STRUCTURAL STUDIES OF REGULATORY AND SIGNAL TRANSDUCTION PROTEINS 
Stephen R. Sprang, Ph.D., Assistant Investigator 
1. Three-dimensional Structure of Tumor Necrosis 
Factor/Cachectin. 
Tumor necrosis factor (TNF) is a cytokine hor- 
mone synthesized primarily by macrophages in re- 
sponse to host infection by gram-negative bacteria, 
sepsis, or invasion by neoplastic tissue. The hor- 
mone binds to a specific 85 kDa receptor expressed 
on the surfaces of nearly all cells. The cytologic ac- 
tion of TNF follows its cellular internalization by 
the classic receptor-mediated endocytotic pathway. 
Although TNF is immediately degraded upon entry 
into the cell, it induces the synthesis of several nu- 
clear oncogenes, including c-myc and c-fos. Subse- 
quent changes in the metabolic state of cells are a 
function of their state of differentiation. These re- 
sult in the induction of the inflammatory response, 
shock, and cachexia. TNF is directly cytotoxic to 
certain cell lines. 
A structural determination of TNF was under- 
taken to elucidate the mechanism of receptor speci- 
ficity and to search for a structural basis of its cyto- 
logical activity. Results from the laboratory of Dr. 
Bruce A. Beutler (HHMI, University of Texas South- 
western Medical Center at Dallas) and from other 
laboratories suggest that receptor binding is itself 
not sufficient for activity, which may be potentiated 
by other structural determinants on the hormone. 
Crystals of TNF that diffract to a resolution ex- 
o 
ceeding 2.5 A were obtained by Michael Eck. Using 
a series of isomorphous heavy-metal derivatives of 
TNF, Eck derived an initial set of crystallographic 
phases. Electron density maps computed at this 
stage revealed that TNF forms a trimer in the asym- 
metric unit of the crystal, confirming reports from 
Dr. Sprang and other laboratories that TNF exists in 
solution and binds receptors as a trimeric species. 
Iterative cycles of symmetry averaging were used to 
refine the crystallographic phases, and an electron 
density map of sufficient quality was computed to 
allow the course of the polypeptide backbone to be 
traced and side-chain atoms to be positioned accu- 
rately. Crystallographic refinement assisted by mo- 
lecular dynamics "simulated annealing" [using the 
program package XPLOR by Dr. Axel T. Briinger 
(HHMI, Yale University)] reduced the crystallo- 
graphic Rvalue to 0.23. 
Subunits of TNF, which are composed entirely of 
antiparallel P-sheet structure, bear a striking topo- 
logical resemblance to coat proteins of icosahedral 
RNA plant and animal viruses. Subunits share an 
extensive interface about the noncrystallographic 
threefold axis of symmetry, in an interaction stabi- 
lized by both polar and hydrophobic contacts. A 
putative receptor-binding site was identified by lo- 
cating, in the three-dimensional atomic model, resi- 
dues that are identically conserved between TNF 
and the related lymphokine, lymphotoxin, which 
also binds to the TNF receptor. This analysis re- 
vealed that lymphotoxin is probably a trimer and 
that the receptor-binding site in both molecules is 
localized to a patch of residues at the base of the 
trimer, near the amino and carboxyl termini of the 
subunits. 
Further studies of TNF mutants with altered re- 
ceptor binding and cytological activities are 
planned, in collaboration with Dr. Beutler, to test 
the present model for the binding site and to delin- 
eate features of the binding site by directed muta- 
genesis. A second, long-range goal is to identify the 
molecular determinants of the hormone transduc- 
tion events subsequent to receptor binding. 
II. Structural Studies of Annexins. 
The annexins comprise a large family of calcium- 
binding proteins associated, in a variety of cell 
types, with the cytoskeleton or with microsomal 
membranes. These proteins, which include the 
calelectrins, endonexins, lipocortins, and calbind- 
ins, appear to have evolved by serial duplication of 
an exon encoding a 70- to 90-residue, possibly 
a-helical, domain. Members of the annexin family 
with molecular weights in the 32-36 kDa range 
comprise four such repeats, while the 67K proteins 
appear to be duplications of the 32-36K protein 
domains. Both molecular weight classes demon- 
strate a phospholipid-dependent calcium-binding 
activity, and several of the annexins are phosphory- 
lated at serine or tyrosine residues. The activity or 
function of none of these proteins has been identi- 
fied, but it is possible that certain members of the 
family are involved in potentiating membrane fu- 
sion in a calcium-regulated manner. 
Using preparative methods developed by Drs. 
Thomas H. Siidhof (HHMI) and Gary Reynolds 
(University of Texas Southwestern Medical Center 
at Dallas), the laboratory has purified both the 32K 
lipocortin III and the 67K calelectrin to homogene- 
ity. Crystals of both proteins have been grown in 
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