Biophysical Studies of Eukaryotic Gene Regulation 
and Molecular Recognition 
Stephen K. Burley, D.Phil., M.D. — Assistant Investigator 
Dr. Burley is also Assistant Professor of Molecular Biophysics at the Rockefeller University. He received a 
B.Sc. degree in physics from the University of Western Ontario, a D.Phil, degree in molecular biophysics 
from Oxford University, and an M.D. degree from Harvard Medical School in the Harvard-MIT Joint Pro- 
gram in Health Sciences and Technology. While a medical student, he carried out research in protein 
crystallography with Gregory Petsko. During his clinical training at Brigham and Women's Hospital, he 
also conducted postdoctoral research in protein crystallography with William lipscomb at Harvard Uni- 
i versity, where he solved the three-dimensional structure of leucine aminopeptidase. 
WE are interested in developing a detailed 
understanding of the physical principles 
that govern the general problem of molecular rec- 
ognition in biological systems. The systems we 
have chosen to study are models for gene regula- 
tion in eukaryotes and slow-binding inhibition of 
enzymes. 
Our approach is to use the methods of x-ray 
crystallography to determine the three-dimen- 
sional structures of biological macromolecules 
and their complexes with DNA or other ligands. 
These structures contain a wealth of atomic detail 
that can be analyzed with biochemical, molecu- 
lar genetic, and theoretical methods to provide a 
functional description of the intra- and intermo- 
lecular interactions responsible for stabilizing 
macromolecular complexes. 
In the long term, we hope that our structural 
studies and analyses will allow us to exploit the 
powerful formalism of physics to classify system- 
atically the interactions between individual 
atoms that effect molecular recognition in biolog- 
ical systems. We believe that such a quantitative 
understanding will ultimately permit us to har- 
ness the machinery of molecular recognition and, 
thereby, make defined interventions into impor- 
tant biochemical processes such as disease states. 
Eukaryotic Gene Regulation 
We have begun to examine two model systems of 
eukaryotic gene regulation, with the goal of im- 
proving our understanding of the structural and 
physical bases of transcriptional control of genes. 
First, we are collaborating with Robert Roeder (the 
Rockefeller University) on x-ray crystallographic 
studies of transcription factor IID (TFIID) and up- 
stream stimulatory factor (USF). These two pro- 
teins are involved in transcription of class II genes 
in eukaryotes. TFIID binds to the TATA consensus 
sequence and functions as a general transcription 
initiation factor. USF is a member of the c-myc-rc- 
lated family of DNA-binding proteins and contains 
both a helix-loop-helix motif and a leucine repeat. 
It binds as a dimer to an upstream activating se- 
quence GGCCACGTGACC. During transcription. 
TFIID and USF bind to DNA in close proximity and 
interact with each other to enhance both DNA bind- 
ing and transcription. In addition to determining 
the three-dimensional structures of these proteins 
complexed to their respective promoter elements, 
we hope to determine the structure of a complex 
consisting of TFIID, USF, and DNA. 
Second, we are collaborating with Eseng Lai 
(Memorial Sloan-Kettering Cancer Center) on 
structural studies of human hepatocyte nuclear 
factor 3- This transcriptionally active protein be- 
longs to a gene family in mammals that is homolo- 
gous to the Drosophila homeotic gene fork 
head. These diverse proteins share a highly con- 
served DNA-binding region, which bears no simi- 
larity to previously defined DNA-binding motifs 
and is thought to represent an entirely new type 
of DNA-binding protein. 
Successful three-dimensional structure deter- 
minations of these model systems will provide 
insights into three different modes of DNA-pro- 
tein interaction and may also give some informa- 
tion about intermolecular interactions between 
proteins bound to the same piece of DNA. 
Slow-binding Enzyme Inhibition 
My previous work in William Lipscomb's 
laboratory (Harvard University) employed x-ray 
crystallography to determine the first three-di- 
mensional structure of an amino-terminal exo- 
protease, a two-zinc metalloenzyme known as 
leucine aminopeptidase (LAP) isolated from bo- 
vine lens. We discovered that the enzyme consists 
of two unequal a/fi domains. In its active form it 
exists as a hexamer, which resembles the alloste- 
ric enzyme aspartate transcarbamoylase in appear- 
ance. The enzyme's active site consists of a novel 
bimetallic cluster, with the two zinc ions sepa- 
rated by 2.9 A and coordinated only by carboxyl- 
ate and carbonyl oxygen atoms. The precise 
structure of the active site allowed us to suggest a 
mechanism by which the enzyme may effect pep- 
tide bond hydrolysis. 
In addition, we determined the isomorphous 
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