1 Strategy to discriminate between high and low affinity bindings of HIV-1 integrase to viral DNA

48 Pages·2003·0.33 MB·English

JBC Papers in Press. Published on March 7, 2003 as Manuscript M211711200
Strategy to discriminate between high and low affinity bindings of HIV-1 integrase
to viral DNA

Running title: Interaction of HIV-1 integrase with viral DNA

Loussinée Zargarian†, Mohamed Salah Benleumi†, Jean-Guillaume Renisio†, Hayate
Merad†, Richard G. Maroun†*, Frédéric Wieber†, Olivier Mauffret†, Horea Porumb†,
Frédéric Troalen‡ and Serge Fermandjian†¶

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Département de Biologie et Pharmacologie Structurales, UMR 8532 CNRS, Institut n
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Gustave Roussy, 94805 Villejuif, France, and Ecole Normale Supérieure de Cachan, fro
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94235 Cachan, France ttp
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* Département des Sciences de la Vie et de la Terre, Faculté des Sciences, Université w
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Saint Joseph, CST - Mar Roukos, B.P. 1514, Beyrouth, Liban brg/
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Laboratoire de Microchimie et d’Immunologie Moléculaire, Département de Biologie t o
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Clinique, Institut Gustave Roussy, 94805 Villejuif, France ril 3
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¶To whom correspondence should be addressed: Dr Serge Fermandjian, Département de
Biologie et Pharmacologie Structurales (CNRS UMR 8532), PR2, Institut Gustave
Roussy, 39, rue Camille Desmoulins, 94805 Villejuif Cedex, France,
Phone: [phone], Fax: [phone], e-mail: [email protected]

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Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
Summary
The last decade has contributed to our understanding of the three-dimensional
structure of the HIV-1 integrase (IN) and to the description of how the enzyme catalyses the
viral DNA integration into the host DNA. Recognition of the viral DNA termini by IN is
sequence-specific and that of the host DNA does not require particular sequence, although in
physicochemical studies IN fails to discriminate between the two interactions. Here, such
discrimination was allowed thanks to a model system using designed oligonucleotides and
peptides as binding structures. Spectroscopic (circular dichroism, NMR and fluorescence
anisotropy) techniques and biochemical (enzymatic and filter binding) assays clearly
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indicated that the amphipatic helix α4 located at the catalytic domain surface, is responsible ow
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for the specific high affinity binding of the enzyme to viral DNA. Analogues of the α4 ed fro
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peptide having increased helicity and still bearing the biologically relevant lysines 156 and ttp
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159 on the DNA binding face, and oligonucleotides conserving an intact attachment site, are w
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required to achieve high affinity complexes (Kd of 1.5nM). Data corroborate previous in vivo rg
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results obtained with mutated viruses. e
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Keywords: Integrase / LTR DNA / α4-helix / interaction / strategy / host-guest

Abbreviations:
LTR, long terminal repeat; CRE, cyclic AMP responsive element; CD, circular dichroism, IN,
integrase; HIV, human immunodeficiency virus; K , dissociation constant.
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Introduction
Nearly 20 years into the human immunodeficiency virus (HIV)/AIDS epidemic, an
estimated 40 million people world wide are currently living with the virus, and some 20
million people have already died (1, 2). If the spreading of HIV (3) continues at the current
rate, even the most devastating scenario someone can anticipate from the present landscape
will look pale compared with the reality. Even through a small number of vaccines are in
clinical tests, none has lived up its early promise (4). Thus, treatment of AIDS still requires
the development of effective inhibitors of HIV replication (5-7). Those targeted to reverse
transcriptase and protease have demonstrated their efficiency in antiviral therapy. New drugs,
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acting on integrase (IN), would be a valuable complement in this therapy. w
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The IN of HIV is essential for the viral replication (8 - 10). As it has no cellular ed
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counterpart, it is considered as a potential target for anti HIV drugs (7, 11, 12). IN uses a http
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multistep reaction to integrate a linear DNA copy (cDNA) of the retroviral genome in the host w
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cell DNA (8, 9, 13). In the first step, termed 3'-end processing, IN removes two nucleotides rg
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from the 3' terminus of each strand of the viral cDNA. In the second step, the free terminal 3'- u
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hydroxyl groups attack the target-host DNA, and the viral cDNA is integrated by a A
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transesterification reaction into the cell genome (14). , 2
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The HIV-1 enzyme (288 amino acid residues) is organized into an N-terminal domain,
a central catalytic domain, or catalytic core (CC), and a C-terminal domain (15 - 17). Several
crystal structures of the catalytic domain fragment and of two two-domain fragments
(catalytic domain linked either to the C-terminal domain or to the N-terminal domain), have
been already resolved by X-ray crystallography (18 - 26). The N-terminal and C-terminal
domains have also been analyzed in solution by NMR spectroscopy (27, 28). The N-terminal
domain includes a conserved HHCC motif that binds zinc and a HTH motif (27, 29). The C-
terminal domain, although less conserved, contains an SH fold (28, 30). The catalytic domain
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contains five β strands surrounded by six α helices, numbered from one to six, as well as a
highly conserved catalytic D, DX E motif embedded in a protein RNase H fold (19 - 22).
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The three domains, taken separately, form dimers, this being also true for the two two-domain
fragments (18 - 30). The dimer of the catalytic domain is organized around a two fold axis
with an interface between the helix α1 of one unit and the helix α5 of the other one (18),
where as the α4 helix is located at the enzyme surface (figure 1).
The 3'-processing and DNA joining reactions can be performed in in vitro assays (31 -
34) generally using a duplex oligonucleotide of 21 base-pairs (bp) length,
d(GTGT........CAGT) d(ACTG........ACAC). This reproduces either the U3 or the U5 end of
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the viral cDNA, and plays the role of both DNA substrate and DNA target. Mutations n
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performed within the viral cDNA ends have proved that the six outermost residues are needed fro
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for catalysis (35, 36), while photo-crosslinking experiments have shown that the 3'-processing ttp
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site, CA↓GT3'/5'ACTG, binds the catalytic residue E152 as well as residues Q148, K156 and w.jb
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K159 of the enzyme (37 - 40). Remarkably, these aminoacids lye on the same side of the brg/
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amphipathic helix α4. es
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The biological relevance of helix α4 has been confirmed by recent studies carried out pril 3
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with several compounds, considered as authentic inhibitors of integrase (6, 7, 41 - 43). For 19
instance, the 5CITEP, i.e. 1-(5-chloroindol-3-yl)-hydroxy-3-(2H-tetrazol-5-yl)-propenone,
which is a diketo-acid derivative, binds the α4 helix residues Q148, E152, N155, K156 and
K159 (20). Moreover, peptides deriving from helix α4 behave as competitive inhibitors of IN
and this is also true for monospecific antibodies raised against a peptide containing α4 (43 -
47).
Yet, despite this ensemble of data, a clear physicochemical demonstration of the
involvement of helix α4 in viral cDNA recognition has not yet been established, undoubtedly
due to the absence of crystallographic or NMR information on the DNA-protein complexes.
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The failure of the spectroscopic methods to discriminate between the high affinity (specific)
and the low affinity (non-specific) binding modes in experiments using the entire enzyme and
several DNA substrates does not help to fill this gap (48, 49).
Here, we carried out a detailed physicochemical study, combining circular dichroism
(CD), NMR, fluorescence anisotropy and filter binding assay, aiming to decipher the role of
the helix α4 of IN in the cDNA recognition events. The principle of our simplified approach
rested on the design of target oligonucleotides and ligand peptides with optimized binding
structures. We assumed that, in order to achieve good binding interactions, the partners would
have to have stable secondary structures resembling the secondary structures of their parent
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segments within the entire cDNA and IN. Actually, our results show that the flexibility and w
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the poor helicity of the synthetic α4 peptide, reproducing the helix α4 sequence, prevent the ed fro
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formation of a high affinity complex with the oligonucleotide target. In contrast, the α4 ttp
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peptide analogue K156, which presents a higher helix content, generated by appropriate w
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helicogenic mutations in the sequence, and still bears the residues K156 and K159, critical for brg/
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the cDNA recognition (37 - 40), expresses a high affinity binding (Kd≈2nM). For the latter to uest o
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take place, it further requires oligonucleotide targets with (i) a stable double-helix structure p
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under the low concentration conditions used in fluorescence anisotropy experiments obtained 01
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by using hairpin folds (monomolecular structures) instead of linear duplexes (dimolecular
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structures) and (ii) an intact attachment sequence (att site): AGC AGT3'/5'ACTGCT .
All in all, our simplified system demonstrates the utility of employing selected protein
and DNA fragments to decipher the mechanisms of interaction of IN with its viral DNA
target. Three criteria have proved to be imperative for the occurrence of primary (specific) or
high affinity binding: (i) an optimized helical content of the peptide involved in the
recognition, this is necessary for a minimal loss of free binding energy during the adjustment
to the DNA partner; (ii) conservation of the basic residues K156 and K159 in the optimized
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peptide structure; and (iii) good stability of the DNA target with integrity of the IN binding
locus att.
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Experimental procedures
Structure Predictions
α helix secondary structure predictions were carried out using the AGADIR and GOR
computer programs (50, 51). The first method considers short range interactions between
residues and provides helicity per residue of peptides lacking tertiary interactions in solution,
selecting the pH and temperature values. The second method provides structure predictions
more suitable for peptide segments fixed in a tertiary structure i.e. within the protein context.
Thus, from the comparison of the AGADIR and GOR predictions one can learn on the
influence of the protein surrounding on the helix α4 stability. The AGADIR predictions can
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also be used for the choice of the mutations improving the helix content in the α4 peptide. wn
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Such mutations are needed to generate a peptide secondary structure resembling as much as fro
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possible the helix α4 folding in the protein tertiary structure. Maximization of the helical ttp
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structure is expected to overcome the entropy problems linked to the otherwise large .jb
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conformational freedom of the peptide in the DNA binding. b/
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Peptide samples pril 3
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The peptides (figure 2) were synthesized according to the Fmoc procedure on an 01
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Applied Biosystems model 432A automatic solid phase synthesizer and were purified by
reverse-phase HPLC on an Aquapore column using a linear gradient from 0 to 100%
acetonitrile, 0.1% trifluoroacetic acid in water. The molecular mass and purity of each peptide
was confirmed by Electrospray Ionisation Mass Spectrometry (ESIMS) on a Platform-
quadruple instrument (VG Biotech). Peptide concentrations were determined from the UV
signal of Tyr and Trp purposely added at the C-terminus, using a molar absorption coefficient
at 280 nm equal to 1197 M-1.cm-1 (K156 and E156) and equal to 5600 M-1.cm-1 (E159):

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Protein samples
Double mutant IN (dmIN). The plasmid encoding dmIN (IN1-288/F185K/C280S) was
kindly provided by R. Craigie (NIH). The enzyme was expressed in Escherichia coli strain
BL21(DE3) as previously described (52). Purification was performed at 4°C under native
conditions without detergent on a His-trap column (Pharmacia) using a Zn-chelate. Cells
expressing dmIN from 250 ml of culture media were resuspended in 10 ml ice-cold lysis
buffer (10 mM imidazole in Buffer A: 20 mM Tris-HCl pH 7.5, 1M NaCl). The cell
suspension was treated with 1 mg/ml lysozyme for 30 min at 4°C and bacteria were disrupted
using a french press. Lysed cells were centrifuged 30 min at 10000g and the supernatant was
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filtered (0.22 µm) and loaded on the column equilibrated with lysis buffer. The column was w
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washed with Buffer A plus 10 to 70 mM imidazole and the protein was eluted with Buffer A ed
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plus 400 to 500 mM imidazole. Fractions containing integrase were pooled and dialysed http
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overnight against storage buffer (20 mM Tris-HCl pH 7.5, 0.8 M NaCl, 2 mM DTT, 50 µM w
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ZnCl2 and 10% (v/v) glycerol). Concentrations of purified dmIN, were determined with the rg
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Bradford kit (Promega) and by UV absorption using a calculated extinction coefficient of ue
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46542 M-1.cm-1 at 280 nm, based on the amino acid composition. Finally, aliquots of purified Ap
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enzyme were stored at –80°C. , 20
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DNA samples
The target oligonucleotides were purchased from CYBERGENE ESGS.
For fluorescence anisotropy experiments, the fluorescein group grafted to
oligonucleotides was used as a reporter (figure 3). Hairpin structures were preferred to linear
duplexes for their higher stability at the low concentrations dictated by fluorescence
spectroscopy. They contain a three-thymine loop and a 17 bp stem reproducing or deriving
from the outermost part of the U5 LTR of HIV-1 cDNA. The fluorescein group was
introduced either at the 5' end or on the central thymine residue, which allows us to assess the
impact of the position of the bulky fluorescein on the peptide - DNA interactions. A hexadeca
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oligonucleotide reproducing the CRE sequence (cAMP responsive element) was also used to w
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determine the non-specific interactions with the peptides. ed
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For the autointegration assays, we used U5b: 5’-GTGTGGAAAATCTCTAGCAGT, and http
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U5a: 3’-CACACCTTTTAGAGATCGTCA and LTR34: 5’- w
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ACTGCTAGAGATTTTCCTTTGGAAAATCTCTAGCAGT (hairpin) as substrate/target rg
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DNAs. 15pmol of U5b and LTR34 were radiolabeled using T4 polynucleotide kinase u
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(Biolabs) and 50 µCi of [γ-32P] ATP (3000 Ci/mmol) (Amersham Pharmacia Biotech). The T4 A
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kinase was heat inactivated. Then 20 pmol of U5a and NaCl at a final concentration of 0.1 M , 20
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were added to U5b. Both samples, LTR34 and U5a/U5b, were heated to 95°C for 3 mn, and
the DNA was then annealed by slow cooling in the case of U5a/U5b and quickly cooled in the
case of LTR34. Unincorporated nucleotides were removed using a Sephadex G-10 column
(Pharmacia).

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