Structural Studies of DNA-binding Proteins 
Carl O. Pabo, Ph.D. — Associate Investigator 
Dr. Pabo is also Professor in the Departments of Molecular Biology and Genetics and of Biophysics at the 
Johns Hopkins University School of Medicine. He received his undergraduate degree from Yale University, 
where he majored in molecular biophysics and biochemistry. He did his graduate work in Mark Ptashne's 
laboratory at Harvard University, where he continued his research as a Jane Coffin Childs fellow in the 
laboratories of Stephen Harrison and Don Wiley. In July 1991, Dr. Pabo will move to the Massachusetts 
Institute of Technology as Professor in the Department of Biology. 
WE are interested in understanding how pro- 
teins recognize specific sites on double- 
stranded DNA and how the bound proteins regu- 
late gene expression. We would like to know 
what structural motifs are used by DNA-binding 
proteins, what side chains make sequence- 
specific contacts, and whether there are any re- 
curring patterns or rules for recognition of sites 
on double-stranded DNA. Much of our current re- 
search has focused on characterizing the major 
structural motifs found in DNA-binding proteins. 
We hope to use this information to design novel 
DNA-binding proteins for research, diagnosis, 
and therapy. 
Prokaryotic repressors provide useful model 
systems for the study of protein-DNA interactions, 
and we are continuing to study several bacterial 
repressors. The repressor from the bacteriophage 
X uses a helix-turn-helix motif and an extended 
amino-terminal arm to contact sites in the major 
groove . The arc repressor from Salmonella bacte- 
riophage P22 uses a (8-sheet for site-specific rec- 
ognition. The major developments in our labora- 
tory during the past year, however, have involved 
studies of two of the key motifs — the homeodo- 
main and the zinc finger — that are used by eukar- 
yotic regulatory proteins. 
Crystal Structures of Homeodomain-DNA 
Complexes 
The homeodomain is a conserved structural 
motif found in many eukaryotic proteins that reg- 
ulate development and cell fate. To understand 
how this motif recognizes DNA and how this is 
related to the helix-turn-helix motif seen in pro- 
karyotic repressors, we have determined the 
crystal structures of two homeodomain-DNA 
complexes. 
We began by studying the homeodomain from 
the engrailed protein, which plays a key role in 
Drosophila development. (This project is a col- 
laboration with Thomas Kornberg at the Univer- 
sity of California, San Francisco.) We were able to 
grow good cocrystals of the homeodomain-DNA 
complex, and Chuck Kissinger solved the struc- 
ture of this complex. The homeodomain makes 
contacts in both the major and minor grooves. 
The helix-turn-helix unit makes critical contacts 
in the major groove, but the orientation of this 
helix-turn-helix unit with respect to the DNA is 
different than the arrangements observed with 
the prokaryotic repressors. Residues near the 
amino-terminal end of the homeodomain form an 
extended "arm" that fits into the minor groove 
and makes additional site-specific contacts. 
We also have been studying a complex contain- 
ing the homeodomain from the a 2 protein, 
which helps to regulate mating type in yeast. 
(This project is a collaboration with Alexander 
Johnson at the University of California, San Fran- 
cisco.) Cynthia Wolberger has recently solved 
this structure. The overall arrangement of the 
helix-turn-helix unit and the amino-terminal arm 
are similar to the arrangement seen in the en- 
grailed complex, but there are a number of dif- 
ferences in the critical side chains used for recog- 
nition. There also is a rich background of 
biochemical and genetic data about a 2 that 
should help us understand the precise role of this 
homeodomain in recognition and regulation. 
Structure of a Zinc Finger-DNA Complex 
The zinc finger domain, which contains about 
30 amino acids, is another key DNA-binding motif 
that is found in a large family of eukaryotic regula- 
tory proteins. Studies from other groups have 
shown that each finger contains an antiparallel 
(8-sheet and an a-helix, but little has been known 
about how these fingers recognize DNA. Nikola 
Pavletich recently solved the structure of a com- 
plex containing three zinc fingers from a murine 
transcription factor. Starting with cDNA for the 
zif 268 gene (provided by Daniel Nathans, HHMI, 
at the Johns Hopkins University) , he cloned and 
expressed a three-finger peptide and crystallized 
the peptide-DNA complex. The zinc fingers rec- 
ognize B-DNA and fit into the major groove. Each 
finger makes its primary contacts with a 3-base 
pair "subsite," and side chains near the amino- 
terminal end of the a-helix make the critical con- 
tacts with the bases. Since the fingers are used in a 
modular fashion, they may be the ideal motif to 
use as we try to design novel DNA-binding 
proteins. 
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