Biochem. J. (2013) 455, 179–184 (Printed in Great Britain)
179
doi:10.1042/BJ20130850
Transient kinetic analyses of the ribonuclease H cleavage activity of HIV-1
reverse transcriptase in complex with efavirenz and/or a β-thujaplicinol
analogue
Brian D. HERMAN* and Nicolas SLUIS-CREMER*1
*Division of Infectious Diseases, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, U.S.A.
EFV (efavirenz) and β-thujaplicinol [2,7-dihydroxy-4-1(methylethyl)-2,4,6-cycloheptatrien-1-one] have contrasting effects on
the RNase H activity of HIV-1 RT (reverse transcriptase).
EFV binds in the non-nucleoside inhibitor-binding pocket and
accelerates this activity, whereas β-thujaplicinol binds in the
RNase H active site and inhibits it. We have used pre-steadystate kinetic analyses to gain an insight into the mechanism
by which EFV and a β-thujaplicinol analogue [19616 (2,7dihydroxy-2,4,6-cyclo-heptatrien-1-one)] modulate RT RNase
H activity. Our data show that EFV and 19616 have no
effect on polymerase-dependent RNase H cleavages. However,
both compounds significantly affected the rates of polymeraseindependent RNase H cleavages. In regard to the latter, we found
no evidence that the bound RNA/DNA template/primer substrate
restricted 19616 from interacting with RT. In light of these data,
we propose a model in which 19616 binds to the RNase H active
site of RT after the primary polymerase-dependent RNase H
cleavage has occurred and stabilizes the 3 -end of the DNA primer
in the polymerase active site thus blocking the enzyme’s ability
to carry out the polymerase-independent cleavages. By contrast,
EFV destabilizes the 3 -end of the DNA primer in the DNA
polymerase active site and promotes RT-mediated polymeraseindependent cleavages. Consistent with this model, we show
antagonism between EFV and 19616.
INTRODUCTION
after the RT-T/P (template/primer) substrate complex is formed,
suggesting that the RNA/DNA substrate may block its access to
the RNase H active site. In contrast, Himmel et al. [3] provided
biochemical evidence that the presence of a bound RNA/DNA
T/P substrate is necessary to achieve its maximal binding to HIV-1
RT. Interestingly, several studies have demonstrated that NNRTIs
such as EFV (efavirenz) can accelerate the enzyme’s RNase H
activity [5–7]. This is surprising given that the NNRTI-binding
pocket is located >50 Å (1 Å = 0.1 nm) away from the RNase
H active site of HIV-1 RT. FRET-based single-molecule studies
suggested that NNRTIs may affect the orientation of RT on the
polypurine tract RNA/DNA hybrid and increase RNase H activity
[8]. Recently, Lapkouski et al. [9] solved the structure of HIV-1
RT containing an RNA/DNA hybrid and EFV. The enzyme and
RNA/DNA structures in this complex differ from all nucleic
acid–RT complexes previously reported. Specifically, the DNA
polymerase active site is disengaged from catalysis, whereas
the RNA/DNA duplex has ready access to the RNase H active
site. Taken together, the single molecule and crystallography
studies suggest that NNRTIs lock the RT enzyme in an RNase H
competent mode.
Cleavage of RNA/DNA hybrids by RNase H can be
accomplished through two different modes of HIV-1 RT binding.
The first mode of cleavage is directed by the 3 -end of the
elongating plus-strand DNA and is thought to occur in concert
with DNA polymerization (i.e. polymerase-dependent RNase H
cleavage). A second mode of RNase H cleavage, directed by
the 5 -end of the RNA in the RNA/DNA hybrid, presumably
allows further degradation of small RNA fragments that remain
annealed to the newly synthesized plus DNA strand after DNA
3 -end-directed RNase H cleavage (i.e. polymerase-independent
RT (reverse transcriptase) facilitates the conversion of the HIV-1
single-stranded RNA genome into double-stranded DNA. HIV1 RT is a multifunctional enzyme that exhibits an RNA- and
DNA-dependent DNA polymerase activity, and RNase H activity.
Owing to its crucial role in viral replication, RT is a major
target for chemotherapeutic intervention. To date, two distinct
groups of RT inhibitors have been approved by the US Food
and Drug Administration for the treatment of HIV-1 infection.
These include: (i) the nucleoside/nucleotide RT inhibitors that
bind to the active site of RT and act as competitive inhibitors
of DNA polymerization; and (ii) the NNRTIs (non-nucleoside
RT inhibitors) that bind to the NNRTI-binding pocket and act as
allosteric inhibitors of DNA polymerization.
Although the RNase H activity of RT has been shown to be
essential for virus infectivity, very few inhibitors of this activity
have been identified and none have progressed to clinical trials.
However, over the past few years, significant progress has
been made in identifying and characterizing new RNase H
inhibitor pharmacophores [1]. For example, β-thujaplicinol [2,7dihydroxy-4-1(methylethyl)-2,4,6-cycloheptatrien-1-one] was
identified as a potent and selective HIV-1 RNase H inhibitor via
high-throughput screening of a National Cancer Institute library
of pure natural products [2]. Importantly, crystal structures of
β-thujaplicinol in complex with RT and an isolated RT–RNase
H domain fragment confirmed that it bound in the RNase H
active site of the enzyme [3]. However, the mechanism by which
β-thujaplicinol inhibits the RNase H activity of HIV-1 RT has
not been well defined. Beilhartz et al. [4] demonstrated that
β-thujaplicinol is unable to inhibit RNase H cleavage if added
Key words: efavirenz, non-nucleoside, reverse transcriptase,
RNase H, RNase H inhibitor.
Abbreviations used: EFV, efavirenz; NNRTI, non-nucleoside reverse transcriptase inhibitor; RT, reverse transcriptase; T/P, template/primer; WT, wild-type.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2013 Biochemical Society
180
B. D. Herman and N. Sluis-Cremer
RNase H cleavage). To gain an insight into the mechanism by
which β-thujaplicinol and EFV modulate the RNase H activity
of HIV-1 RT, we have systematically assessed their affect on the
polymerase-dependent and -independent cleavages of HIV-1 RT
using the pre-steady-state kinetic approach.
EXPERIMENTAL
Materials
WT (wild-type) and N348I HIV-1 RT were overexpressed
and purified to homogeneity as described previously [10,11].
RT concentration was determined spectrophotometrically at
280 nm using an absorbance coefficient of 260 450 M − 1 ·cm − 1 .
The 7-hydroxy tropolone derivatives 19616 (2,7-dihydroxy2,4,6-cyclo-heptatrien-1-one), 19617 (2,7-dihydroxy-4-methyl2,4,6-cyclo-heptatrien-1-one), β-thujaplicinol and 19619 (2,7dihydroxy-6-methyl-2,4,6-cyclo-heptatrien-1-one) were purchased from Molecular Diversity Preservation International.
EFV was obtained through the NIH (National Institutes of
Health) AIDS Reagent Program [Division of AIDS, NIAID
(National Institute of Allergy and Infectious Diseases), NIH,
Bethesda, MD, U.S.A.]. T4 polynucleotide kinase and [γ -32 P]ATP
were acquired through Fisher Scientific and PerkinElmer Life
Sciences respectively. The DNA and RNA oligonucleotides were
synthesized and purified by Integrated DNA Technologies. All
other reagents were of the highest quality available and were used
without further purification.
T/P substrates
The following RNA/DNA T/P substrates were used in the
present study: first, an 18 nt RNA template (RNA-18T: 5 GAUCUGAGCCUGGGAGCU-3 ) annealed to an 18 nt primer
(DNA-18T: 5 -AGCTCCCAGGCTCAGATC-3 ), as described
previously [12]. For FRET-based assays, fluorescein and
IowaBlack FQTM were covalently attached to the 3 - and 5 termini of the RNA and DNA oligonucleotides respectively. For
denaturing gel-based assays, the RNA was 5 -radiolabelled with
[γ -32 P]ATP as described previously [7]. Secondly, a 26 nt DNA
primer (DNA-26P; 5 -CCTGTTCGGGCGCCACTGCTAGAGAT-3 ) annealed to a 35 nt 5 -32 P-labelled RNA template (RNA35T; 5 -AGAAUGGAAAAUCUCUAGCAGUGGCGCCCGAACAG-3 ). Thirdly, DNA-26P annealed to a 27 nt 5 -32 P-labelled
RNA template (RNA-27T; 5 -AGAAUGGAAAAUCUCUAGCAGUGGCGC-3 ).
FRET-based RNase H cleavage assays
The FRET-based RNase H cleavage assays were carried out
as described previously [7]. Fluorescence was measured with
a SpectraMax M2 microplate spectrofluorometer (Molecular
Devices) using an excitation wavelength of 490 nm and an
emission wavelength of 528 nm. Data were analysed using
SoftMax Pro software (Molecular Devices). The concentration
of each 7-hyroxy-tropolone analogue required to inhibit RNase
H cleavage by 50 % (i.e. IC50 ) was calculated using non-linear
regression analyses (SigmaPlot Software Version 11, Systat
Software) from at least three independent experiments.
Steady-state RNase H cleavage assays
Steady-state RNase H cleavage was examined using both
fixed-time dose-response assays and time-course experiments.
c The Authors Journal compilation c 2013 Biochemical Society
In a typical experiment, 400 nM RT was diluted in 50 mM
Tris/HCl, pH 7.5, 50 mM KCl and 20 mM MgCl2 . Reactions
were initiated by the addition of an equal volume of 40 nM T/P
in 50 mM Tris/HCl, pH 7.5, and 50 mM KCl. Reactions were
quenched at appropriate times with gel-loading buffer. RNase
H cleavage products were resolved using denaturing PAGE.
Product formation was analysed by phosphorimaging using a
GS-525 Molecular Imager and Quantity One Software (Bio-Rad
Laboratories). The concentration of 19616 required to inhibit
RNase H cleavage by 50 % or the concentration of EFV required
to enhance RNase H cleavage by 50 % was calculated by nonlinear regression analyses (SigmaPlot Software Version 11, Systat
Software).
Pre-steady-state RNase H cleavage assays
Pre-steady-state experiments were carried out using a Kintek
RQF-3 rapid quench instrument (Kintek Corporation). In a
typical experiment, 400 nM RT was pre-incubated with 40 nM
5 -[32 P]RNA/DNA T/P in 50 mM Tris/HCl, pH 7.5, 50 mM KCl
and 2 mM EDTA prior to mixing with an equivalent volume
of 50 mM Tris/HCl, pH 7.5, 50 mM KCl and 22 mM MgCl2 .
Reactions were quenched with 50 mM EDTA, pH 8.0, at times
ranging from 25 ms to 120 s. Samples were further analysed as
described above.
RESULTS AND DISCUSSION
Structure–activity relationships for the inhibition of HIV-1 RT
RNase H activity by 7-hydroxy-tropolone analogues
We first assessed the inhibitory activity of β-thujaplicinol and
three related 7-hydroxy-troplone analogues (19616, 19617
and 19619) towards HIV-1 RT RNase H activity (Figure 1)
using a FRET-based assay as described previously [12]. βThujaplicinol inhibited RT RNase H activity with an IC50 value of
1.0 +
− 0.3 μM. The IC50 values of 19616 and 19617 were similar
to β-thujaplicinol (1.1 +
− 0.3 μM respectively).
− 0.2 μM and 2.0 +
In contrast, 19619 was less active (IC50 = 15.6 +
− 5.6 μM). The
available crystal structure data show that β-thujaplicinol primarily
interacts with the RNase H active site of RT through co-ordination
of the metal ions (all three oxygen atoms of the tropolone ring of
β-thujaplicinol co-ordinate the metal ions) and that there are very
few interactions between the tropolone ring and the amino acid
residues of HIV-1 RT [3]. Consequently the chemical changes
at position 5 of the tropolone ring in 19616 and 19617 have a
minimal effect on the activity of these analogues. However, there
is a non-covalent interaction between the carbon at position 3 of
the tropolone ring and His539 of RT [3], and the introduction of a
methyl group at this position probably impedes inhibitor binding
via a steric clash. Owing to the availability of samples, 19616 was
used in all further experiments.
Pre-steady-state kinetic analyses of HIV-1 RT RNase H activity in
the absence or presence of EFV or 19616
There is discrepancy in the literature in regard to the mechanism
by which β-thujaplicinol inhibits HIV-1 RT RNase H activity.
Beilhartz et al. [4] suggested that the inhibitor is unable to bind to
a preformed RT–RNA/DNA complex. In contrast, Himmel et al.
[3] revealed that β-thujaplicinol can bind to the RT-RNA/DNA
complex to form an RT–DNA/RNA–inhibitor complex. The T/P
substrate used in these studies differed. Beilhartz et al. [4] used
a RNA/DNA substrate with a 22 nt DNA primer recessed at
NNRTI and RNase H inhibitors affect secondary RNase H cleavages of HIV-1 reverse transcriptase
181
Figure 1 Chemical structures and HIV-1 RT RNase H inhibitory properties
of the compounds used in the present study
The structures of 19616, 19617, β-thujaplicinol and 19619 are shown. IC50 values were
determined using the FRET assay as described in the Experimental section.
both 5 - and 3 -ends annealed to a 52 nt RNA template. HIV-1
RT can simultaneously engage this substrate at both the DNA
polymerase and RNase H active sites of RT and the primer can
be extended if the next correct dNTP is added. As such, the
first RNase H cleavage is polymerase-dependent. Himmel et al.
[3] used the double-stranded 18 nt RNA/DNA duplex substrate
described in our FRET assays and depicted in Figure 2(A). This
T/P substrate cannot simultaneously engage both active sites
of RT and therefore the first RNase H cleavage is polymeraseindependent. In light of these studies, we next assessed the ability
of 19616 and EFV to affect the RNase H activity of HIV-1
RT on different RNA/DNA T/P substrates using pre-steady-state
kinetics, as described below.
First, we assessed HIV-1 RT RNase H activity on the bluntended 18 nt RNA/DNA duplex substrate (Figure 2). Our data
show that 19616 can inhibit cleavage of the RNA irrespective of
the order of substrate addition (Figures 2B–2D). Similarly, EFV
enhances RNA cleavage in a manner that is also independent
of the order of substrate addition. These findings are consistent
with those of Himmel et al. [3] and strongly suggest the
formation of RT–RNA/DNA–inhibitor complexes under the assay
conditions.
We further assessed HIV-1 RT RNase H activity on a T/P with
an RNA/DNA duplex length of 25 nt (Figure 3A). We have used
this T/P substrate in multiple studies and have clearly delineated
the RT RNase H-mediated cleavage events in both the absence
and the presence of EFV under steady-state assay conditions
[7,13,14]. However, we had not previously characterized the
ability of an RNase H inhibitor to block the polymerase-dependent
and -independent cleavages of HIV-1 RT on this T/P substrate.
Therefore we first assessed the ability of 19616 to inhibit
HIV-1 RT RNase H activity using a fixed time assay under
steady-state conditions (Figure 3B). Our data show that 19616
inhibited the secondary RNase H cleavages that reduced the RNA
template to 19 nt in length. However, we found no evidence of
inhibition of the primary RNase H cleavage event (i.e. there
was no accumulation of the full-length 35 nt RNA template
with increasing drug concentration). Quantitative analysis of the
formation of the 19 nt product yielded an IC50 value of 0.27 μM
for 19616 (results not shown). Consistent with previous studies
[7,13,14], EFV enhanced the formation of the 19 nt product
(IC50 = 14.1 nM; results not shown). To assess directly the affect
of 19616, EFV and combinations of both inhibitors on the primary
and secondary RNase H cleavages of RT we performed presteady-state kinetic analyses using a timescale from 25 ms to
90 min (Figure 4). The primary polymerase-dependent RNase H
cleavage occurred rapidly and more than 80 % of the substrate
was cleaved within 25 ms (Figures 4A and 4B). Despite this, we
saw no evidence that either 19616 or EFV affected the primary
Figure 2 Pre-steady-state kinetic analysis of the RNase H activity of HIV-1
RT, in the absence and presence of 19616 and EFV, on a blunt-ended 18 nt
T/P substrate
(A) Sequence of the RNA/DNA T/P substrate. (B) The RT–T/P–1 mM EDTA complex was mixed
with an equal volume of solution containing MgCl2 , MgCl2 –19616 or MgCl2 –EFV. (C) The
RT–T/P–1 mM EDTA–19616 complex or the RT–T/P–EDTA–EFV complex was mixed with
an equal volume of MgCl2 . (D) An RT–MgCl2 complex was mixed with an equal volume of
solution containing T/P–1 mM EDTA, T/P–1 mM EDTA–19616 or T/P–1 mM EDTA–EFV. The
final concentration of RT, T/P, free MgCl2 , 19616 and EFV in all experiments was 200 nM, 20 nM,
10 mM, 20 μM and 1 μM respectively. Molecular masses are indicated on the right-hand side
in kDa.
cleavage event (Figures 4A and 4B). We also carried out presteady-state kinetics experiments in which the RT–MgCl2 –EFV
(or RT–MgCl2 –19616) complex was mixed with the T/P substrate.
Under these conditions, the initial rate of RNase H cleavage was
slowed; however, we again saw no evidence that either 19616 or
EFV affected RNase H cleavage (results not shown). However, the
rates of the secondary RNase H cleavage events were significantly
affected by both 19616 and EFV (Figure 4C). In reactions carried
out with 19616, there was accumulation of the primary RNase
H cleavage products (28 and 27 nt respectively) and the rates of
formation of the secondary RNase H cleavage products at 25 nt,
21 nt and 19 nt were significantly decreased compared with the
c The Authors Journal compilation c 2013 Biochemical Society
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B. D. Herman and N. Sluis-Cremer
control (i.e. no drug). In contrast, all of these secondary cleavage
events occurred much faster when EFV was added to the reaction.
Interestingly, when both drugs were combined there was evidence
of antagonism: the final RNase H cleavage pattern was similar to
the control reaction (Figure 4C). Consistent with the data shown
in Figure 2, the ability of 19616 and/or EFV to modulate the
polymerase-independent RNase H cleavage activity of HIV-1 RT
was independent of the order of substrate addition (results not
shown).
Taken together, these data show that both 19616 and EFV have
a minimal effect on the primary (DNA polymerase-dependent)
RNase H cleavage events of HIV-1 RT, but that both compounds
significantly affect the secondary (DNA polymerase-independent)
cleavages.
Figure 3 Effect of 19616 and EFV on the RNase H activity of HIV-1 RT on
a T/P substrate that supports both polymerase-dependent and polymeraseindependent cleavages
Effect of the N348I mutation in HIV-1 RT on the activity of 19616
and EFV
(A) Sequence of the RNA/DNA T/P substrate. (B) Effect of different concentrations of 19616
or EFV on the RNase H cleavage pattern of HIV-1 RT. Molecular masses are indicated on the
right-hand side in kDa.
The N348I mutation in the connection domain of HIV-1
RT confers zidovudine and EFV resistance [15–17]. Previous
studies have demonstrated that the N348I mutation decreases
Figure 4 Pre-steady-state kinetic analysis of the RNase H activity of HIV-1 RT, in the absence and presence of 19616 and EFV, on a T/P substrate that
supports both polymerase-dependent and polymerase-independent cleavages
(A) Kinetics of polymerase-dependent RNase H cleavage in the absence or presence of 20 μM 19616, 1 μM EFV or 20 μM 19616 plus 1 μM EFV. Reactions were carried out by mixing an
RT–T/P–1 mM EDTA complex with MgCl2 , MgCl2 –19616, MgCl2 –EFV or MgCl2 –19616–EFV. Reactions were stopped at times ranging from 25 ms to 15 s. (B) Isotherm showing the rates of
polymerase-dependent RNase H cleavage in the absence or presence of 20 μM 19616, 1 μM EFV or 20 μM 19616 plus 1 μM EFV. (C) Kinetics of polymerase-independent RNase H cleavage in
the absence or presence of 20 μM 19616, 1 μM EFV or 20 μM 19616 plus 1 μM EFV. Reactions were carried out as described above, except reactions were stopped at times ranging from 20 s to
90 min. Molecular masses are indicated on the right-hand side in kDa.
c The Authors Journal compilation c 2013 Biochemical Society
NNRTI and RNase H inhibitors affect secondary RNase H cleavages of HIV-1 reverse transcriptase
183
Figure 5 Effect of 19616 and EFV on polymerase-independent RNase H
cleavages of WT and N348I HIV-1 RT
(A) Sequence of the RNA/DNA T/P substrate. (B) Inhibition of RNase H cleavage by 19616. Data
were obtained by quantifying the loss of the 27 nt RNA template in the absence or presence of
inhibitor. (C) Enhancement of RNase H cleavage by EFV. Data were obtained by quantifying the
appearance of the 19 nt RNA product (see Figure 4C) in the absence or presence of inhibitor.
the frequency of secondary polymerase-independent RNase H
cleavages by promoting RT to preferentially bind T/P substrates
with an RNA/DNA duplex length of 18 nt or less in a polymerasecompetent mode (i.e. the 3 -end of the DNA primer is in
the DNA polymerase active site and the RNA/DNA duplex
does not extend into the RNase H active site and therefore
polymerase-independent RNase H cleavages cannot occur)
[15,17]. Importantly, previous studies have shown that the N348I
mutation in HIV-1 RT significantly decreases the ability of NNRTI
to accelerate the rates of the polymerase-independent RNase
H cleavages [13,14]. To assess whether 19616 preferentially
inhibited N348I RT (compared with WT RT) we performed 19616
and EFV RNase H cleavage dose–response assays using a T/P
substrate identical in sequence with that described in Figure 3(A),
except that the 3 -end of the RNA was recessed, mimicking
the product that results from polymerase-dependent RNase H
cleavage (Figure 5A). The data (Figure 5B) show that the IC50
value for 19616 for N348I HIV-1 RT (6.1 +
− 0.44 μM) is 2.5fold less than the IC50 for WT HIV-1 RT (15.17 +
− 3.20 μM). In
contrast, N348I HIV-1 RT exhibited significantly less polymeraseindependent RNase H cleavage products (as quantified by the
19 nt band) than the WT enzyme (Figure 3C). Taken together,
these data suggest that 19616 exhibits a greater capacity to inhibit
the N348I enzyme that binds recessed RNA/DNA T/P substrates
in a DNA polymerase competent mode.
Mechanism by which 19616 and EFV modulate HIV-1 RT RNase H
cleavage activity
The results of the present study clearly demonstrate that 19616
does not inhibit the primary polymerase-dependent RNase H
cleavage activity of HIV-1 RT. Structural overlay of the crystal
structure of HIV-1 RT in complex with β-thujaplicinol (PDB code
3IG1) with RT in complex with an RNA/DNA T/P that occupies
the RNase H active site (PDB code 4B3O) shows that the inhibitor
and RNA/DNA duplex cannot simultaneously bind in the RNase
H active site of RT. The RNA strand in the RNA/DNA duplex
occupies the same space as β-thujaplicinol (Figure 6A). This
finding is consistent with that of Bielhartz et al. [4] who reported
Figure 6 Proposed model by which 19616 and EFV modulate the RNase H
activity of HIV-1 RT
(A) Structural overlay of the RNase H domain of HIV-1 RT in complex with β-thujaplicinol
(white; PDB code 3IG1) with RT in complex with an RNA/DNA T/P that occupies the RNase H
active site (grey; PDB code 4B3O). (B) Schematic diagram illustrating the affect of 19616 and
EFV on the polymerase-dependent and -independent cleavages of HIV-1 RT.
that the RNA/DNA substrate may block β-thujaplicinol access
to the RNase H active site. However, once the primary RNase
H cleavage event has occurred, 19616 can bind to the RT–T/P
complex and form an RT–T/P–inhibitor complex that prevents
RT from translocating (or dissociating and re-binding) to bind
the T/P in a manner that supports the secondary polymeraseindependent RNase H cleavages (Figure 6B). This model is
consistent with the finding that β-thujaplicinol binds to HIV-1
RT in complex with a blunt-ended 18 nt RNA/DNA T/P substrate
[3], and with the results of the present study which show that
the order of substrate addition does not affect the ability of
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B. D. Herman and N. Sluis-Cremer
19616 to inhibit the polymerase-independent cleavages of HIV1 RT and that N348I RT is hypersusceptible to inhibition by
19616. In contrast, EFV binding to RT destabilizes the 3 -end
of the DNA primer in the polymerase active site, favouring
polymerase-independent RNase H cleavage [7,9]. Consistent with
this model, we have shown antagonism between EFV and 19616
(Figure 4C).
AUTHOR CONTRIBUTION
Nicolas Sluis-Cremer and Brian Herman planned the experiments. Brian Herman performed
all of the experiments and the data analysis. Nicolas Sluis-Cremer wrote the paper.
FUNDING
This work was supported by the National Institutes of Health [grant number GM068406
(to N.S.-C.)].
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