Structural Studies of DNA-binding Proteins 
Carl O. Pabo, Ph.D. — Investigator 
Dr. Pabo is also Professor of Biophysics and Structural Biology at the Massachusetts Institute of 
Technology. 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. Prior to accepting a position at MIT, Dr. Pabo was Professor of Molecular 
Biology and Genetics and of Biophysics at the Johns Hopkins University School of Medicine. 
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-spe- 
cific contacts, and whether there are any recur- 
ring 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 «rc repressor from 5a/mo«e//a bacte- 
riophage P22 uses a |8-sheet for site-specific rec- 
ognition. The major developments in our labora- 
tory during the past two years, however, have 
involved studies of two of the key motifs — the 
homeodomain and the zinc finger — that are used 
by eukaryotic 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 a2 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 recently solved this 
structure, and comparison with engrailed re- 
vealed that 1 ) the structures of these two homeo- 
domains are very similar (despite a 3 -residue in- 
sertion in a2 and despite significant amino acid 
sequence differences) and 2) the orientation of 
the helix-turn-helix unit with respect to the DNA 
also is conserved. This conserved docking ar- 
rangement is maintained by side chains that are 
identical in a2 and engrailed. Because these resi- 
dues tend to be conserved among all homeodo- 
mains, these structures may provide a general 
model for homeodomain-DNA interactions. 
Our studies of the homeodomain are supported 
by a grant from the National Institutes of Health. 
Crystal Structures of Zinc Finger-DNA 
Complexes 
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 
jS-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, 
Johns Hopkins University), he cloned and ex- 
pressed a three-finger peptide and crystallized 
the peptide-DNA complex. The zinc fingers rec- 
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