Chemical Details of Cellular Regulation 
Paul B. Sigler, M.D., Ph.D. — Investigator 
Dr. Sigler is also Professor of Molecular Biophysics and Biochemistry at Yale University. He studied chem- 
istry at Princeton University and received his M.D. degree from Columbia University. He then spent two 
years as a house officer in the Department of Medicine at Columbia- Presbyterian Medical Center, New 
York. He began his work on crystallography with David Davies at the NIH. He studied as a Helen Hay 
Whitney Fellow at the MRC Laboratory of Molecular Biology in Cambridge, England, where he received 
his Ph.D. degree in biochemistry. Before accepting his present position. Dr. Sigler was Professor of Bio- 
chemistry and Molecular Biology at the University of Chicago. 
OUR goal is to understand how the cell re- 
sponds to regulatory signals. We have fo- 
cused on two processes: the transmission of sig- 
nals across the membrane, and the regulation of 
gene expression. The spirit of the work is reduc- 
tionist, rather than descriptive; we aim to under- 
stand the key steps of these processes in terms of 
basic physical and chemical principles. To do 
this, we must visualize the detailed structure of 
the relevant macromolecules and the complexes 
they form with other molecules. The best way to 
do this is to crystallize these complexes and de- 
termine their structure by x-ray diffraction. 
The stereochemical underpinning for the 
transmission of regulatory signals through cell 
membranes is a broad and varied subject. We 
have begun by focusing on the clinically impor- 
tant question of inflammation. When the signals 
are transmitted, phospholipases are stimulated to 
attack phospholipids, the main substance of 
membranes. The breakdown products are used to 
supply precursors or signals for subsequent reac- 
tions inside the cell. The enzyme phospholipase 
A2 (PLA2) is thought to be responsible for pro- 
ducing arachidonic acid, which is the precursor 
of most of the small compounds that mediate 
inflammation. 
Recently we defined the elusive mechanism by 
which PIA2 hydrolyzes phospholipids and re- 
leases arachidonic acid. We worked out the mech- 
anism by solving the crystal structures of enzyme- 
inhibitor complexes in which the inhibitor was 
designed — by Michael Gelb of the University of 
Washington — to simulate the crucial catalytic in- 
termediate, or "transition," state. Enzymes can 
speed reactions by stabilizing such states, and 
these crystal structures show clearly how PLA2 
does this. We now have three crystallographically 
independent structures of these complexes, and 
all show the same characteristic relationship be- 
tween the enzyme's active surface and the transi- 
tion-state analogue. 
These structures have additional and special in- 
terest to a wide scientific audience. We have de- 
fined, in atomic detail, the mechanism of a cal- 
cium ion's regulatory activity at the cell surface. 
Moreover, we have shown how a soluble protein 
interacts with the face of the cell membrane. Be- 
sides answering general scientific questions, 
these studies afford an immediate practical gain. 
Our structures may provide a basis for the ratio- 
nal design of therapeutic agents that block an in- 
appropriate or exceptionally severe inflamma- 
tory response. To this end, we have recently 
obtained suitable crystals of PLA2 found in the 
inflamed joints of patients with acute rheumatoid 
arthritis and the serum of women with toxic 
shock syndrome. The crystal structure of this en- 
zyme should provide a starting point for the ratio- 
nal design of drugs aimed at these and related 
inflammatory diseases. 
Ultimately most regulatory signals control the 
expression of genes, turning some off and others 
on. Much of this occurs by regulating transcrip- 
tion (the synthesis of messenger RNA) . An essen- 
tial element of transcriptional regulation is to tar- 
get the regulatory proteins to the genes they are 
designed to control. For example, the estrogen 
receptor, which is an activator of transcription, is 
obviously targeted to different genes than its 
counterpart, the testosterone receptor. This is 
usually accomplished by a recognition process 
whereby the regulatory protein binds to a spe- 
cific DNA sequence associated with the genes to 
be controlled. Recognition involves the forma- 
tion of a strong interaction between the protein 
and the correct DNA sequence and weak interac- 
tions with other DNA. We have focused our atten- 
tion on the basic chemistry responsible for stabi- 
lizing specific protein-DNA interactions. 
Our results in this effort are most advanced in 
the case of the trp repressor-operator complex, 
which regulates the expression of genes responsi- 
ble for tryptophan biosynthesis in Escherichia 
colt. The structure was refined to unusually high 
resolution (1.9 A). This refinement, coupled to 
the fact that the same structure is found in four 
independent representations, provides us with a 
model of exceptional clarity and detail. 
The chemistry of the interface was unex- 
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