(Received for publication, December 4, 1995; and in revised form, March 4, 1996)
From the
Residues 259-284 of HIV-1 reverse transcriptase exhibit
sequence homology with other nucleic acid polymerases and have been
termed the ``helix clamp''' (Hermann, T., Meier, T.,
Götte, M., and Heumann, H.(1994) Nucleic Acids
Res. 22, 4625-4633), since crystallographic evidence
indicates these residues are part of two -helices (
H and
I) that interact with DNA. Alanine-scanning mutagenesis has
previously demonstrated that several residues in
H make important
interactions with nucleic acid and influence frameshift fidelity. To
define the role of
I (residues 278-286) during catalytic
cycling, we performed systematic site-directed mutagenesis from
position 277 through position 287 by changing each residue, one by one,
to alanine. Each mutant protein was expressed and, except for L283A and
T286A, was soluble. The soluble mutant enzymes were purified and
characterized. In contrast to alanine mutants of
H, alanine
substitution in
I did not have a significant effect on
template
primer (T
P) binding as revealed by a lack of an
effect on K
, K
for 3`-azido-2`,3`-dideoxythymidine
5`-triphosphate, k
, and processivity.
Consistent with these observations, the fidelity of the mutant enzymes
was not influenced. However, alanine mutagenesis of
I lowered the
apparent activity of every mutant relative to wild-type enzyme.
Titration of two mutants exhibiting the lowest activity with T
P
(L282A and R284A) demonstrated that these mutant enzymes could bind
T
P stoichiometrically and tightly. In contrast, active site
concentrations determined from ``burst'' experiments suggest
that the lower activity is due to a smaller population of enzyme bound
productively to T
P. The putative electrostatic interactions
between the basic side chains of the helix clamp and the DNA backbone
are either very weak or kinetically silent. In contrast, interactions
between several residues of
H and the DNA minor groove, 3-5
nucleotides from the 3`-primer terminus, are suggested to be critical
for DNA binding and fidelity.
The type 1 human immunodeficiency (HIV-1) ()reverse
transcriptase (RT) is a DNA polymerase that utilizes both RNA and DNA
templates to accomplish genomic replication. Crystallographic
structures of RT complexed with a non-nucleoside inhibitor (1, 2) or DNA (3) have been solved. Reverse
transcriptase is a heterodimer of 66- and 51-kDa polypeptides. The p51
subunit is a carboxyl-terminal truncation of the p66 subunit and
although the amino-terminal sequence of p66 is identical to p51, the
tertiary organization of the two subunits differ(4) . The
polymerase domain of the p66 subunit forms a nucleic acid binding
cleft, and by analogy with a right hand, its three subdomains are
referred to as fingers, palm, and thumb(4) . Each subunit of
the heterodimer also has a ``connection'' subdomain that
forms the ``floor'' of the nucleic acid binding
site(5) . The carboxyl terminus of the p66 subunit has a fifth
subdomain with RNase H activity that cleaves the RNA strand of hybrid
duplex. As the nucleic acid binding cleft in the p51 polymerase domain
is occluded by the connection subdomain, it lacks a functional
``active site'' in the heterodimer.
Crystallographic data
suggest that two antiparallel -helices in the thumb subdomain
interact 3-5 (
H) and 6-9 (
I) nucleotides from the
polymerase active site with the primer and template strands,
respectively (Fig. 1). Within this region, the DNA is observed
to be bent 40-45° and undergoes a transition from A- to
B-form(3) . The primary sequence in the vicinity of these
-helices has been found to have sequence homology with several
other nucleic acid polymerases and has been termed the ``helix
clamp''(6) . Alanine-scanning mutagenesis has demonstrated
that
H plays an important role in template
primer (T
P)
binding and fidelity(7, 8) . The periodicity of the
effects observed suggest that a short segment of one face of
H (i.e. Gln
, Gly
, Trp
)
interacts with the T
P. Kinetic parameters for T
P observed
with the alanine mutants of Gly
and Trp
indicate that interactions in the minor groove of the duplex,
several nucleotides from the primer-terminus, influence processivity
and fidelity through T
P slippage(8) . These results
support earlier observations that processive synthesis by RT is
dependent on the DNA sequence of the first 6 base pairs of the duplex
primer stem and that single base pair differences in this region can
affect both processivity (9) and frameshift
fidelity(10) .
Figure 1:
Interaction between
templateprimer and
H
I. The position of the
phosphates of the DNA and the C
s of
H and
I are from the
coordinates obtained from the Protein Data Bank (PDB file 1HMI, see (3) ). The DNA phosphates and C
s of the basic residues are highlighted as spheres, and the 3`-end of the primer
strand is indicated. The bonds closest to the viewer are
thickest.
The DNA in the crystal structure of the
RTDNA
Fab complex was too short to determine whether nucleic
acid interactions occur in other subdomains of p51(3) .
However, modeling longer stretches of nucleic acid into the binding
cleft has suggested that residues of both
H and
I of the p51
thumb subdomain may also interact with nucleic acid (5, 11) . Recent modeling of a RNA
template
primer into the nucleic acid binding cleft has suggested
that interactions may occur between the RNA primer strand and
I(6) . The primary sequence in the stretch of residues
comprising
H and
I is highly basic (Lys
,
Lys
, Lys
, Arg
,
Lys
, Arg
, Lys
), and most of
these residues are observed to be facing the DNA phosphate backbone (Fig. 1). To define the role of
I during catalytic cycling,
we performed systematic site-directed mutagenesis from position 277
through 287 by changing each residue, one by one, to alanine. Each
mutant enzyme was expressed and purified to near-homogeneity, and their
specific activities, substrate kinetic parameters, and fidelity
surveyed.
Enzyme concentrations were determined from protein determinations (16) which had been calibrated by amino acid analysis.
The dissociation rate constant (k)
for poly(rA)
oligo(dT)
was determined as
described(15) . Briefly, enzyme was preincubated with T
P
for 5 min before challenging free polymerase with heparin (zero time).
At time intervals after adding challenge, 20-µl aliquots were
removed and mixed with 20 µl of dTTP/Mg
to
determine the concentration of RT remaining bound to T
P. After an
additional 10-min incubation, the reaction was stopped with EDTA. The
final reaction conditions were 150 nM RT, 150 nM T
P (expressed as primer 3` termini), 50 mM Tris-HCl, pH 7.4, 10 mM MgCl
, 30 µM [
-
P]dTTP, and 1 mg/ml heparin.
Titration of enzyme with TP (i.e. poly(rA)
oligo(dT)
) was performed as
before(15) . To determine the concentration of RT bound to
T
P, a 40-µl solution of 32 nM enzyme, expressed as
concentration of dimer, was preincubated with varying concentrations of
poly(rA)
oligo(dT)
. After 10 min, the reaction was
initiated with 10 µl of 5 mg/ml heparin and 150 µM [
-
P]dTTP. After an additional 10 min,
the reaction was stopped by the addition of 20 µl of 0.5 M EDTA and the amount of incorporation was determined as above.
Under these conditions, total incorporation is proportional to the
concentration of RT-T
P complex.
To determine the active site
concentration of enzyme, synthesis was limited to a single
deoxynucleotide by using a 47/24-mer heteropolymeric
templateprimer and including only the complementary dTTP to the
first template base. The sequence of the primer and template were
5`-GCTTGCATGCCTGCAGGTGTACGT-3` and
3`-CGAACGTACGGACGTCCACATGCAATTGCCTAGGGGCCCATGGCTCG-5`, respectively.
When product release is the rate-limiting step during catalytic
cycling, then a burst of product formation is observed upon initiation
of the reaction (, k
k
).
The amount of product formed during the burst should be
equivalent to the concentration of active enzyme and has been observed
previously(17, 18, 19, 20) .
Reactions were initiated by the addition of enzyme and 30-µl
aliquots withdrawn at time intervals, stopped with EDTA, and
incorporation determined by filter binding as described above. The
final reaction conditions were 560 nM dimeric RT, 2 µM TP (expressed as primer 3` termini), 50 mM Tris-HCl, pH 7.4, 150 mM KCl, 5 mM MgCl
, 10 µM [
-
P]dTTP.
Figure 2: SDS-polyacrylamide gel electrophoresis analysis of HIV-1 mutant polypeptides. Photograph of Coomassie Blue-stained gel is shown. The location of the purified recombinant p66 polypeptides for wild-type (wt) and alanine mutant enzymes, and the top of the gel, are indicated on the right. Molecular weight markers (M) and their masses were: phosphorylase b, 97.4 kDa; bovine serum albumin, 69 kDa; ovalbumin, 46 kDa; carbonic anhydrase, 30 kDa; soybean trypsin inhibitor, 21.5 kDa; lysozyme, 14.3 kDa. The p66 subunit represents at least 90% of the total protein as determined from the integrated densitometry signals of each band measured using Millipore BioImage Visage Software. Some preparations of mutant enzyme also contained a small amount of p51 (<10%), which was assumed to form heterodimer.
Figure 3:
Steady-state Michaelis constant for dNTP
binding and AZTTP inhibition for alanine mutants of I. Assays were
performed as described under ``Experimental Procedures.'' The
poly(rA)
oligo(dT)
concentration was 1 µM 3`-primer-termini. The equilibrium constant for dNTP binding was
estimated from K
determinations (left panel). The dTTP concentration was
typically varied from 0.3
K
to 5
K
, and velocities were fitted to
the Michaelis equation by nonlinear least squares methods. The apparent
binding affinity for AZTTP (right panel) was determined as
described previously(7) . The dTTP concentration was present at
a concentration equivalent to the K
,
for each mutant RT and
the AZTTP concentration varied. Dixon plots were examined to assure
simple competitive behavior. The wild-type RT kinetic parameters are
indicated with a dashed line. NS indicates that the
alanine mutant was insoluble.
Inhibition of dTTP incorporation with
poly(rA)oligo(dT)
by AZTTP can be a sensitive assay
to probe polymerase and dNTP, as well as template
primer
interactions (7, 20) . Since AZTTP is a substrate, the K
determined when measuring the inhibition of dTTP
incorporation is equivalent to the K
for AZTTP
incorporation (), where K
= K
(k
/k
) (17) . The K
for AZTTP inhibition is,
therefore, sensitive to K
for AZTTP,
template
primer dissociation rate constant (k
), and rate constant for nucleotide
incorporation (k
).
In contrast to several
alanine mutants of H (cf. Fig. 5of (7) ),
alanine mutation of
I did not change the sensitivity of the
mutants to AZTTP (Fig. 3, right panel). Because K
is influenced by k
and k
, as
discussed above, these results suggest that T
P binding (i.e.
k
), as well as catalytic activity (i.e.
k
) should not be altered (see below). Although there
are several potential electrostatic interactions between
I, of
both the p51 and p66 subunits, and the sugar-phosphate backbone
suggested by modeling studies(5, 6, 11) ,
individual removal of these basic side chains appears to have little
influence on apparent DNA binding affinity as revealed from the
sensitivity of the mutant enzymes to AZTTP.
Figure 5:
Apparent steady-state turnover number (k) for alanine mutants of
I. Assays
were performed as described under ``Experimental
Procedures.'' The apparent turnover number was determined by
fitting velocities as described in the legend to Fig. 3A, where k
= V
/[Enzyme]. The apparent turnover
number for wild-type RT is indicated with a dashed line. NS indicates that the alanine mutant was
insoluble.
Figure 4:
Steady-state Michaelis constant for
templateprimer binding and dissociation of
RT-template
primer complexes for alanine mutants of
I. Assays
were performed as described under ``Experimental
Procedures.'' For the K
(left
panel), the dTTP concentration was 30 µM, and the
poly(rA)
oligo(dT)
concentration was varied from 0.3
K
to 5
K
. Velocities were fitted to the
Michaelis equation by nonlinear least squares methods. The dissociation
rate constant for T
P (right panel) was determined as
described(15) . Alanine mutant RT was preincubated with
poly(rA)
oligo(dT)
and at t = 0,
heparin was added. At time intervals, Mg
/dTTP was
added to determine the amount of RT-T
P complex remaining. Data
were fitted to a single exponential model (I
/I
= Ae
),
where I
/I
=
pmol of dTMP incorporated at time t relative to time 0, A = amplitude, and k = apparent first-order
rate constant. The wild-type RT kinetic parameters are indicated with a dashed line. NS indicates that the alanine mutant was
insoluble.
Figure 6:
Termination analysis for the alanine
mutants of the basic residues of I. Processivity was measured on
M13mp2 DNA primed with a
P-5`-end-labeled 15-mer primer
complementary to lacZ positions 105-120(8) . DNA
is in excess severalfold over enzyme to limit synthesis on a primer to
a single cycle. Products were analyzed by electrophoresis on 16%
denaturing polyacrylamide gels, quantified by phosphorimagery, and
termination probability at each site expressed in percent, as the ratio
of products at a site to the products at that site plus all greater
length products. Termination results from a kinetic competition between
further extension, k
, and dissociation of the
enzyme from DNA, k
(see ).
To directly
assess whether I interacts with nucleic acid, the dissociation
rate constant for the RT-T
P complex (k
or k
in ) was measured. As
demonstrated with
H(7) , the dissociation rate constant
for the RT-T
P complex is a sensitive index of the nature of the
interactions between RT and T
P. The dissociation of the
RT-T
P complex can be monitored by challenging the complex with
heparin, to trap RT dissociating from T
P, and then assaying for
the concentration of complex remaining after increasing periods of
challenge(15) . The dissociation rate constant for T
P
with several alanine mutants of
I was increased modestly for
several alanine mutants, but the increase was less than 4-fold when
compared with wild-type enzyme (Fig. 4, right panel).
The increase in k
was generally
observed when a basic side chain was replaced with alanine. T
P
bound much weaker to G262A and W266A of
H relative to wild-type
enzyme, as monitored by the dissociation rate constant for T
P (cf. Fig. 7of (7) ).
Figure 7:
Templateprimer and active site
titration of alanine mutants of
I. Assays were performed as
described under ``Experimental Procedures.'' Top
panel, heparin challenge assay was used to determine the
concentration of RT-T
P complex(15) . The concentration of
dimeric R284A in the reaction mixture was 32 nM. Assuming an
infinitely small equilibrium constant, the data were fitted to the
quadratic equation by nonlinear least squares methods. The fit (solid line) indicated that the concentration of T
P
binding sites was 27 ± 3 nM. Bottom panel,
biphasic time courses for wild-type (
) and R284A (
) on a
heteropolymeric DNA template
primer (47
24-mer). The enzyme
concentration was 560 nM, and only the complementary
nucleotide (i.e. dTTP) for correct incorporation of the next
nucleotide was included in the reaction mixture. The rapid burst of
product formation represents the active fraction of enzyme, whereas the
slow linear portion represents the dissociation of enzyme from the
extended DNA (i.e. v/[E]
= k
). The burst amplitude
(P/E) was 0.58 ± 0.04 and 0.05 ± 0.01 and the
dissociation rate constant (k
) was 0.008
± 0.001 and 0.024 ± 0.005 s
for
wild-type and R284A p66, respectively.
Since processivity is determined by kinetic
competition between further extension (k) and
dissociation of the enzyme from nucleic acid (k
),
termination probability is a measure of the ratio of these rate
constants. The processivity of the alanine mutants of
I was
measured on M13mp2 DNA primed with a
P-5`-end-labeled
15-mer primer(8, 9) . Primer extension reactions were
performed using conditions that minimize reinitiation on previously
extended primers(8, 9, 10, 23) .
Quantitative analysis of the probability of termination with the lacZ template indicated that the processivity of the alanine
mutants of the basic residues of
I was similar to wild-type enzyme (Fig. 6). Additionally, for all the other mutants of
I
altered by alanine substitution, there was no change in processivity as
compared with wild type (data not shown). This is in contrast to G262A
and W266A of
H, which had elevated K
, K
, k
(7) , and reduced processivity
on the lacZ template(8) . The processivity
measurements support the minimal effect observed on the dissociation
rate constant for the RT-T
P complex observed for the
I
mutants, but also suggests a lack of an effect on k
as indicated by the sensitivity of these mutants toward AZTTP (Fig. 3, right panel).
Since I in the p51
subunit is near the subunit interface, the alanine mutation may be
perturbing dimerization of the respective subunits leading to an
apparent loss of activity. The dimeric concentration of enzyme would be
overestimated from the total protein concentration, resulting in an
underestimation of the catalytic rate (i.e. k
< k
). Since dimeric enzyme binds
poly(rA)
oligo(dT)
tightly and
stoichiometrically(15) , we titrated R284A (6% of wild-type
activity) with T
P (Fig. 7, top panel). The
titration indicates that all the protein in the reaction mixture can
bind nucleic acid tightly (K
< 1 nM)
and that this mutant enzyme is dimeric. Therefore, it is unlikely that
the alanine substitution altered the concentration of dimeric RT.
T
P titration of L282A (<1% of wild-type activity) gave similar
results (data not shown).
An alternative explanation for the
apparent lower activity is that alanine substitution results in a lower
fraction of active enzyme. When product release is the rate-limiting
step during catalytic cycling, then a burst of product formation is
observed upon initiation of the reaction (, k
k
). The amount of
product formed during the burst should be equivalent to the
concentration of active enzyme and has been observed
previously(17, 18, 19, 20) . R284A
has only 6% the activity of wild-type enzyme (Fig. 5). The
active fraction of enzyme as determined from the burst of product
formation on heteropolymeric DNA indicates that 9% of the mutant enzyme
is active, relative to wild-type, and that k
is 3-fold higher than with wild-type enzyme (Fig. 7, bottom panel), as observed with a RNA template (Fig. 4, right panel). Increasing the enzyme concentration gave a
proportional increase in the burst amplitude (data not shown). It
should be noted that the wild-type enzyme exhibited a burst amplitude
60% of that expected from protein determination and T
P titration.
This is similar to the burst amplitude measured by Kati et al.(18) using rapid mixing and quenching techniques.
Figure 8:
Forward mutation frequency of the alanine
mutants of I. The mutational target for polymerase errors was the
258-nucleotide lacZ
-complementation sequence present as
a single-stranded template within a 390-nucleotide gap in an otherwise
double-stranded M13mp2 DNA. The background mutant frequency (M.F.) for uncopied DNA in the forward mutation assay was 6
10
. The forward mutation frequency of the
wild-type enzyme is indicated with a dashed line. NS indicates
that the alanine mutant was insoluble.
The overall
average forward mutant frequency for the C280A derivative was elevated
approximately 1.5-fold when compared with the wild-type RT. Because
error rates are highly sequence context-dependent, we examined the
error specificity of the lacZ complementation mutants
generated in the forward assay. Fifty independent lacZ
complementation mutants were isolated and sequenced to determine
the distribution and types of errors produced by this derivative. These
data were compared with a collection of 189 independent wild-type lacZ
complementation mutants. No significant differences
in quantitative error rates were found for both frameshift and base
substitution errors, indicating the C280A derivative has a similar
error specificity as the wild-type RT (data not shown).
From examination
of the 3.0-Å crystallographic structure(3) , HIV-1 RT
appears to make numerous protein-nucleic interactions in the vicinity
of the polymerase active site. Interactions near the RNase H active
site are deduced from enzymatic footprinting (11) and molecular
modeling(5, 6, 11) , since the duplex of the
bound DNA (1918-mer) is too short to deduce interactions with the
distant RNase H domain. The distance between the polymerase and RNase H
active sites within the DNA binding channel is approximately
15-19 nucleotides as determined by chemical footprinting (25) or kinetic coupling between polymerase and RNase H
activities(18) .
Interactions near the polymerase active
site of the p66 subunit are contributed by the fingers, palm, and thumb
subdomains. The 3`-OH at the primer terminus may be positioned by the
12-
13 hairpin (residues 227-235) and has been termed
the ``primer grip.'' A ``template grip'' has also
been described which interacts with the DNA sugar-phosphate backbone of
the first four nucleotides of the template strand. These include
4
and
B (residues 73-83) of the fingers subdomain and
5a
(residues 86-90),
5a-
5b connecting loop (residues
91-93), and
8-
E (residues 148-154) connecting
loop of the palm subdomain(3, 5) .
Although the DNA
in the crystallographic structure has only a single nucleotide
overhang, important single-stranded template interactions have been
suggested by site-directed mutagenesis analysis of the fingers
subdomain(26) . RT residues that have been implicated in HIV-1
viral sensitivity toward nucleoside inhibitors have been suggested to
interact with the single-stranded template(4) . Since
nucleoside inhibitor sensitivity is dependent on TP binding
affinity, the lack of wild-type RT sensitivity to these inhibitors with
short template overhangs (1-3 nucleotides) is consistent with a
lower DNA binding affinity for DNA substrates with short
single-stranded template overhangs(27) . The low sensitivity of
the mutant RTs (i.e. L74V and E89G) with longer
single-stranded templates is consistent with a lower affinity of these
polymerases for T
P(26) .
In the thumb subdomain of RT,
H and
I appear to interact with the sugar-phosphate backbone.
Alanine-scanning mutagenesis of
H has revealed that several
residues on one side of
H interacts with DNA and substitution of
these side chains with alanine drastically lowers the affinity of the
polymerase for nucleic acid resulting in a lower in vitro sensitivity to AZTTP and processivity(7, 8) .
Additionally, two alanine mutants of
H, G262A and W266A, exhibit a
lower fidelity due to an increase in template
primer
slippage-initiated errors, indicating that protein-duplex nucleic acid
interactions 3-5 base pairs from the active site can influence
polymerase fidelity(8) . These results suggest that contacts
within the minor groove are critical for processive polymerization of
high fidelity.
In contrast to the critical role H plays in
binding DNA, alanine-scanning mutagenesis failed to reveal a
significant role for
I in binding nucleic acid as revealed by a
lack of an effect on K
,
(Fig. 4, left panel), K
(Fig. 3, right
panel), k
(Fig. 4, right panel), and processivity (Fig. 6). Consistent
with these observations, the fidelity of the mutant enzymes was not
influenced (Fig. 8). Although the side of
I that faces the
DNA has several basic residues (Fig. 1), only modest effects,
3-fold, were observed on the dissociation rate constant for T
P (i.e. k
) when several of these residues were
replaced with alanine (Fig. 4, right panel, e.g. Arg
and Lys
; Fig. 7, bottom
panel). This is similar to what has been observed when Lys
and Lys
of
H was replaced with
alanine(7) . These results suggest that these putative
electrostatic interactions may be weak. It has also been suggested that
similar interactions occur between
H and
I in the p51 subunit
and modeled nucleic acid(5, 6, 11) . Weak
electrostatic interactions may indicate that unfavorable changes in
solvation of the charged groups occur upon binding resulting in a lower
overall binding free energy(28) . Alternatively, it has been
suggested that nucleic acid binding to RT is at least a two-step
reaction and that the slow rate-limiting step for dissociation of the
T
P from the complex is the reversal of an isomerization, k
in (15, 20, 29) . This would mean
that any effect on intrinsic binding (i.e. k
or k
) may be kinetically silent since
tight nucleic acid binding is dominated by the slow reversal of this
isomerization.
RT subunit dimerization has been
suggested to occur in at least two steps(30) . The first step
forms the TP binding site but enzyme is inactive. This is
followed by slow structural rearrangements leading to active enzyme. To
determine if the alanine mutants may represent a dimerization
intermediate form, the active fraction of enzyme was determined from
the magnitude of the burst of product formation occurring in the first
turnover. When product release is slower than chemistry (k
k
, ),
the amplitude of the burst represents the active fraction of
enzyme(17, 18, 19, 20) . Time
courses for the incorporation of a single nucleotide demonstrated that
the burst amplitude for R284A was lower than wild-type enzyme to the
same extent as observed for k
( Fig. 5and Fig. 7, bottom panel). When one
calculates the turnover number for the linear steady-state portion of
the time courses using the active enzyme concentrations determined from
the burst, the mutant enzyme has a 3-fold higher activity than
wild-type enzyme (k
= 0.008
s
). In this case, k
is
limited by the dissociation rate constant for nucleic acid (k
in ) which was observed to be
elevated 3-fold for a RNA template (Fig. 4, right
panel). Alanine mutation of
I, therefore, appears to
influence the concentration of inactive dimeric enzyme. Even in the
case of wild-type enzyme, a population of inactive enzyme which could
bind template
primer tightly was observed.
Since the alanine
substitution occurs in both subunits of the homodimer. The thumb
subdomain of the p66 subunit analogous to p51 (p51`) forms part of the
subunit interface near the RNase H domain of p66. It is tempting to
suggest that the alanine substitution in the p51` subunit was
responsible for the altered population of inactive dimeric enzyme,
since these positions are near the subunit interface in the
``mature'' heterodimer. However, mutagenesis of a leucine
repeat motif, residues 282-310(31) , indicated that these
mutants also express very low activity(13) . In this case,
subunit-specific mutagenesis indicated that mutagenesis of Leu in p66, but not Leu
of p51, abolished dimerization.
The p66 thumb appears to be conformationally mobile. In one form of the
apoenzyme it is in a closed conformation(32) , and in the
complex with DNA (3) or nevirapine(1) , it is open. At
the tip of the thumb in the closed form, Leu
makes a
hydrophobic contact with the fingers subdomain. Therefore, movements in
the thumb subdomain of p66 may also play an important role in
dimerization or enzyme ``activation.'' It has been suggested
that the monomeric subunits (p66 and p51) are in a compact conformation
where the thumb is open and the connection subdomain is folded into the
polymerase active site to remove hydrophobic surface area(33) .
Alterations in the p66 thumb subdomain which favor this compact
structure requiring the thumb to be in an open structure may influence
dimerization.