Structural Studies of Proteins Involved in 
Hormonal Signal Transduction 
Stephen R. Sprang, Ph.D. — Associate Investigator 
Dr. Sprang is also Associate Professor of Biochemistry at the University of Texas Southwestern Medical 
Center at Dallas. He received a B.S. degree from California State University at Los Angeles, and a Ph.D. 
degree from the University of Wisconsin, Madison, where Muttaiya Sundaralingam was his advisor. His 
postdoctoral training was at the University of Alberta, Edmonton, with Robert Fletterick. Again with Dr. 
Fletterick, he was an assistant research biochemist at the University of California, San Francisco, before 
assuming his present position. 
CELLS communicate with one another by se- 
creting growth factors, cytokines, or hor- 
mones and by responding to factors produced by 
other cells. These chemical messengers act by 
changing patterns of gene expression within the 
target cell, thereby altering its metabolic or devel- 
opmental program. One of the major unsolved 
problems in biology is to define the mechanisms 
by which cells specifically recognize these chem- 
ical messengers and to understand how the mes- 
sages are transduced intracellularly. 
In our laboratory, we are using x-ray crystallo- 
graphic techniques to define the molecular na- 
ture of the interactions between specific growth 
factors and their cognate receptors. These recep- 
tors are themselves protein molecules distributed 
on the outer surface of the plasma membrane 
surrounding the target cell. The goal of our re- 
search is to learn how receptors specifically rec- 
ognize growth factors and how the receptor mol- 
ecules' structure and chemical properties are 
changed, if at all, as a consequence of this 
interaction. 
Tumor Necrosis Factors 
One family of factors we study, called tumor 
necrosis factors (TNF), are produced by cells of 
the immune system. Macrophages — white blood 
cells responsible for engulfing cellular debris — 
produce large quantities of TNF-a (also known as 
cachectin) when stimulated by toxins carried on 
the surface of bacteria, as would occur in the 
event of infection. The avid attachment of this 
cytokine to receptors present on a variety of cells 
in the body triggers a series of events that dramati- 
cally alter fat and triglyceride metabolism and 
mobilize neutrophils. These events mediate in- 
flammation, endotoxic shock, and the wasting 
phenomenon cachexia, from which the cytokine 
takes one of its names. TNF-a also induces a num- 
ber of enzymes that effect the destruction and re- 
modeling of tissues. 
A related cytokine, TNF-/?, is produced by T 
lymphocytes. It has many properties in common 
with TNF-a, including the ability to bind to the 
same receptors, but the two cytokines are only 30 
percent identical in amino acid sequence. It is of 
interest to learn how such distantly related mole- 
cules can interact with the same receptor. 
The three-dimensional structure of TNF-a, de- 
termined by graduate student Michael Eck, re- 
vealed the molecule to be composed of three 
identical protein subunits packed about a three- 
fold axis of symmetry. We speculated that TNF 
may be capable of binding three receptors at once 
and that the TNF-induced aggregation of recep- 
tors might trigger the cascade of molecular events 
within the cell. The solution of the structure of 
TNF- (8 is still in progress, but initial results indi- 
cate that it has the same overall subunit tertiary 
structure and trimeric quaternary structure as 
TNF-a. Of particular interest, once more struc- 
tural information is available about the nature of 
the TNF-receptor interactions, will be to deter- 
mine whether different or similar chemical 
groups are used by the two cytokines in recogniz- 
ing the TNF receptor. The proteins used in 
our crystallographic studies were provided by 
George Kuo of Chiron, Inc. (TNF-a), and Mark 
Ultsch, Bart DeVos, and Anthony Kossiakofif of 
Genentech, Inc. (TNF-/?). 
Recently several researchers have succeeded in 
isolating the gene encoding the receptors that en- 
gage TNF-a and -0. Two different types of TNF 
receptor were found. Surprisingly, their amino 
acid sequences bear little identity to each other. 
Both receptors, however, contain a structural 
"motif" composed of four inexact copies of a re- 
peat rich in the sulfur-containing amino acid cys- 
teine. The cysteine residues may form crosslinks 
to give each repeat a common and distinctive 
three-dimensional structure. Preliminary data 
from other laboratories suggest that each trimer 
of TNF-a interacts with three receptor molecules. 
Collaborating with Bruce Beutler (HHMI, Uni- 
versity of Texas Southwestern Medical Center at 
Dallas), we have initiated experiments to express 
the extracellular domains of TNF receptors in 
both mammalian and bacterial cells, which we 
will then crystallize in both the presence and ab- 
sence of TNF-a and TNF-/?. We expect that the 
three-dimensional structures will show how the 
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