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References listed in PDB file
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Key reference
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Title
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Crystal structures of clinically relevant lys103asn/tyr181cys double mutant HIV-1 reverse transcriptase in complexes with ATP and non-Nucleoside inhibitor hby 097.
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Authors
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K.Das,
S.G.Sarafianos,
A.D.Clark,
P.L.Boyer,
S.H.Hughes,
E.Arnold.
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Ref.
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J Mol Biol, 2007,
365,
77-89.
[DOI no: ]
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PubMed id
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Abstract
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Lys103Asn and Tyr181Cys are the two mutations frequently observed in patients
exposed to various non-nucleoside reverse transcriptase inhibitor drugs
(NNRTIs). Human immunodeficiency virus (HIV) strains containing both reverse
transcriptase (RT) mutations are resistant to all of the approved NNRTI drugs.
We have determined crystal structures of Lys103Asn/Tyr181Cys mutant HIV-1 RT
with and without a bound non-nucleoside inhibitor (HBY 097,
(S)-4-isopropoxycarbonyl-6-methoxy-3-(methylthio-methyl)-3,4-dihydroquinoxalin-2(1H)-thione)
at 3.0 A and 2.5 A resolution, respectively. The structure of the double mutant
RT/HBY 097 complex shows a rearrangement of the isopropoxycarbonyl group of HBY
097 compared to its binding with wild-type RT. HBY 097 makes a hydrogen bond
with the thiol group of Cys181 that helps the drug retain potency against the
Tyr181Cys mutation. The structure of the unliganded double mutant HIV-1 RT
showed that Lys103Asn mutation facilitates coordination of a sodium ion with
Lys101 O, Asn103 N and O(delta1), Tyr188 O(eta), and two water molecules. The
formation of the binding pocket requires the removal of the sodium ion. Although
the RT alone and the RT/HBY 097 complex were crystallized in the presence of
ATP, only the RT has an ATP coordinated with two Mn(2+) at the polymerase active
site. The metal coordination mimics a reaction intermediate state in which
complete octahedral coordination was observed for both metal ions. Asp186
coordinates at an axial position whereas the carboxylates of Asp110 and Asp185
are in the planes of coordination of both metal ions. The structures provide
evidence that NNRTIs restrict the flexibility of the YMDD loop and prevent the
catalytic aspartate residues from adopting their metal-binding conformations.
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Figure 1.
Figure 1. Effects of the two mutations (Lys103Asn and
Tyr181Cys) on the structure of unliganded HIV-1 RT. (a) A stereo
view of the NNIBP region of the double mutant RT/ATP structure.
The composite simulated annealing omit map (2|F[o]|–|F[c]|)
electron density (cyan) contoured at 1.2σ defines the
coordination of a Na ion at the NNIBP region; OW1 and OW2 are
two water molecules. (b) The NNIBP region of the double mutant
(Lys103Asn/Tyr181Cys) HIV-1 RT. The mutated amino acids have
altered interactions with the surrounding amino acids. (c) The
NNIBP region of the wild type unliganded HIV-1 RT structure.^13
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Figure 2.
Figure 2. Binding mode of HBY 097 to the
Lys103Asn/Tyr181Cys double mutant RT. (a) Stereo view of the
(2|F[o]|–|F[c]|) electron density (contoured at 1.2σ)
covering HBY 097 (cyan) and Cys181 (magenta). The dotted line
represents the hydrogen bond between the thiol group of Cys181
and HBY 097. Electrostatic potential surface^62 showing the
NNIBP region of (b) the double mutant RT/HBY 097 and (c)
wild-type RT/HBY 097^13 structures.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2007,
365,
77-89)
copyright 2007.
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Secondary reference #1
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Title
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Structure of unliganded HIV-1 reverse transcriptase at 2.7 a resolution: implications of conformational changes for polymerization and inhibition mechanisms.
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Authors
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Y.Hsiou,
J.Ding,
K.Das,
A.D.Clark,
S.H.Hughes,
E.Arnold.
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Ref.
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Structure, 1996,
4,
853-860.
[DOI no: ]
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PubMed id
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Figure 3.
Figure 3. Superposition of (a) unliganded RT and RT–DNA–Fab
complex and (b) unliganded RT and RT–α-APA
(α-anilinophenylacetamide) complex based on 89 Cα atoms in the
p66 palm subdomain, including the β6–β10–β9 region. The
unliganded RT is shown in red, RT–α-APA in blue, and
RT–DNA–Fab in green. A comparison of the two superpositions
reveals that NNRTI binding appears to be accompanied by a
long-range distortion that is coupled with a hinge motion
(indicated by curved arrows) between the β6–β10–β9 and
β12–β13–β14 sheets at the p66 palm subdomain (within the
circle). The different positions of the thumb in different HIV-1
RT structures supports the idea that this subdomain could play
a role during polymerization. Figure 3. Superposition of (a)
unliganded RT and RT–DNA–Fab complex and (b) unliganded RT
and RT–α-APA (α-anilinophenylacetamide) complex based on 89
Cα atoms in the p66 palm subdomain, including the
β6–β10–β9 region. The unliganded RT is shown in red,
RT–α-APA in blue, and RT–DNA–Fab in green. A comparison
of the two superpositions reveals that NNRTI binding appears to
be accompanied by a long-range distortion that is coupled with a
hinge motion (indicated by curved arrows) between the
β6–β10–β9 and β12–β13–β14 sheets at the p66 palm
subdomain (within the circle). The different positions of the
thumb in different HIV-1 RT structures supports the idea that
this subdomain could play a role during polymerization.
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Figure 4.
Figure 4. Stereoview of a portion of a (2mF[obs]–F[calc])
difference Fourier map at the p66 connection subdomain, at 2.7
å resolution. The phases were computed from the current
atomic model and the map is contoured at 1.4σ. The side chain
and the carboxyl groups are well defined in the electron density
map. Figure 4. Stereoview of a portion of a
(2mF[obs]–F[calc]) difference Fourier map at the p66
connection subdomain, at 2.7 å resolution. The phases were
computed from the current atomic model and the map is contoured
at 1.4σ. The side chain and the carboxyl groups are well
defined in the electron density map.
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The above figures are
reproduced from the cited reference
with permission from Cell Press
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Secondary reference #2
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Title
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Crystal structures of 8-Cl and 9-Cl tibo complexed with wild-Type HIV-1 rt and 8-Cl tibo complexed with the tyr181cys HIV-1 rt drug-Resistant mutant.
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Authors
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K.Das,
J.Ding,
Y.Hsiou,
A.D.Clark,
H.Moereels,
L.Koymans,
K.Andries,
R.Pauwels,
P.A.Janssen,
P.L.Boyer,
P.Clark,
R.H.Smith,
M.B.Kroeger smith,
C.J.Michejda,
S.H.Hughes,
E.Arnold.
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Ref.
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J Mol Biol, 1996,
264,
1085-1100.
[DOI no: ]
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PubMed id
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Figure 1.
Figure 1. Chemical structures
with the numbering scheme used
and distances (E3.6 Å ) between
atoms of the TIBO inhibitor and of
the amino acid residues of the
NNIBP for: (a) 8-Cl TIBO (R86183,
tivirapine) complexed with wild-
type HIV-1 RT; (b) 8-Cl TIBO
complexed with Tyr181Cys mutant
HIV-1 RT; and (c) 9-Cl TIBO
(R82913) complexed with wild-type
HIV-1 RT. An NNIBP residue is
shown only if atoms of that residue
are E3.6 Å from an inhibitor atom
with the exception of Cys181 in (b).
The wings I and II portions of the
inhibitors in the butterfly-like anal-
ogy for NNRTIs (Ding et al., 1995a)
are indicated here and in sub-
sequent Figures by Roman nu-
merals I and II. The dotted line in (a)
indicates the subdivision of atoms
between wings I and II.
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Figure 5.
Figure 5. A stereoview of the superposition (based on the C
a
atoms of the b6-b10-b9 sheet) of the HIV-1 RT/DNA/Fab
complex structure (in gray) (Jacobo-Molina et al., 1993) on the HIV-1 RT/9-Cl TIBO complex structure (in cyan) in the
regions near the NNIBP and the polymerase active site showing the disposition of the b12-b13-b14 sheet containing
the primer grip. Bound 9-Cl TIBO in the HIV-1 RT/9-Cl TIBO complex is shown in gold and the two 3'-terminal
nucleotides 17 and 18 of the primer strand in the HIV-1 RT/DNA/Fab complex are shown with a yellow ball-and-stick
model. The broken line represents interactions between the primer grip and the primer terminal phosphate in the HIV-1
RT/DNA/Fab complex and the arrow indicates the movement of the primer grip that accompanies NNRTI binding.
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The above figures are
reproduced from the cited reference
with permission from Elsevier
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Secondary reference #3
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Title
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Structures of tyr188leu mutant and wild-Type HIV-1 reverse transcriptase complexed with the non-Nucleoside inhibitor hby 097: inhibitor flexibility is a useful design feature for reducing drug resistance.
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Authors
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Y.Hsiou,
K.Das,
J.Ding,
A.D.Clark,
J.P.Kleim,
M.Rösner,
I.Winkler,
G.Riess,
S.H.Hughes,
E.Arnold.
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Ref.
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J Mol Biol, 1998,
284,
313-323.
[DOI no: ]
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PubMed id
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Figure 1.
Figure 1. Diagram of HBY 097 (a quinoxaline derivative)
contacts with protein residues around the NNIBP in both (a)
wild-type HIV-1 RT/HBY 097 and (b) Tyr188Leu mutant HIV-1 RT/HBY
097 complexes. Distances (  slant
3.6 Å) between protein and inhibitor atoms are indicated.
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Figure 4.
Figure 4. (a) Stereoview of a difference Fourier m(F[obs] -
F[calc]) map showing the electron density of HBY 097 in the
wild-type HIV-1 RT/HBY 097 complex. The map is calculated at 3.1
Å resolution with 2s contours (in magenta). The phases
were computed from the protein model prior to inclusion of the
inhibitor. The green density corresponds to the difference
Fourier map (3.7 Å resolution) between HBY 097 and S-0483
complexed with wild-type HIV-1 RT (bromine in S-0483 replaces
the methoxy group of HBY 097), contoured at the 5s level,
showing the position of the bromine atom and confirming the
orientation and placement of the inhibitor. Difference Fourier
2mF[obs] - F[calc] map at 3.3 Å resolution, contoured at
1.2s, (b) of the Tyr188Leu mutant HIV-1 RT/HBY 097 complex at
the NNIBP region in p66 showing the absence of any density for
the side-chain of Leu188; clear density for HBY 097 is seen in
the binding pocket; and of a (c) similar region in the p51
subunit, showing clear electron density for the side-chain at
Leu188.
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The above figures are
reproduced from the cited reference
with permission from Elsevier
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Secondary reference #4
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Title
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The lys103asn mutation of HIV-1 rt: a novel mechanism of drug resistance.
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Authors
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Y.Hsiou,
J.Ding,
K.Das,
A.D.Clark,
P.L.Boyer,
P.Lewi,
P.A.Janssen,
J.P.Kleim,
M.Rösner,
S.H.Hughes,
E.Arnold.
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Ref.
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J Mol Biol, 2001,
309,
437-445.
[DOI no: ]
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PubMed id
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Figure 3.
Figure 3. Stereoview of a SIGMAA weighted 2mFo
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DFc difference Fourier map showing the electron density in
the NNIBP region of p66 in the unliganded Lys103Asn mutant HIV-1 RT structure. The phases were computed from
the final model at 2.7 Å resolution and the map was contoured at 2s. Selected hydrogen-bonding interactions are
indicated with broken lines.
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Figure 4.
Figure 4. Energy diagram for the binding of an
NNRTI to wild-type and Lys103Asn mutant RT. The
Lys103Asn mutant RT with the additional hydrogen
bonding network in the NNIBP region is assumed to be
more stable than wild-type HIV-1 RT. The relative stab-
ility of wild-type and Lys103Asn mutant HIV-1 RT com-
plexes with NNRTIs will depend on the inhibitor.
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The above figures are
reproduced from the cited reference
with permission from Elsevier
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Secondary reference #5
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Title
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Roles of conformational and positional adaptability in structure-Based design of tmc125-R165335 (etravirine) and related non-Nucleoside reverse transcriptase inhibitors that are highly potent and effective against wild-Type and drug-Resistant HIV-1 variants.
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Authors
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K.Das,
A.D.Clark,
P.J.Lewi,
J.Heeres,
M.R.De jonge,
L.M.Koymans,
H.M.Vinkers,
F.Daeyaert,
D.W.Ludovici,
M.J.Kukla,
B.De corte,
R.W.Kavash,
C.Y.Ho,
H.Ye,
M.A.Lichtenstein,
K.Andries,
R.Pauwels,
M.P.De béthune,
P.L.Boyer,
P.Clark,
S.H.Hughes,
P.A.Janssen,
E.Arnold.
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Ref.
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J Med Chem, 2004,
47,
2550-2560.
[DOI no: ]
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PubMed id
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