HIV-1 reverse transcriptase complexed
with FAB-28 monoclonal antibody fragments
Oliver Benes '03, Rebecca Burke '03,
Rebecca Palacios '03
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
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)
. The other chain is a 51kD subunit (p51),
which is related to p66. However, the C-terminal RNase domain present
in p66 is absent in p51
. These two domains are responsible for the binding of the DNA:RNA
(Sarafianos et al, 2001; Kohlstaedt
et al, 1992).
III. p66 and p51 subunits
The p66 subunit
is the larger of the two subunits within HIV-1 reverse transcriptase
. This subunit contains the finger,
subdomains as well as the RNase H
. The p51
subunit is the smaller of the two subunits
in HIV-1 reverse transcriptase
. This subunit also contains the finger,
(Kohlstaedt et al, 1992). The p51
subunit is a product of the same gene as the p66 subunit, however, the RNase
H domain is absent in the p51 subunit as a result of proteolytic cleavage.
to see a detailed schematic of the two subunits interacting with DNA (Flint,
IV. DNA/RNA binding to Reverse Transcriptase
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
. In addition, residues glutamate 89
91 of the template grip in the p66 palm are involved
in the RNA-RT interactions
. The p51 subunit also plays a role in the interactions between the
RNA:DNA duplex and reverse transcriptase. In particular residues lysine
395, glutamate 396,
lysine 22, and lysine
390 of the p51 subunit interact with the DNA:RNA
There are four regions in the nucleic acid that have characteristic
geometrical conformations. Structural analysis reveals that none of
the four regions displays canonical A- or B-type geometry. Region
I assumes a conformation more closely related
to that of A-form geometry.
. Regions II,
and IV exhibit a
conformation that is intermediate between A-and B-form
. This conformation is known as the H-form, and is characterized by
angles of the base pairs with respect to the helical axis, the dislocation
of the base pairs from the helix axis, and the helical rise (Sarafianos
et al, 2001).
V. The Polypurine Tract
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
. The left end of the upstream long terminal repeat (LTR) of the polypurine
tract and the downstream LTR form the substrate on which the viral integrase
enzyme acts. The viral integrase enzyme inserts its linear viral DNA
into the host genome at this site. A key feature of the PPT
are the A-tracts
, which are stretches of four or more consecutive adenines.
Characteristics of the A-tracts include their extreme resistance to reconstitution
around nucleosome cores, their straight chain conformation, and their narrow
minor groove and large propeller twist. During DNA binding, the integration
host factor protein recognizes the A-tracts. In particular, it recognizes
the phosphates of the narrow minor groove. In this way, the integration
host factor recognizes the adenine regions based on conformation rather
than on base-specific contacts (Sarafianos
et al, 2001).
VI. Unzipping of the PPT
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
. 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
The HIV-1 Reverse Transcriptase is complexed
with two monoclonal antibody fragments, the FAB-28
light chain and the FAB-28
. 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).
PDB from Molecules
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