STRUCTURAL STUDIES OF VIRUSES, RECEPTORS, AND TRANSCRIPTIONAL CONTROL PROTEINS 
Stephen C. Harrison, Ph.D., Investigator 
Structural studies of macromolecular complexes 
are aimed at discovering basic molecular mecha- 
nisms in cell organization. Dr. Harrison's laboratory 
has targeted three broad areas for x-ray crystallo- 
graphic analysis of assembly and recognition: vi- 
ruses and their interactions with cells, pro- 
tein/nucleic-acid complexes, and cell-surface 
receptors. 
I. Viruses. 
The structure of SV40, one of the simplest of the 
double-stranded DNA viruses, has been determined 
at 3.8 A resolution. This is the largest of the icosa- 
hedral viruses to be visualized in such detail, and its 
structure differs in important ways from the com- 
mon design seen in various positive-strand RNA vi- 
ruses. The polyomaviruses, of which SV40 is an ex- 
ample, are composed of 360 copies of an —45 kDa 
protein, VPl; —30-60 copies each of two minor 
proteins, VP2 and VP3; and a closed, circular DNA 
chromosome complexed with histones. The sub- 
units of VPl form pentamers; 72 such pentamers 
pack into the icosahedrally symmetric coat. The ge- 
ometry of this coat requires that identical VPl pen- 
tamers assemble with either five or six neighbors. 
The pentamers are elongated, roughly cylindrical 
objects, packing tightly together at a radius of —200 
o o 
A and projecting outward to a radius of —250 A. 
The VPl subunit fold is based on two opposed 3- 
sheets with radially directed strands. Each sheet has 
four principal strands, connected according to the 
"Swiss roll" pattern found in a number of other 
proteins, including the subunits of the icosahedral 
RNA viruses. The sheets are extended by additional 
strands contributed from neighboring subunits in 
the VPl pentamer. Loops connecting the strands 
extend outward to form the tip of the cylindrical 
pentamer, and one large loop covers its lateral sur- 
face. These loops are the principal loci of variability 
among sequences of related polyomaviruses. In 
particular, one of them has an eight-residue inser- 
tion in the VPl of murine polyomavirus, and muta- 
tional evidence suggests that this loop forms a sialic 
acid-binding site. A 6 A resolution structure of mu- 
rine polyomavirus, computed using the SV40 map 
for initial phase determination, shows that this 
loop must form a shallow pocket at the outer mar- 
gin of the VPl pentamer. Polyoma requires sialic 
acid for initial attachment to cells; SV40 does not. 
Efforts are in progress to obtain well-ordered crys- 
tals of polyoma with a bound sialic acid derivative. 
The SV40 structure also shows how VPl pentamers 
can accommodate either five or six nearest neigh- 
bors. Each subunit contains 4 short a-helices, so 
disposed that they contribute to a palisade of 20 
such helices, radially directed around the base of 
the cylindrical pentamer. Alternative packings of 
these helices enable adjacent pentamers to pack in 
distinct, yet specific, ways. The capacity of a-helices 
to pack with others of somewhat variable se- 
quences may be generally important in assemblies 
where alternative specific contacts must occur. 
The SV40 electron density map reveals part of a 
subunit of VP2 and/or VP3 bound from within 
along the axis of the hollow VPl pentamer. These 
internal proteins thus appear to form links be- 
tween the pentameric assembly units of the outer 
shell and the compact but spatially disordered 
minichromosome . 
II. Protein/Nucleic-Acid Complexes. 
Crystals of bacteriophage repressor/operator 
complexes have offered the first view of how regu- 
latory proteins recognize specific DNA sequences. 
In temperate bacteriophages of Escherichia coli, 
such as 434 and lambda, the different affinities of 
the repressor for each of a set of DNA operator sites 
ensure efficient regulation, and the inversely 
graded affinities of the Cro protein for the same 
sites create a sharply controlled binary switch. The 
binding domain of 434 repressor (Rl-69) and 434 
Cro have been studied as free proteins and in com- 
plex with a series of synthetic operators. The struc- 
ture of Rl-69 in complex with a 20 base pair DNA 
fragment containing the sequence of operator site 
Oj^l shows in detail how this representative helix- 
turn-heltx protein interacts with its target. Direct, 
noncovalent contacts between amino acid side 
chains and the edges of base pairs in the major 
groove can account for the principal specificity. 
Nonpolar interactions (complementary, hydropho- 
bic van der Waals surfaces) are as important as hy- 
drogen bonds. Because the protein imposes a pre- 
cise conformation on DNA when it binds, the 
relative ease with which the operator can adopt the 
required conformation affects its affinity. One way 
in which DNA can adjust to conformational strain is 
through propeller twisting of its base pairs. The 
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