The Connection Domain Is Implicated in Metalloporphyrin Binding
and Inhibition of HIV Reverse Transcriptase*
Elias G.
Argyris
§,
Jane M.
Vanderkooi§¶,
P. S.
Venkateswaran
,
Brian K.
Kay**, and
Yvonne
Paterson
§
From the Departments of
Microbiology and
¶ Biochemistry and Biophysics and the § Johnson
Research Foundation, University of Pennsylvania School of Medicine and
the
Institute for Antiviral Research, University City Science
Center, Philadelphia, Pennsylvania 19104 and the ** Department of
Pharmacology, University of Wisconsin,
Madison, Wisconsin 53706-1532
 |
ABSTRACT |
We have shown that heme and zinc protoporphyrin
inhibit both human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) reverse transcriptases (RTs) and, in combination with other
nucleoside and non-nucleoside inhibitors, exert an additive effect on
HIV-1 RT inhibition. Screening of a phage peptide library against heme resulted in the isolation of a peptide with sequence similarity to
sequence 398-407 from the connection subdomain of both HIV-1 and HIV-2
RTs, suggesting that this highly conserved region of HIV RTs
corresponds to the binding site for metalloporphyrins and a new site
for inhibition of enzyme activity. Inclusion of a synthetic peptide
corresponding to the exact sequence 398-407 of HIV-1 RT in RT
inhibition assays had a protective effect on metalloporphyrin
inhibition, as it was able to reverse the inhibitory effect of both
metalloporphyrins on HIV-1 RT activity. Furthermore, intrinsic
fluorescence assays indicated that these metalloporphyrins bind to
synthetic peptide 398-407 as well as to intact dimeric HIV-1 RT. The
identification of this novel inhibition site will help to expand our
understanding of the mode of action of metalloporphyrins in RT
inhibition and will assist in the design and development of more potent
metalloporphyrin RT inhibitors for the management of HIV infection.
 |
INTRODUCTION |
Human immunodeficiency virus type 1 (HIV-1)1 reverse
transcriptase (RT) is a major target for chemotherapeutic agents used in the treatment of AIDS. Currently, two major classes of anti-AIDS chemotherapeutic drugs have been developed that target the enzyme: the
nucleoside RT inhibitors (NIs), which are chain-terminating nucleoside
analogs, and the non-nucleoside inhibitors (NNIs), which are chemically
and structurally diverse and appear to inhibit HIV-1 RT by binding to a
common site distinct from the binding site of the nucleoside compounds
(for a review, see Refs. 1 and 2).
Recently, it has been shown that heme (iron protoporphyrin IX) has an
additive effect on murine hematopoietic recovery after treatment with
the nucleoside analog AZT-TP (3). Furthermore, heme and certain other
metalloporphyrins stimulate the immune system and enhance
erythropoiesis (4, 5). Heme, as well as the synthetic heme analog tin
protoporphyrin, significantly enhances bone marrow erythroid progenitor
proliferation and differentiation in lymphoproliferative disorders (6).
It has also been shown that heme and other synthetic heme analogs
significantly inhibit HIV-1 RT activity in a noncompetitive manner with
respect to deoxythymidine triphosphate and markedly enhance the
inhibitory effect of AZT-TP on HIV-1 RT (7). We have extended these
studies and demonstrated that heme and zinc protoporphyrin (ZnPP)
inhibit both HIV-1 and HIV-2 RTs in a
concentration-dependent manner, using in vitro RT inhibition assays. Furthermore, these metalloporphyrins, in combination with other nucleoside (AZT-TP) and non-nucleoside (BHAP)
HIV-1 RT inhibitors, exert an additive effect on HIV-1 RT inhibition,
indicating an independent mode of action by binding to a distinct
inhibition site. We thus used a novel approach to investigate the sites
on HIV RT and eventually other proteins that might be the target of
heme binding and modulation. In recent years, random peptide libraries
displayed on the surface of filamentous bacteriophage M13 have been
used successfully for the identification of peptide ligands that
interact with molecular targets. Phage libraries have been generated to
display peptides of variable lengths ranging from 6 to 38 random amino
acids, protein fragments, or fully active folded proteins (for a
review, see Ref. 8). Such libraries have been used to identify peptide
epitopes for monoclonal (9-12) and polyclonal (13-15) antibodies as
well as ligands for a variety of other molecules, both proteinaceous
and non-proteinaceous, including those that are known to interact with
small peptide ligands (HLA-DR1 (16), BiP (17), and calmodulin (18)) and
those that had previously undefined specificity for peptides
(concanavalin A (19), streptavidin (20), and others).
Here we report the use of a 12-amino acid residue random peptide
library displayed on bacteriophage M13 to identify peptide ligands that
interact with heme. Sequence determination of the displayed peptides,
found to be specific for heme, revealed that they lack amino acid
sequence similarity, yet are highly enriched in Trp and Tyr residues
and have homology to a wide variety of proteins. Among the different
Trp-rich peptides displayed by heme-selected clones, one in particular
was found to be partially homologous to sequence 398-407 from the
connection domain of both HIV-1 and HIV-2 RTs as well as SIV RT,
suggesting that this sequence corresponds to the distinct binding site
for heme and a site for inhibition of enzyme activity. Intrinsic
fluorescence assays indicated that heme and ZnPP bind to a synthetic
peptide corresponding to the exact sequence 398-407 of HIV-1 RT as
well as to intact dimeric HIV-1 RT. We have also demonstrated the
ability of synthetic peptide 398-407 to reverse the inhibitory effect
of both metalloporphyrins on HIV-1 RT activity. Molecular modeling
indicated that sequence 398-407 from the connection subdomain of HIV-1
RT is sufficiently exposed to accommodate heme binding. These studies
have thus identified a new site for inhibition on HIV-1 and HIV-2 RTs
that facilitates the design and preparation of new porphyrin-based
inhibitors of RT that may have clinical potential in the management of AIDS.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Heme was purchased as the chloride salt, hemin
chloride (Calbiochem), and prepared as described (21). In certain
experiments, hemin chloride was conjugated through its propionyl groups
to BSA by EDC-mediated hapten carrier protein formation (Imject
Immunogen EDC Conjugation kit, Pierce), and the conjugated protein was
purified by gel filtration. In other experiments, heme was directly
solubilized in methanol. Bovine hemin (type I) immobilized on
cross-linked 4% beaded agarose was purchased from Sigma. ZnPP was
obtained from Porphyrin Products (Logan, UT) and prepared as described (21).
Construction of the Phage Peptide Library--
The construction
of the phage peptide library is described elsewhere (12, 22). The total
complexity of the peptide library was 2 × 109. The
combinatorial gene sequences displayed at the N terminus of the M13
pIII gene were of the form TCG AGN (NNK)12 TCT AGA, where N represents all four nucleotides (A, C, G, and T) in an equal
molar ratio, and K represents an equal molar ratio of G and T. The
encoded amino acid scheme was S(S/R)(X)12SR. The
library had a working titer of 1 × 1013 pfu/ml.
Affinity Purification of Binding Phage--
Affinity
purification was performed utilizing three different attachment
strategies for the heme. In the first strategy, hemin chloride,
EDC-coupled to BSA (20 µg), was directly bound on microtiter wells of
a 96-well microtiter plate (Corning Inc., Oneonta, NY) in 100 mM NaHCO3, pH 8.5, at ambient temperature for
2-3 h or at 4 °C overnight. Wells were then blocked with 30 mg/ml
BSA in 100 mM NaHCO3 (blocking solution) for
2 h at ambient temperature and finally washed three to five times
with washing buffer (phosphate-buffered saline and 0.5% (v/v) Tween
20, pH 7.4). Phage (1011 pfu) were added to each well and
allowed to bind for 3 h at ambient temperature. Unbound phage were
removed by extensive washing of the wells, and adherent phage were
eluted with 50 mM glycine HCl, pH 2.2, and neutralized in
200 mM NaPO4 buffer, pH 7.5. An aliquot of the
neutralized phage was liquid-amplified in Escherichia coli DH5
F' (Life Technologies, Inc.) as described (23). This panning protocol was then repeated twice. After three rounds of selection, affinity-purified phage stocks were plated to determine titers and to
yield isolated plaques from which clonal cultures were produced for
further analysis. In the second strategy, where hemin chloride was
directly dissolved in methanol at a concentration of 1 mg/ml, 50 µl
(50 µg) of the solution were added to the microtiter wells and dried
out after incubation for 1-2 h at 37 °C. The wells were then
blocked, and screening of the library was performed as described above.
In the third strategy, commercially available hemin (bovine, type I)
immobilized on cross-linked 4% beaded agarose was used as a target for
screening the CW peptide library. In brief, 500 µg of target-coupled
beads were incubated in a microcentrifuge tube in 1 ml of
phosphate-buffered saline/Tween 20 with phage (1011 pfu)
for 2 h at ambient temperature. Beads were then washed
extensively, and bound phage were eluted by resuspending the beads in
300 µl of elution buffer, pH 2.2, for 15 min at ambient temperature. The beads were pelleted, and eluted phage were neutralized by transferring the solution to a new tube containing 300 µl of
neutralization buffer, pH 7.5. The recovered phage were amplified, and
the affinity purification was repeated for a total of three rounds.
Evaluation of the Specificity of Selected Clones--
To
evaluate the specificity of each selected clone against heme, we
performed plaque assays as described (23). In brief, equal titers
(1011 input pfu) of each phage clone were incubated with
the heme target, immobilized on a solid support by the three different
attachment strategies. In parallel, equal titers of phage stock were
added to microtiter wells coated with blocking solution or to
cross-linked beaded agarose in tubes, which served as negative
controls. Microtiter wells and tubes were incubated and washed as
described above. Eluted and neutralized phage were serially diluted and
plated onto LB plates. The binding specificity was determined by
comparing the calculated percentage of input pfu recovered from the
phage bound to heme with that bound to BSA or to cross-linked beaded agarose.
Phage Sequencing--
Aliquots of the heme-bound phage output,
after the third round of selection, were plated to isolate individual
plaques (clones), from which phage stocks were produced for the
purification of double-stranded DNA (Wizard Plus Minipreps DNA
Purification system, Promega, Madison, WI). Purified double-stranded
DNA was sequenced by automated cycle sequencing (ABI 377 or 373A
Stretch sequencers, with Taq FS dye terminator or dye primer
chemistry). An oligonucleotide primer with the sequence 5-AGC GTA ACG
ATC TAA A-3' was used to determine the nucleotide sequence of the
unique region of the M13 phage.
Peptide Synthesis and Purification--
The P1 peptide
(WETWWTEYWQ), which corresponds to sequence 398-407 from the
connection domain of HIV-1 RT (based on the HIV-1 BH10 molecular clone
sequence), was purchased as a crude cleavage product from the
Biopolymer Synthesis Center, California Institute of Technology
(Pasadena, CA). The P2 control peptide (ASQEVKNWM, from HIV-1
glycosaminoglycan protein) was synthesized in our laboratory using the
RaMPS multiple peptide synthesis system procedure on Wang resin
(DuPont) using Fmoc (N-(9-fluorenyl)methoxycarbonyl) nitrogen terminal protected amino acids (BACHEM Biotech Co.,
Philadelphia, PA). Both peptides were purified in our laboratory by
reverse-phase high performance liquid chromatography (Perkin-Elmer
Series 400) using a Vydac C18 column, and their identity and purity
were verified by matrix-assisted laser desorption ionization/mass
spectroscopy (24).
Reverse Transcriptase Assay--
Recombinant HIV-1 and HIV-2 RTs
were donated by the Center for AIDS Research at Case Western Reserve
University. The NI AZT-TP was obtained from Moravek Biochemicals, Inc.
(Brea, CA), and the NNI U-90152T, which is a BHAP analog, from
Pharmacia Upjohn Co. [methyl-3H]Deoxythymidine
triphosphate ([3H]dTTP), with a specific activity of 17.5 Ci/mmol, was purchased from NEN Life Science Products, and the
primer-template poly(rA)·p(dT)12-18 was from Pharmacia
Biotech. The reaction mixture for measuring polymerase activity, using
poly(rA)·p(dT)12-18 (10 units/ml), contained 100 mM Tris-HCl, pH 8.3, 5 mM MgCl2, 80 mM KCl, 1 mM dithiothreitol, 2.5 units of
recombinant HIV-1 RT, 5 µM dTTP, and 1 µCi of
[3H]dTTP as a substrate in a total volume of 200 µl.
Reaction mixtures were incubated for 1 h at 37 °C and
terminated by the addition of 0.5 ml of 5% trichloroacetic acid with
2% (w/v) sodium pyrophosphate. Then 100 µl of a solution containing
BSA (2.5%) and calf thymus DNA (5%) were added. Acid-precipitable
material was collected on glass-fiber filters and washed with 5%
trichloroacetic acid and 95% ethanol, and radioactivity retained on
the filters was measured by scintillation counting.
Fluorescence Assays--
Conventional fluorescence spectra were
recorded using a Perkin-Elmer LS-5 luminescence spectrometer. HIV-1 RT
and the peptides were routinely excited at 280 nm, and the fluorescence
emission spectrum was integrated between 300 and 460 nm. The maximum
emission intensity for the RT was measured at 340 nm and for the
peptides at 360 nm. The intrinsic fluorescence emission of HIV-1 RT
(200 nM protein) and of peptides P1 and P2 (1 µM) was measured in fluorescence buffer containing 0.1 Tris-HCl, pH 8.3. The average intrinsic association constant or
affinity (Ko) was calculated on the basis of the
logarithmic form of the Sips distribution function as described (25).
Analytical Ultracentrifugation--
Sedimentation equilibrium
measurements were made using a Beckman XL-I analytical ultracentrifuge
with interference optics. Samples of RT alone and RT with heme were
prepared using the same mixture composition as described under
"Reverse Transcriptase Assay," but all reagents were 50-fold more
concentrated. Sedimentation of the samples was measured against buffer
blanks in standard 1.2-cm path length, six-channel cells with sapphire
windows. Data were obtained at 10 °C at 15,000 rpm for 36 h,
followed by another 30 h at 12,000 rpm. Equilibrium concentration
profiles at each speed were analyzed using equilibrium sedimentation
equations for reversible heterodimerization with Igor-Pro®
programs (Wavemetrics, Lake Oswego, OR).
Molecular Graphics Representations of HIV-1 RT and Heme--
The
structure of HIV-1 RT (BH10 isolate) was obtained from the Protein Data
Bank at Brookhaven National Laboratory (PDB file 3HVT). The heme
structure was extracted from PDB file 1MYG. Model building was
performed using the SYBYL molecular modeling package (Tripos
Associates, St. Louis, MO) on a Silicon Graphics display system as well
as the molecular visualization program RasMol Version 2.5 (50) on a
Macintosh display system.
 |
RESULTS |
Heme and ZnPP Inhibit HIV-1 RT in a Noncompetitive/Nonexclusive
Manner with NIs and NNIs and Exert an Additive Effect on Enzyme
Inhibition--
To confirm the findings of other investigators and to
further our understanding of the mode of action of metalloporphyrins in
HIV-1 RT inhibition, we conducted RT inhibition assays as described under "Experimental Procedures." As shown in Table
I, both metalloporphyrins inhibited RT in
a concentration-dependent manner. Heme or ZnPP (both at 10 µM) resulted in >80% inhibition, which is significantly greater than the findings of other workers (7). Our results also
indicate that ZnPP is a slightly better inhibitor than heme. The
calculated IC50 values for inhibition of HIV-1 RT were 6 µM for heme and 4.8 µM for ZnPP. In
addition, we compared the inhibitory effects on HIV-1 RT of heme, ZnPP,
and heme or ZnPP in combination with AZT-TP with those of AZT-TP alone.
As shown in Table I, AZT-TP (10 nM) inhibited the enzyme by
19%. The inclusion of heme or ZnPP markedly enhanced the inhibition of
RT by AZT-TP, thus confirming the findings of Staudinger et
al. (7) that the combination of heme with AZT-TP synergistically
inhibits the activity of HIV-1 RT. Furthermore, our results indicate
that metalloporphyrins in combination with the chain terminator AZT-TP
exert a strictly additive effect on the inhibition of the enzyme. The
studies of Staudinger et al. have also suggested a distinct
but allosterically linked binding site for heme and its analogs on
HIV-1 RT with respect to the nucleoside-binding site. To investigate
whether metalloporphyrins bind to the other known site of inhibition on RT, the common binding site of other NNIs that is located in the p66
palm subdomain of the enzyme (2), we compared the inhibitory effect of
heme, ZnPP, and heme or ZnPP in combination with the well characterized
NNI, BHAP (26-30). As shown in Table I, heme and ZnPP in combination
with the NNI inhibited RT in a noncompetitive and nonexclusive manner.
Indeed, they enhanced the inhibitory effect of BHAP by wielding an
additive effect on the enzyme inhibition, indicating that
metalloporphyrins bind to a distinct binding site on HIV-1 RT that is
not shared by either NIs or NNIs.
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Table I
Inhibitory effects of metalloporphyrins alone and in combination with
AZT-TP and BHAP on HIV-1 RT activity
The RT was assayed as described under "Experimental Procedures."
Results are the mean of five experiments.
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Identification of a Heme-binding Site on HIV-1 and HIV-2 RTs Using
a Phage Peptide Library--
To investigate the sites on HIV-1 RT that
might be the target of heme binding, we screened a phage-displayed
12-mer peptide library against heme. After three rounds of selection, a
total of 43 heme-specific clones were isolated, which represented 40 different clones (Table II). As shown,
the displayed peptides shared no obvious sequence similarity; however,
examination of the frequencies of individual amino acids indicated that
they are greatly enriched in Trp and Tyr residues and are completely devoid of Cys residues. Statistical analysis of the frequency of
individual amino acids in the displayed peptides indicated that the Trp
and Tyr enrichment, with frequencies of 17.5 and 7.29%, respectively,
was highly significant (Trp, p = 0.000001; Tyr,
p = 0.018). Among the 40 different peptide sequences,
only two did not include any Trp or Tyr residues (clones A-3 and A-15), whereas the remainder contained at least one Trp or Tyr residue. No
significant differences were found in the amino acid composition and
frequency among the peptides displayed by heme-selected phage from the
different attachment strategies for heme. The evaluation of the
specificity of the 40 recovered clones by the plaque assay showed that
all phage clones were heme-specific, with a typical 10
1
to 100% recovery of input pfu as compared with the
negative controls, which gave 10
6 to 10
5%
recovery of input pfu. Overall, all 40 different clones bound at least
>104 times better to heme targets than to the negative
controls (wells coated with BSA alone or with cross-linked beaded
agarose).
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Table II
Deduced amino acid sequences of peptides displayed by heme-selected
phages after three rounds of selection
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To determine any homology between our peptide sequences and registered
protein sequences, including HIV-1 RT, we performed a search of protein
data bases using the advanced BLAST Sequencing Similarity Searching,
from the National Center for Biotechnology Information Services. The
advanced searching revealed complete or partial homology between almost
all of our peptide sequences and a large number of different protein
sequences registered in protein data bases, indicating that the random
library did generate authentic protein sequences. It is noteworthy that
the advanced searching also partially matched several of our
heme-specific peptide sequences with registered heme-binding proteins
(our clone A-8 with cytochrome b5, clone A-14
with heme exporter protein D, clone A-16 with cytochrome c
oxidase, and clone B-3 with probable cytochrome c
biosynthesis protein). Since our objective was to identify peptide
ligands reflecting regions of HIV-1 RT that might be the target of heme
binding, we were gratified to find that heme-specific clone A-7
(WGEWWRNGWQRD) partially matched a sequence from the connection domain
of HIV-1 RT (sequence 398-407, WETWWTEYWQ) as well as WEQWWDNYWQ
(corresponding to sequence 398-407 of HIV-2 RT) and WEQWWADYWQ
(sequence 398-407 of SIV RT). The partial homology of the Trp-rich
clone A-7, from the screening of the phage library against heme, with
sequence 398-407 from the connection domain of the HIV and SIV RTs
suggested that this sequence corresponds to at least a part of the
binding site for metalloporphyrins on RT. It should be noted that clone
A-7, which was originally selected from the screening of the phage
library against heme coupled to BSA, also bound 104 times
better to heme immobilized on cross-linked agarose than to the negative
control, cross-linked beaded agarose (data not shown).
A Synthetic Peptide (P1) Corresponding to the Exact Sequence
398-407 of HIV-1 RT (WETWWTEYWQ) Protects HIV-1 RT from
Metalloporphyrin Inhibition--
To test our hypothesis that sequence
398-407 of HIV-1 RT corresponds to the binding site for heme and
eventually other heme analogs, we examined the ability of the P1
peptide to reverse the inhibitory effect of heme and ZnPP on RT
activity, perhaps by competing with HIV-1 RT for binding to the
porphyrin compounds, using in vitro RT inhibition assays.
For this purpose, we included the peptide in the reaction mixture with
the metalloporphyrins at different molar ratios prior to the addition
of the enzyme and initiation of the reaction. To control for the direct
effects of the P1 peptide on the activity of HIV-1 RT, we also tested the peptide alone. In addition, we tested a peptide (P2) from HIV-1
glycosaminoglycan (ASQEVKNWM), as a negative control, in combination
with the metalloporphyrins and alone. The effects of the P1 and P2
peptides on the enzyme activity, either in the presence or absence of
heme and/or ZnPP, are shown in Fig. 1. Inclusion of the P1 peptide at an equimolar ratio (5 µM)
with heme did not alter the inhibitory effect of heme on the enzyme activity. However, at a peptide concentration of 50 µM,
which translates to a molar ratio of 10:1 for P1 peptide/heme, the
enzyme activity was almost completely restored. The P1 peptide exerted a similar "protective" effect on the enzyme activity when
used in combination with ZnPP, although to a lesser extent. As
expected, the P2 peptide at similar concentrations in the presence of
heme and/or ZnPP did not have any effect on the enzyme activity. Both peptides, when tested alone, did not show any effect on the activity of
HIV-1 RT. Our results strongly suggest that sequence 398-407 of HIV-1
RT is implicated as the binding site for heme and eventually other heme
analogs.

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Fig. 1.
Effects of the P1 and P2 peptides on the
enzyme activity in the presence or absence of heme and/or
ZnPP.
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Metalloporphyrins Are Potent Inhibitors of HIV-2 RT--
Given
that the heme-specific phage clone A-7 matched not only sequence
398-407 of HIV-1 RT, but also the same highly conserved region in
HIV-2 and SIV RTs, we wished to examine the effect of heme and ZnPP on
HIV-2 and SIV RT activities. The ability of metalloporphyrins to
inhibit other immunodeficiency viral RTs has not previously been
investigated. Because of limited availability of these reagents (recombinant HIV-2 and SIV RTs), we were able to test the effect of
heme and ZnPP only on HIV-2 RT. As shown in Table
III, heme and ZnPP were also potent
inhibitors of HIV-2 RT. Our results indicate that ZnPP is a better
inhibitor than heme. The calculated IC50 values for
inhibition of HIV-2 RT were 23 µM for heme and 7 µM for ZnPP. Our findings strongly support our suggestion
that metalloporphyrins inhibit both HIV-1 and HIV-2 (and perhaps SIV) RTs by binding to the highly conserved region 398-407 from the connection domain of these enzymes.
Intrinsic Fluorescence Assays Confirm the Binding of
Metalloporphyrins to Synthetic Peptide P1 and Intact Dimeric HIV-1
RT--
To verify the specific binding of metalloporphyrins to the
HIV-1 P1 peptide (WETWWTEYWQ), we used fluorescence quenching
spectroscopy. We measured changes in tryptophan fluorescence on
metalloporphyrin binding to examine the binding of the P1 peptide to
heme and ZnPP using a fixed peptide concentration (1 µM)
and increasing concentrations of heme and ZnPP. The results of the
intrinsic tryptophan fluorescence changes are shown in Fig.
2A and demonstrate that both
porphyrin derivatives bound to the peptide, with ZnPP binding slightly
better (Ko = 0.14 × 106
M
1) than heme (Ko = 0.10 × 106 M
1). Overall,
our data confirm that the HIV-1 RT sequence 398-407 binds to heme and
ZnPP and support our suggestion that this sequence corresponds to the
binding site for metalloporphyrins on HIV-1 RT. To examine the RT-heme
and RT-ZnPP interactions, we measured the fluorescence changes using a
fixed enzyme concentration of 200 nM and increasing
concentrations of heme and ZnPP (Fig. 2B). The binding of
ZnPP to the enzyme was greater (Ko = 0.36 × 108 M
1) than that of heme
(Ko = 0.15 × 108
M
1). These results are consistent with the RT
inhibition assays, in which we showed that ZnPP is a more effective
inhibitor of HIV-1 RT than heme. They are also consistent with the
requirement for high concentrations of peptide to abrogate heme
inhibition of RT (Fig. 1) in that the affinity of
peptide for heme is 2 logs lower than that of heme for RT.

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Fig. 2.
Fluorescence properties of synthetic P1 and
control P2 peptide (A) and HIV-1 RT (B) binding
to heme and ZnPP.
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Heme Does Not Inhibit RT by Promoting Dissociation of the
Dimer--
It should be noted that the addition of heme or ZnPP to
HIV-1 RT did not alter the maximum of the emission spectrum, suggesting that no new Trp residues are exposed when metalloporphyrins bind to the
enzyme. This argues against a mechanism for heme inhibition in which
the metalloporphyrin promotes dissociation of the RT dimer by binding
to the connection domain. To test this hypothesis, we also performed
sedimentation equilibrium measurements by analytical ultracentrifugation, and no dissociation of the RT dimer in the presence of heme was observed (data not shown).
The Crystal Structure of HIV-1 RT Indicates That Sequence 398-407
Is Sufficiently Exposed to Provide a Site for Heme Binding--
The
recent availability of several crystallographic structures of the
enzyme complexed with double-stranded DNA as well as with
non-nucleoside inhibitors has promoted our understanding of the
structure-function relationship of the biochemical reaction catalyzed
by HIV-1 RT and facilitated the rational design of more specific and
potent inhibitors. Crystal structures of non-nucleoside inhibitors such
as nevirapine (31),
1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (32),
-anilinophenylacetamide (32), and
tetrahydroimidazo(4,5,1-jk)(1,4)-benzodiazepin-2(1H)-one
(33) complexed with HIV-1 RT revealed that these structurally diverse
molecules all bind at a common site in the p66 subdomain, although each
exhibits slightly different interactions with the enzyme. The binding
site for the NNIs is located close to the p66 polymerase active site
and is more of a hydrophobic cavity than a pocket. All inhibitors are
able to bind to this site in a "butterfly" shape that is a native
structural feature of the molecule or one that the molecule can adopt
with little energy penalty. In the native enzyme, the drug-binding
cavity does not exist; it is created by the movement of the side chains
of Trp229, Tyr181, and Tyr188
(34-36). Our study indicates that heme has a completely different binding site on HIV-1 RT as compared with the known binding site of the
NNIs. As shown in Figs. 3 and
4, molecular modeling indicates that the
highly conserved sequence 398-407 from the connection domain of HIV-1
RT is sufficiently exposed to provide surfaces of appropriate size for
heme binding. Given that the two subunits have the same amino acid
sequence throughout the polymerase region, sequence 398-407 occurs
twice in the RT dimer and once in each of the p66 and p51 subunits.
This sequence overlaps
-helix L from the connection domains,
sequence 395-404 in p66 and sequence 1395-1404 in p51, as defined in
the RT three-dimensional structure (37). Each sequence 398-407 is
partially exposed on the front surface, passes through the interface of
the p66 and p51 dimer, and has another exposed surface on the other
side. The largest exposed area, which could accommodate a heme molecule
bound with the tetrapyrrole groups planar to the RT surface, occurs on
the back surface of the p66 domain (Fig. 3C and Fig.
4B). The other three exposed surfaces are narrower and would
require heme to bind perpendicular to the RT surface (Fig. 3,
A, B, and D; and Fig. 4,
A and B) perhaps through hydrophobic contact with
the vinyl groups.

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Fig. 3.
Molecular graphic representations of the
proposed binding site for heme to HIV-1 RT, with the heme molecule
docked onto the enzyme. The representations were prepared as
described under "Experimental Procedures." A and
B, "front" side of HIV-1 RT; C and
D, "back" side of HIV-1 RT. The p66 subunit is shown in
blue, and the p51 subunit is shown in green. Heme
is shown in red. The small yellow regions
represent the conserved residues between the peptide sequence of the
heme-selected clone A-7 and sequence 398-407 from the connection
domain of HIV-1 RT, whereas the residues that vary are shown in
cyan.
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Fig. 4.
Molecular representations of an enlarged view
of the proposed binding site for heme to the HIV-1 RT dimer. The
representations were prepared as described under "Experimental
Procedures." A, "front" side of HIV-1 RT;
B, "back" side of HIV-1 RT. The p66 subunit is shown in
dark gray, and the p51 subunit is shown in light
gray. The exposed residues in sequence 398-407 from the
connection domain of both the p66 and p51 subunits are shown in
white.
|
|
 |
DISCUSSION |
At present, RT represents one of the major targets in the
development of chemotherapeutic drugs against HIV, the etiologic agent
responsible for the development of AIDS. Inhibitors that have already
been developed can be divided into nucleoside analogs that are active
in the triphosphorylated form and non-nucleosides that interact
directly with RT (1, 2). There have also been attempts to develop other
classes of RT inhibitors, such as antisense RNA and sense
oligonucleotides (38-40), ribozymes (41, 42), aptamers (43), and
oligopeptides (44), that exert an inhibitory effect by interfering with
the dimerization process of RT.
Metalloporphyrins have been shown to represent potent non-nucleoside
inhibitors of HIV-1 RT and viral replication. In a recent study,
noncompetitive inhibition of HIV-1 RT activity with respect to the
nucleoside-binding site was demonstrated (7), suggesting a distinct but
allosterically linked binding site for heme and its analogs as compared
with that for AZT. In the present work, we have further investigated
the mode of heme and ZnPP inhibition of HIV-1 RT. Using in
vitro RT inhibition assays, we have shown that metalloporphyrins,
in combination with the well characterized NNI, BHAP, inhibit the
enzyme in a noncompetitive and nonexclusive manner and enhance the
inhibitory effect of BHAP by wielding an additive effect on the enzyme
inhibition, which indicates that heme and its analogs inhibit HIV-1 RT
by binding to a distinct site as compared with the common binding site
of NNIs.
To investigate the sites on HIV-1 RT that might be the target of heme
binding, we screened a phage peptide library against heme and isolated
heme-specific peptides that were highly enriched in Trp and Tyr
residues, indicating that these residues are important for heme
binding. It is well documented that the specific structure of heme
enables this small molecule to interact with proteins and peptides in
various ways. The fifth and sixth coordination sites of the iron can
ligand with various amino acids, whereas the propionyl and vinyl side
chains of the tetrapyrrole ring are able to interact with appropriate
amino acids via ionic and hydrophobic interactions, respectively (45).
We believe that the high frequency of Trp and Tyr residues in the
peptides identified by phage display arises due to the interaction of
the Trp and Tyr residues in the heme-specific displayed peptides with
the heme molecule via
-
aromatic stacking. Nevertheless,
comparison of our heme-specific peptide sequences with those in protein
sequence data bases revealed complete or partial homology between
almost all of our peptide sequences and a large number of different
protein sequences, including known heme-binding proteins. It was also
not surprising that we found a match between one clone, A-7, and the
highly conserved sequence 398-407 from the connection domain of HIV-1,
HIV-2, and SIV RTs, indicating that this region corresponds to the
binding site for heme and for other heme analogs on RT. It should be
noted that the identification of a ligand-binding site in a protein by
random peptide libraries is not unprecedented. Similar studies have
described the isolation of phage-displayed peptides specific for
different proteinaceous targets, which led to the identification of
ligand-binding sites in proteins known to interact with those targets
(46, 47).
This paper also provides the first demonstration that heme and its
synthetic analog, ZnPP, are potent inhibitors of HIV-2 RT, thus
strongly supporting our suggestion that metalloporphyrins inhibit both
HIV-1 and HIV-2 (and perhaps SIV) RTs by binding to region 398-407
from the connection domain of the enzymes, which is conserved between
these species. The ability of a synthetic peptide corresponding to the
exact sequence 398-407 of HIV-1 RT to reverse the inhibitory effect of
both heme and ZnPP on HIV-1 RT activity is consistent with our
findings. Intrinsic fluorescence assays also indicated that
metalloporphyrins bind to the HIV-1 RT synthetic peptide 398-407,
although with much lower affinity than to intact dimeric enzyme. In
addition, sedimentation equilibrium measurements by analytical
ultracentrifugation also demonstrated that heme binds to the dimeric
form of RT, which argues against a mechanism for heme inhibition in
which porphyrin promotes dissociation of the RT dimer. Support of our
suggestion that metalloporphyrins inhibit RT from HIV-1 and HIV-2, by
binding to region 398-407 from the connection domain of the enzymes,
is provided by the finding that heme does not inhibit RT from feline
immunodeficiency virus, where region 398-407 (WESNLINSPY) (48) from
the connection domain of the enzyme is dissimilar to that of HIV-1,
HIV-2, and SIV.2
As indicated by molecular modeling, the highly conserved sequence
398-407 from the connection domain of HIV-1 RT is sufficiently exposed
to provide surfaces of appropriate size for heme binding (Figs. 3 and
4). There are several possible modes for heme binding to the RT
heterodimer. Heme may bind to sequence 398-407 either at the front or
back of the p66 or p51 connection domain at either exposed site.
Alternatively, it may bind to both subunits on a larger site including
surface residues not identified by the phage-displayed peptide library,
such as Trp410, which is also highly exposed and links the
p66 and p51 peptide sites on the back of the molecule (Fig.
3B). Only one of these four potential heme-binding sites
could obviously interfere with RT enzyme activity. This is the p51
front exposed surface shown in Fig. 3A, in which
Trp398, Glu399, Thr400, and
Trp402 are partially exposed. Given that
-helix L of the
p51 subunit has been shown to make contact with the primer strand (37), binding to this site could interfere with the binding of the
primer-template. If the region shown in Fig. 3A is indeed
the heme-binding site, then this is a rare example of a site of
inhibition in the p51 monomer of RT. To our knowledge, the only other
residue in the p51 monomer implicated in a site of inhibition is
Glu138, which may be involved in inhibition by NNIs (49).
Heme binding to the other three sites, however, could only inhibit RT
activity by indirectly disturbing the conformation of the molecule in
the active-site region.
The mechanism of heme inhibition clearly merits further study. The
identification, here, of a possible site or sites of inhibition will
facilitate this process. Site-directed mutagenesis to generate single
or extended amino acid substitutions within the coding region of HIV-1
RT, with specific emphasis on region 398-407 from the connection
subdomains of the enzyme, will facilitate the determination of the
mechanism of heme binding to HIV-1 RT and its inhibitory effect on the
enzyme. In addition, p66 and/or p51 could be subjected to
subunit-specific mutagenesis to distinguish which of the four possible
heme-binding sites on RT (Fig. 3, A and B) are
sites of inhibition. This work demonstrates that heme and ZnPP are
indeed true HIV-1 and HIV-2 RT inhibitors by binding to a distinct
binding site on the enzyme and encourages the design and preparation of a new class of porphyrin-based systems that will have a more potent inhibitory effect on HIV-1 RT, especially in soluble forms.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Stuart Le Grice (Case Western
Reserve Center for AIDS Research) for providing recombinant HIV-1 and
HIV-2 RTs, helpful comments, and critical reading of the manuscript. We
also thank Dr. Les Dutton (Johnson Research Foundation) for allowing us
to use the molecular modeling facility and Dr. James D. Lear (Johnson
Research Foundation) for excellent assistance with sedimentation equilibrium measurements.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant GM31841 (to Y. P.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.

To whom correspondence and reprint requests should be
addressed: Dept. of Microbiology, University of Pennsylvania School of
Medicine, Rm. 323, Johnson Pavilion/6076, 36th and Hamilton Walk,
Philadelphia, PA 19104. Tel.: 215-898-3461; Fax: 215-573-4666; E-mail:
yvonne{at}mail.med.upenn.edu.
The abbreviations used are:
HIV-1, human
immunodeficiency virus type 1; HIV-2, human immunodeficiency virus type
2; RT, reverse transcriptase; NIs, nucleoside inhibitors; NNIs, non-nucleoside inhibitors; AZT-TP, 3'-azido-3'-deoxythymidine
triphosphate; ZnPP, zinc protoporphyrin; BHAP, bisheteroarylpiperazine; SIV, simian immunodeficiency virus; BSA, bovine serum albumin; pfu, plaque-forming units; EDC, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride.
2
S. Le Grice, personal communication.
 |
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