Structural Studies of Regulatory Proteins 
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 each other by se- 
creting growth factors, cytokines, or hor- 
mones into the extracellular space, and by re- 
sponding to factors produced by other cells. 
These chemical messengers act by changing pat- 
terns of gene expression within target cells, 
thereby altering the cells' metabolic or develop- 
mental program. Among the major unsolved ques- 
tions in biology is how cells specifically recog- 
nize these chemical messengers and how the 
messages are transduced within cells. 
We are using x-ray crystallographic techniques 
to define the molecular nature of the interaction 
between specific growth factors and their cog- 
nate receptors. Receptors are themselves protein 
molecules distributed on the outer surface of the 
plasma membrane surrounding the target cell. 
The goal of our research is to learn how receptor 
molecules specifically recognize growth factors, 
and how, if at all, the receptors alter their struc- 
ture and chemical properties as a consequence of 
their interaction with the factors. 
Tumor Necrosis Factors and Fibroblast 
Growth Factors 
Tumor necrosis factors (TNF) are produced by 
cells of the immune system. Macrophages — the 
white blood cells responsible for engulfing cel- 
lular debris — produce large quantities of TNF-a 
(also known as cachectin) when stimulated by 
toxins carried on the surface of bacteria, as oc- 
curs in an infection. The avid attachment of this 
cytokine to receptors present on a variety of cells 
in the body triggers a series of events that mediate 
inflammation, endotoxic shock, and the wasting 
phenomenon, cachexia, from which the cytokine 
takes one of its names. 
A related cytokine, TNF-|8, is produced by T 
lymphocytes and has many properties in common 
with TNF-a, including the ability to bind to the 
same receptors. Both molecules are toxic to many 
types of tumor cells. 
Two different types of TNF receptor have been 
found which, surprisingly, bear little amino acid 
sequence identity to each other. Both receptors, 
however, contain a structural "motif" composed 
of four inexact copies of a repeat rich in the sul- 
fur-containing amino acid cysteine. Preliminary 
data from our own and other laboratories suggest 
that each trimer of TNF-a interacts with three re- 
ceptor molecules. We hope to learn how these 
distantly related molecules interact with the 
same receptors and how this interaction might 
mediate a cellular response. 
The three-dimensional atomic structures of 
TNF-a, and most recently of TNF-/?, have been 
determined by Michael Eck, a graduate student in 
the laboratory (and now a Research Associate 
with Don Wiley and Stephen Harrison at HHMI, 
Harvard University). Despite the differences in 
amino acid composition and sequence, TNF-a 
and TNF-/3 have the same three-dimensional 
structure and assemble to form trimers composed 
of three identical protein subunits packed about 
a threefold axis of symmetry. However, because 
of the differences in the amino acid composition 
of the two cytokines, the surface of the TNF-a 
trimer is chemically quite different from that of 
the TNF-/3 trimer. We hope to determine how the 
same receptor can recognize these different mo- 
lecular surfaces with equal affinity. 
More importantly from a pharmacological per- 
spective, we would like to be able to design TNF 
molecules that interact with one, but not the 
other receptor, or receptor antagonists that inter- 
act with only one of the two TNF species. Toward 
this goal, we are now attempting to determine the 
structure of theTNF-receptor complexes. The lab- 
oratory has been successful in growing crystals of 
the TNF-binding fragment of one of the two re- 
ceptors, and crystallographic studies are now 
under way. We are also attempting to produce 
crystals of the complex formed between TNF-a 
and one of the receptors in order to study directly 
the interaction between the two molecules. 
Last year we reported the three-dimensional 
structure of basic fibroblast growth factor 
(bFGF), one of a group of seven structurally re- 
lated proteins that promote cell division. This 
factor is present in the space between cells, par- 
ticularly in endothelial tissue, where it stimulates 
the movements of cells to sites of tissue genera- 
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