HIV-1 reverse transcriptase complexed with FAB-28
monoclonal antibody fragments
Oliver Benes '03, Rebecca Burke '03,
Rebecca Palacios '03
Contents:
I. Introduction
The retrovirus HIV, and its
subsequent progression to AIDS, is a rapidly growing worldwide epidemic.
HIV-1 reverse transcriptase is one of the key players in the mechanism
of infection by this retrovirus. The HIV-1 reverse transcriptase
enzyme is responsible for copying a single-stranded viral RNA genome into
double-stranded DNA (Sarafianos et al,
2001). The newly created DNA can then be incorporated into the
host genome; the host is mainly the human in the case of HIV. The
HIV-1 reverse transcriptase enzyme contains two main domains: a
DNA polymerase
domain and a
ribonuclease H (RNase H) domain. The
DNA polymerase is able to copy either an RNA or DNA template. The
function of the RNase H domain is to cleave and degrade the template RNA
after DNA synthesis so that the newly made DNA can generate a second DNA
strand. The RNase H domain is also responsible for the integration
of the duplex DNA into the host cell chromosome.
Here we describe the crystal
structure of HIV-1 reverse transcriptase complexed with two FAB-28 monoclonal
antibody fragments and an DNA:RNA
hybrid. The FAB-28 heavy chain
and FAB-28 light chain
are not actual components of the reverse transcriptase enzyme. The
antibody fragments are complexed with the enzyme during the crystallization
procedure in order to stabilize the enzyme structure. This allows
for a higher resolution crystallographic structure (Sarafianos
et
al, 2001).
II. General Structure
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HIV-1 reverse transcriptase is a dimer composed
of two distinct, but related chains. The first of these two chains
is a 66-kD subunit (p66)
<
p51),
which is related to p66. However, the C-terminal RNase domain present
in p66 is absent in p51 <
DNA:RNA
substrate <
III. p66 and p51 subunits
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The p66 subunit
is the larger of the two subunits within HIV-1 reverse transcriptase <
finger,
palm,
thumb,
and connection
subdomains
as well as the RNase H
subdomain<
p51 subunit
is the smaller of the two subunits in HIV-1 reverse transcriptase
<
finger,
palm,
thumb
and connection
subdomains <
IV. DNA/RNA binding to Reverse Transcriptase
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There are multiple interactions between the
2'-OH groups of the RNA template and the reverse transcriptase enzyme.
Residues serine 280
and arginine 284
of helix I in the p66 thumb are involved in the RNA-RT
interactions <
glutamate 89
and glutamine 91
of the template grip in the p66 palm are involved in the RNA-RT
interactions <
lysine
395, glutamate
396, lysine
22, and lysine
390 of the p51 subunit interact with the DNA:RNA
duplex <
Region
I assumes a conformation more closely related
to that of A-form geometry. <
II, III,
and IV exhibit
a conformation that is intermediate between A-and B-form <
V. The Polypurine Tract
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The RNA polypurine tract (PPT) acts
as the primer for (+) strand synthesis by resisting RNase H cleavage.
It is located at the 5' end of U3 in the RNA
strand <
VI. Unzipping of the PPT
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In one section of the PPT, a departure
from Watson-Crick base pairing is observed. This particular region
is referred to as the "unzipping of the PPT." Features of this region
include the melting of the first two base pairs of the 5'- end of the PPT,
which results in an unpaired template base, Adenine
15 <
>.
Following this unpaired nucleotide is a frame-shifted A-T base pair, Tempate
A-16 and Primer T-15, and a GT mismatch, Template
G-17 and Primer T-16 <
>.
The nucleotide primer following this is the unpaired
C-17, compensating for the unpaired nucleotide on the template strand<
>.
This marks the return to Watson-Crick base pairing (Sarafianos
et
al, 2001).
VII. FAB-28 Monoclonal Antibody Fragments
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The HIV-1 Reverse Transcriptase is complexed
with two monoclonal antibody fragments, the FAB-28
light chain and the FAB-28
heavy chain <
>.
The light chain contains 214 amino acids whereas the heavy chain is composed
of 220 amino acids. The heavy
chain interacts with the p55
subunit of HIV-1 reverse transcriptase
<
>
but has no interations with the p66 subunit or the RNA-DNA hybrid.
These fragments do not play a role in the function of the HIV-1 reverse
transcriptase and are merely present to stabilize the structure for crystallization.
A stablized structure allows for better resolution in the crystallization
procedure. It has been suggested that the fragments serve as a molecular
clamp to constrain the conformational freedom of the enzyme (Sarafianos
et
al, 2001; Ding et al, 1998; Jacobo-Molina
et
al, 1991).
VIII. References
PDB from Molecules
R Us file 1hys
Ding, Jianping, Kalyan Das,
Yu Hsiou, Stefan G. Sarafianos, Arthur D. Clark, Jr., Alfredo Jacobo-Molina,
Chris Tantillo, Stephen H. Hughes, and Edward Arnold. 1998. Strucutre and
functional implications of the polymerase active site region in a complex
of HIV-1 RT with a double-stranded DNA template-primer and an antibody
fab fragment at 2.8 A resolution. Implications of the Polymerase Active
Site Region. 1095-1111.
Flint, S.J. Principles of
Virology. 2000. ASM Press.
Jacobo-Molina, Alfredo, Arthur
D. Clark Jr., Roger L. Williams, Raymond G. Nann, Partick Clark, Andrea
L. Ferris, Stephen H. Hughes, and Edward Arnold. 1991. Crystals of a ternary
complex of human immunodeficiency virus type 1 reverse transcriptase with
a monoclonal antibody Fab fragment and double-stranded DNA diffact x-rays
to 3.5-A resolution. Proc. Natl. Acad. Sci. 88:10895-10899.
Kohlstaedt, L.A., J. Wang, J.M. Friedman, P.A. Rice,
and T.A. Steitz. 1992. Crystal strucutre at 3.5 A resolution of HIV-1 reverse
transcriptase complexed with an inhibitor. Science 256: 1783-1790.
Safarianos, Stefan G., Kaylan Das, Chris Tantillo,
Arthur D. Clark, Jr., Jianping Ding, Jeanette M. Whitcomb, Paul L. Boyer,
Stephen H. Hughes, and Edward Arnold. 2001. Crystal strucutre of HIV-1
reverse transcriptase in complex with a polypurine tract RNA:DNA. The
EMBO Journal 20:1449-1461.
Biomolecules
Index