TNF-)
mutant (M3S) with low systemic toxicity in vivo was
designed, and its structures in two different crystal packings were
determined crystallographically at 1.8 and 2.15-Å resolution,
respectively, to explain altered biological activities of the mutant.
M3S contains four changes: a hydrophilic substitution 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-. Compared with wild-type TNF-, it exhibits 11- and
71-fold lower binding affinities for the human TNF-R55 and TNF-R75
receptors, respectively, and in vitro cytotoxic effect and
in vivo systemic toxicity of M3S are 20 and 10 times lower,
respectively. However, in a transplanted solid tumor mouse model, M3S
suppresses tumor growth more efficiently than wild-type TNF-. M3S is
highly resistant to proteolysis by trypsin, and it exhibits increased
thermal stability and a prolonged half-life in vivo. The
L29S mutation causes substantial restructuring of the loop containing
residues 29-36 into a rigid segment as a consequence of induced
formation of intra- and intersubunit interactions, explaining the
altered receptor binding affinity and thermal stability. A mass
spectrometric analysis identified 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 not induce a noticeable conformational
change. The side chain of Phe56 projects into a hydrophobic
cavity, while Ile52 is exposed to the bulk solvent.
Ile52 should be involved in hydrophobic interactions with
the receptors, since a mutant containing the same mutations as in M3S
except for the L29S mutation exhibits an increased receptor binding
affinity. The low systemic toxicity of M3S appears to be the effect of
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-type TNF-. It is also expected that the 1.8-Å resolution structure will serve as an accurate model for explaining the
structure-function relationship of wild-type TNF- and many TNF-
mutants reported previously and for the design of new TNF-
mutants.
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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