Báo cáo khoa học: Structural insights into mechanisms of non-nucleoside drug resistance for HIV-1 reverse transcriptases mutated at codons 101 or 138 pot

Structural insights into mechanisms of non-nucleoside
drug resistance for HIV-1 reverse transcriptases mutated
at codons 101 or 138
Jingshan Ren
1
, Charles E. Nichols
1
, Anna Stamp
1
, Phillip P. Chamberlain
1
, Robert Ferris
2
,
Kurt L. Weaver
2
, Steven A. Short
2
and David K. Stammers
1
1 Division of Structural Biology, The Wellcome Trust Centre for Human Genetics, Henry Wellcome Building for Genomic Medicine,
University of Oxford, UK
2 Glaxo Smith Kline Inc., Research Triangle Park, NC, USA
The emergence of resistant viruses resulting from drug
treatment of HIV-infected patients poses one of the
most significant problems in countering AIDS in West-
ern countries [1]. Two virus specific proteins, reverse
transcriptase (RT) and protease, have been the main
targets for the development of anti-HIV drugs used
in multidrug combination therapy regimens [2]. HIV-1
RT contains two distinct inhibitor binding sites for

Keywords
drug resistance; HIV-1 reverse transcriptase
mutants; Lys101Glu, Glu138Lys; non-
nucleoside inhibitors; X-ray crystallography
Correspondence
D.K. Stammers, Division of Structural
Biology, The Wellcome Trust Centre for
Human Genetics, University of Oxford,
Roosevelt Drive, Oxford OX3 7BN, UK
Fax: +44 1865 287 547
Tel: +44 1865 287 565
E-mail:
(Received 23 March 2006, revised 20 June
2006, accepted 22 June 2006)
doi:10.1111/j.1742-4658.2006.05392.x
Lys101Glu is a drug resistance mutation in reverse transcriptase clinically
observed in HIV-1 from infected patients treated with the non-nucleoside
inhibitor (NNRTI) drugs nevirapine and efavirenz. In contrast to many
NNRTI resistance mutations, Lys101(p66 subunit) is positioned at the sur-
face of the NNRTI pocket where it interacts across the reverse transcrip-
tase (RT) subunit interface with Glu138(p51 subunit). However, nevirapine
contacts Lys101 and Glu138 only indirectly, via water molecules, thus the
structural basis of drug resistance induced by Lys101Glu is unclear. We
have determined crystal structures of RT(Glu138Lys) and RT(Lys101Glu)
in complexes with nevirapine to 2.5 A
˚
, allowing the determination of water
structure within the NNRTI-binding pocket, essential for an understanding
of nevirapine binding. Both RT(Glu138Lys) and RT(Lys101Glu) have
remarkably similar protein conformations to wild-type RT, except for sig-

nificant movement of the mutated side-chains away from the NNRTI
pocket induced by charge inversion. There are also small shifts in the posi-
tion of nevirapine for both mutant structures which may influence ring
stacking interactions with Tyr181. However, the reduction in hydrogen
bonds in the drug-water-side-chain network resulting from the mutated
side-chain movement appears to be the most significant contribution to
nevirapine resistance for RT(Lys101Glu). The movement of Glu101 away
from the NNRTI pocket can also explain the resistance of RT(Lys101Glu)
to efavirenz but in this case is due to a loss of side-chain contacts with the
drug. RT(Lys101Glu) is thus a distinctive NNRTI resistance mutant in
that it can give rise to both direct and indirect mechanisms of drug resist-
ance, which are inhibitor-dependent.
Abbreviations
NRTI, nucleoside analogue inhibitors of RT; NNRTI, non-nucleoside reverse transcriptase inhibitor; NtRTI, nucleotide analogue; PETT,
phenethylthiazolylthiourea; RT, reverse transcriptase; TSAO, (2¢,5¢-bis-O-(tert-butyldimethylsilyl)-b-d-ribofuranosyl]-3¢-spiro-5¢¢-(4¢¢-amino-
1¢¢,2¢¢-oxathiole-2¢¢,2¢¢-dioxide).
3850 FEBS Journal 273 (2006) 3850–3860 ª 2006 The Authors Journal compilation ª 2006 FEBS
different classes of drugs within the larger subunit of
the p66 ⁄ p51 heterodimer [3,4]. Nucleoside analogue
inhibitors of RT (NRTIs), such as zidovudine, lamivu-
dine, stavudine and the nucleotide analogue (NtRTI)
tenofovir, bind, respectively, as activated tri- or di-
phosphate forms at the RT polymerase active site.
NRTIs and NtRTIs, as well as competing with cognate
nucleotide substrates, are also incorporated into the
primer strand causing termination of the growing
DNA chain [5]. Non-nucleoside inhibitors (NNRTIs)
form a second class of compounds that target HIV-1
RT [6]. NNRTIs bind at an allosteric site about 10 A
˚

from the polymerase active site. The structural mech-
anism of NNRTI inhibition involves distortion of the
catalytically essential triad of aspartic acid residues
[7,8]. The non-nucleoside site, although almost entirely
contained within the p66 subunit, also has Glu138
from the p51 subunit located at the edge of the inhib-
itor pocket [3,4]. First-generation NNRTI drugs such
as nevirapine (Scheme 1) and delavirdine generally
show significant reduction in potency in the presence
of a wide range of single point mutations [9]. Second
generation compounds, including efavirenz, have
greater resilience to many such mutations [10,11].
Whilst the majority of the mutation sites encoding
resistance to NRTIs are distal to the dNTP site within
RT [12,13], those which result in NNRTI drug resist-
ance are generally proximal to the inhibitor binding
pocket [3,4].
Many of the most commonly observed NNRTI
resistance mutations are located at hydrophobic resi-
dues at the centre of the drug binding pocket (e.g.
Leu100, Val106, Val108, Tyr181 and Tyr188). How-
ever, charged residues positioned towards the surface
of the drug pocket are also known sites of drug
resistance mutations. Indeed, Lys103Asn is the muta-
tion most frequently observed in clinical use of
NNRTIs. Other NNRTI resistance mutations at sur-
face residues in RT include Lys101 and Glu138 (the
latter residue from the p51 subunit). Mutations at
residues 101 and 138 that give resistance to NNRTIs
usually involve a change in the sign of the side-chain

charge, e.g. Lys101Glu or Glu138Lys ⁄ Arg. Pyridinone
NNRTIs can select for the Lys101Glu mutation in
HIV-1 RT, giving approximately eight-fold resistance
to compounds in this series [14]. The Lys101Glu
mutation in RT also gives eight-fold resistance to
nevirapine, based on the mean of four published
values [15–18]. Significantly, Lys101Glu in RT is
observed in the clinic for drug regimens which include
nevirapine [19,20] or efavirenz [21] as the NNRTI
component. The Lys101Glu mutation in RT is also
selected by passaging HIV in tissue culture in the
presence of carboxanilide NNRTIs such as UC-38
[22], giving greater than 100-fold resistance [23]. In
the crystal structure with wild-type HIV-1 RT, UC-38
has a van der Waals contact with the side-chain of
Lys101 [24]. Consistent with the reports of the selec-
tion of Lys101Glu in clinical use of efavirenz, the
crystal structure of RT with this NNRTI shows a
contact of the inhibitor with the CD atom of Lys101
[25]. In the case of nevirapine, however, there are
only indirect contacts of Lys101 in the complex with
wild-type RT, via a series of three water molecules
that link the drug, the side-chain of Glu138 and the
main-chain carbonyl of Lys101 [4]. The molecular
mechanism of drug resistance for nevirapine induced
by Lys101Glu mutant is thus unclear, although an
indirect mechanism is present.
The RT(Glu138Lys) mutation in HIV is selected
in tissue culture by inhibitors of the TSAO (2¢ ,5¢-bis-
O-(tert-butyldimethylsilyl)-b-d-ribofuranos yl]-3¢-spiro-

5¢¢-(4¢¢-amino-1¢¢,2¢¢-oxathiole-2¢¢,2¢¢-dioxide) series [26].
TSAO compounds have been shown to be capable of
disrupting the p66 ⁄ p51 heterodimer [27], indeed model-
ling studies suggest that certain TSAO NNRTIs may
bind at a novel site between the subunits of the RT
heterodimer [28]. It is proposed that mutation of
Glu138Lys causes loss of a key interaction of the gluta-
mic acid carboxyl group to an amino group of TSAO
inhibitors. Mutations in RT at codon 138 (to Lys ⁄ Arg)
have been shown to give cross resistance to a number
of NNRTIs including members of the PETT series [29]
as well as to emivirine [17]. In contrast, RT(Glu138Lys)
is reported as having minimal cross resistance to nevi-
rapine [18,30]. PETT-2 (Scheme 1) forms a van der
Waals contact with the carboxyl group of Glu138 in
the complex with wild-type RT [31].
To date, crystal structure determinations of
NNRTI resistant HIV-1 RTs have mainly focussed
on mutations at positions 103, 181 and 188 [25,32–
36]. More recently structures of RT with drug resist-
ance mutations Leu100Ile, Val106Ala and Val108Ile
have been reported, which indicate an indirect com-
ponent in the mechanism of resistance via modulation
N
N
O
N
N
Nevirapine
N

N
H
N
H
N
Cl
O
S
F
PETT-2
H
1
10
Scheme 1. Structures of nevirapine and PETT-2 are shown.
J. Ren et al. HIV-1 RT drug resistance mutations at 101 and 138
FEBS Journal 273 (2006) 3850–3860 ª 2006 The Authors Journal compilation ª 2006 FEBS 3851
of interactions with the key aromatic side-chains of
Tyr181 and Tyr188 [37]. For RT(Lys103Asn) it has
been shown that the second generation NNRTI efavi-
renz can bind to the enzyme in both a wild-type con-
formation as well as with Tyr181 in a rearranged
position [25,36]. The side-chain of Asn103 can form a
hydrogen bond to the Tyr188 hydroxyl thereby stabil-
izing the unliganded RT structure [7,35]. In work
reported here, crystal structures of HIV-1 RTs either
containing the mutation Lys101Glu or Glu138Lys in
complexes with nevirapine and PETT-2 have been
determined in order to investigate structural effects
of such mutants on the drug binding pocket and
how these might relate to resistance mechanisms.

RT(Glu138Lys) and RT(Lys101Glu) mutants are logi-
cal to study as a pair as they are both charged sur-
face residues interacting across the p66 ⁄ p51 interface.
The availability of a range of structures of NNRTI-
resistant RTs drugs will provide greater understanding
of the molecular basis of drug resistance mechanisms.
Such data will contribute to the design of novel inhibi-
tors, which are still needed for containing the effects of
HIV infection.
Results
Overall RT structure
Details of the X-ray data collection and the
refinement statistics are shown in Table 1. Of the
wide range of NNRTIs tested for co-crystallization
with RT(Glu138Lys) and RT(Lys101Glu), three
complexes gave crystals suitable for structure
determination: RT(Lys101Glu)-nevirapine (to 2.5-A
˚
resolution), RT(Glu138Lys)-nevirapine (2.5 A
˚
) and
RT(Glu138Lys)-PETT2 (3.0 A
˚
). The relatively high
resolution of the mutant RT-nevirapine structures
allowed details of the water structure associated with
the protein relevant to NNRTI binding to be deter-
mined. Water molecules are important for the interac-
tions of nevirapine with RT, particularly via Lys101
and Glu138, as there are no direct contacts of the

inhibitor with these side-chains. Fo-Fc omit maps
show clear electron density for the bound NNRTI,
some water molecules, as well as for the mutated side-
chains (Fig. 1) in each case. Side-chain electron density
relevant to NNRTI drug resistance is also shown in
Table 1. Statistics for crystallographic structure determinations.
Data collection details
Data set K101E-nevirapine E138K-nevirapine E138K-PETT-2
Data collection site PF BL-6 A APS SBC-2 SRS PX14.2
Detector Fuji BAS III CCD SBC CCD ADSC-Q4
Wavelength (A
˚
) 1.000 0.82657 0.979
Unit cell dimensions (a,b,c in A
˚
)
a
138.9, 114.9, 65.6 139.6, 115.0, 65.6 138.8, 114.8, 65.9
Resolution range (A
˚
) 30.0–2.5 30.0–2.5 30.0–3.0
Observations 149821 251696 57571
Unique reflections 35057 37104 19095
Completeness (%) 94.4 99.5 88.0
Average I ⁄ r(I) 9.8 12.6 11.8
R
merge
b
0.100 0.089 0.083
Outer resolution shell

Resolution range (A
˚
) 2.59–2.50 2.59–2.50 3.11–3.00
Unique reflections 3371 3627 1537
Completeness (%) 92.5 99.5 72.6
Average I ⁄ r(I) 1.0 1.1 1.4
Refinement statistics:
Resolution range (A
˚
) 30.0–2.5 30.0–2.5 20.0–3.0
Number of reflections(working ⁄ test) 33264 ⁄ 1740 35199 ⁄ 1856 18153 ⁄ 929
R-factor
c
(R
work
⁄ R
free
) 0.198 ⁄ 0.270 0.216 ⁄ 0.288 0.226 ⁄ 0.277
R-factor
c
(all data) 0.194 0.203 0.226
Number of atoms (protein ⁄ inhibitor ⁄ water) 7570 ⁄ 20 ⁄ 80 7614 ⁄ 20 ⁄ 79 7554 ⁄ 23 ⁄ –
Rms bond length deviation (A
˚
) 0.008 0.008 0.009
Rms bond angle deviation (°) 1.4 1.4 1.5
Mean B-factor (A
˚
2
)

d
59 ⁄ 65 ⁄ 37 ⁄ 44 62 ⁄ 68 ⁄ 39 ⁄ 48 73 ⁄ 81 ⁄ 60 ⁄ –
Rms backbone B-factor deviation (A
˚
2
) 4.4 4.2 3.4
a
All crystals belong to space group P2
1
2
1
2
1
[40,41];
b
R
merge
¼ S|I –<I > | ⁄S < I > ;
c
R factor ¼ S|F
o
-F
c
| ⁄SF
o
;
d
mean B factor for main-chain,
side-chain, inhibitor and water molecules.
HIV-1 RT drug resistance mutations at 101 and 138 J. Ren et al.

3852 FEBS Journal 273 (2006) 3850–3860 ª 2006 The Authors Journal compilation ª 2006 FEBS
Fig. 1, i.e. for Lys101Glu in the p66 subunit (Fig. 1A)
and Glu138Lys in the p51 subunit (Fig. 1B,C).
Comparison of wild-type and mutant RT structures
Lys101Glu
In the crystal structure of RT(Lys101Glu) in complex
with nevirapine, electron density for the mutant gluta-
mic acid side-chain is well defined (Fig. 1A). The struc-
ture of the NNRTI pocket is overall remarkably
similar for mutant and wild-type, with the exception of
the mutated side-chain and adjacent interacting resi-
due. There is a significant movement of Glu101 relat-
ive to the position of the wild-type Lys101, such that
the side-chain carboxyl group points away from the
NNRTI site (distance of the carboxyl group to the
equivalent wild-type lysine side-chain amino group of
3.4 A
˚
) (Fig. 2A). There is also a smaller displacement
of the Glu138 side-chain with a shift of 0.5 A
˚
relative
to wild-type. The result of these changes is the loss of
the salt bridge that links the protonated amino group
of Lys101 in the p66 subunit to the negatively charged
carboxyl of the Glu138 side-chain from the p51 sub-
unit in the wild-type RT nevirapine complex [4].
Accompanying the side-chain movement related to the
Lys101Glu mutation there is also a shift in the posi-
tion of nevirapine, with an average atom displacement

of 0.3 A
˚
and a maximum movement of 0.5 A
˚
. The
side-chains of Tyr181 and Tyr188 are however, main-
tained in a wild-type conformation. In spite of the
observed rearrangements of inhibitor and the side-
chains of residues 101 and 138, the three water mole-
cules that link nevirapine to Glu138 in the wild-type
complex are retained in the mutant structure albeit
with shifts in position in the same direction as the
nevirapine, i.e. away from the binding pocket.
Glu138Lys
The structures of HIV-1 RT(Glu138Lys) in complexes
with nevirapine and PETT-2 complex have well defined
side-chain density for Lys138 (p51) (Fig. 1B,C). As
with the Lys101Glu mutant, the overall structure of
the NNRTI pocket in RT(Glu138Lys) closely matches
that of the equivalent nevirapine complex with wild-
type RT, with the exception of the mutated side-chain
itself, which together with Lys101 undergoes large
positional changes. Lys138 moves away from the
NNRTI pocket in the complex with nevirapine as indi-
cated by the shift in position of the CD atom of 2.8 A
˚
(Fig. 2B), whilst Lys101 becomes much less ordered
than in the wild-type structure. The best fit of the side-
chain to electron density indicates a rotation of close
to 150° about the Lys101 CA–CB bond, positioning it

away from the NNRTI pocket (Fig. 2B) and resulting
in the loss of interaction of residue 138 in p51 with
Lys101 in the p66 subunit. There is also a shift in the
position of nevirapine outwards from the binding
pocket with an average atom displacement of 0.4 A
˚
and a maximum displacement of 0.6 A
˚
. The three
water molecules that bridge between Glu138 and
Fig. 1. Simulated annealing omit electron density maps contoured
at 3.5r showing bound inhibitors, waters and mutated residues
within one of the HIV-1 RT subunits as indicated. (A) Lys101Glu in
p66 and nevirapine. (B) Glu138Lys in p51 and nevirapine. (C)
Glu138Lys in p51 and PETT-2.
J. Ren et al. HIV-1 RT drug resistance mutations at 101 and 138
FEBS Journal 273 (2006) 3850–3860 ª 2006 The Authors Journal compilation ª 2006 FEBS 3853
nevirapine in the wild-type complex remain in the
Glu138Lys mutant but are displaced in position and
the network of hydrogen bonds is altered. The remain-
ing side-chains within the mutant NNRTI binding site
are essentially positioned the same as for the wild-type
enzyme.
Fig. 2. Stereo-diagrams comparing the NNRTI binding sites of wild-type and mutant RTs for the following complexes: (A) Lys101Glu and
nevirapine, (B) Glu138Lys and nevirapine, and (C) Glu138Lys and PETT-2. The thinner and thicker bonds show the CA backbone and side-
chains with wild-type RT coloured orange and the mutant RTs coloured blue, respectively. Inhibitors and water molecules are shown in ball-
and-stick representation and coloured red for wild-type RT and cyan for mutant RTs. For clarity, the side-chains at the site of mutation are
shown in magenta for wild-type and green for the mutants. Hydrogen bonds linking drug to protein and drug to water molecules are marked
in dashed lines, coloured yellow for wild-type and blue for mutant RTs.
HIV-1 RT drug resistance mutations at 101 and 138 J. Ren et al.

3854 FEBS Journal 273 (2006) 3850–3860 ª 2006 The Authors Journal compilation ª 2006 FEBS
Comparison of RT wild-type and RT(Glu138Lys)
complexes with PETT-2 shows that there appears to
be a perturbation in the positions of a number of side-
chains, with the largest changes at the mutation site.
The wild-type Glu138 and mutant Lys138 residues
overlap as far as the CB atoms, the remainder of the
side chain of Lys138 is swung away from the NNRTI
pocket by a rotation of 170° about the CA–CB bond
giving a difference in position of amino and carboxyl
groups of 6.3 A
˚
(Fig. 2C). The difference in conforma-
tion of Lys138 relative to Glu138 results in the loss of
the van der Waals contact between the carboxyl and
the pyridine nitrogen ring of PETT-2 in the wild-type
complex. Additionally, there is a movement in the
Lys101 side chain which involves a rotation about the
CD–CE bond resulting in the amino group moving
3.4 A
˚
relative to the wild-type structure, positioning it
further away from the NNRTI pocket. The changes in
side-chain conformations are also accompanied by a
shift in the position of PETT-2 within the binding site
with an average atom displacement of 0.4 A
˚
and a
maximum displacement of 0.7 A
˚

(Fig. 2C).
Potency of PETT-2 against RT(Glu138Lys) in
viral and enzyme assays
The results of EC
50
determinations for PETT-2 in
the HIV assay together with fold-resistance for the
Glu138Lys mutant are shown in Table 2. IC
50
values
determined from inhibition assays of recombinant
HIV-1 RT by PETT-2 for wild-type and
RT(Glu138Lys) are also shown in Table 2. In both
antiviral and enzyme assays, no significant difference
in potency was observed for PETT2 between wild-type
and the Glu138Lys mutant, hence no cross-resistance
was detectable.
Discussion
The effect of many of the drug resistance mutations
reported for NNRTIs can be rationalized at the
molecular level in terms of direct alterations in the ste-
reochemistry of inhibitor–RT interactions. Thus, in the
case of both RT(Tyr181Cys) and RT(Tyr188Cys), the
loss of aromatic ring stacking with first generation
NNRTIs, such as nevirapine, explains the observed
drug resistance [32,34]. The mechanism whereby
RT(Lys101Glu) gives resistance to nevirapine is more
complex as no direct contacts between the drug and
Lys101 exist, rather a network of three water mole-
cules links the inhibitor to the side-chains of Lys101

and Glu138 in wild-type RT [4]. We were fortunate
in being able to solve both RT(Glu138Lys) and
RT(Lys101Glu) nevirapine complexes to the relatively
high resolution for HIV-1 RT crystals of 2.5 A
˚
,
thereby allowing full structural refinement and deter-
mination of the associated water structure, of key
importance in understanding the interaction of the
drug with these residues. There are some similarities in
the structural changes seen in the nevirapine complexes
for both the RT(Glu138Lys) and RT(Lys101Glu)
mutants. Thus significant movements of side-chains
away from the NNRTI pocket are observed in both
structures due to the juxtaposition of like charges
caused by the charge inversion resulting from these
mutations. Although three water molecules, which link
Glu138, Lys101 and nevirapine in wild-type RT are
also present in both RT(Glu138Lys) and RT(Lys101-
Glu) structures, they are shifted in position and hydro-
gen bonding patterns are perturbed. A set of H-bonds
linking waters to the main-chain carbonyl and amide
groups of residue 101 to one of the nevirapine pyridine
rings is retained in each case. However the side-chain
movements away from the NNRTI pocket observed
for RT(Lys101Glu) and RT(Glu138Lys) nevirapine
complexes in both cases abolish the H-bond and salt
bridge linking Lys101 to Glu138 in wild-type RT. Such
movement leads to a reduction in the extent of the
H-bond network so that there is no longer a water

molecule H-bonded to residue 138 in the Glu138Lys
mutant. Paradoxically the greater loss of H-bonding
seen in RT(Glu138Lys) does not lead to a bigger
reduction in binding potency for nevirapine, clearly
indicating that there must be some compensatory
changes. Detailed inspection of overlaps of the
RT(Lys101Glu) and RT(Glu138Lys) structures with
Table 2. Potency of PETT-2 against HIV-1 RT(Glu138Lys).
EC
50
virus assay (nM)IC
50
enzyme assay (nM)
WT E138K Fold resistance WT E138K Fold resistance
PETT-2 3.2 ± 2.0 2.8 ± 0.5 0.9 2.8 ± 0.3 2.7 ± 0.4 1.0
Nevirapine 81 ± 12 97 ± 21 1.2 1955 ± 376
a
1165 ± 375
a
0.6
a
Data from reference [30].
J. Ren et al. HIV-1 RT drug resistance mutations at 101 and 138
FEBS Journal 273 (2006) 3850–3860 ª 2006 The Authors Journal compilation ª 2006 FEBS 3855
the equivalent nevirapine complex of wild-type RT
indicates the NNRTI site protein conformation is
remarkably similar in both mutants and wild-type RT,
thus limiting possible options for compensatory chan-
ges. Indeed, apart from movements of the mutated
and immediately interacting side-chains and associated

water molecules, the only other changes visible are
shifts in the position of nevirapine for both
RT(Lys101Glu) and RT(Glu138Lys), with the biggest
movement observed for the drug in the latter mutant.
Previous structural work has shown that a shift of
nevirapine relative to Tyr181 can compromise drug to
side-chain aromatic ring stacking interactions, thereby
contributing to weaker drug binding [37]. There
are, however, distinctive features in the case of
RT(Glu138Lys), in which the movement of nevirapine
relative to the Tyr181 side-chain is by a translation in
one direction rather than a rotation, thus potentially
optimizing the favourable parallel offset ring stacking
interactions with Tyr181. Normally, where changes of
the interactions of nevirapine with the tyrosine side-
chain leads to reduced binding, e.g. Val106Ala, there is
a rotation of the nevirapine and significant movement
of the side-chain [37]. Thus the nevirapine movement
in RT(Glu138Lys) appears in this case to lead to an
optimization of interactions rather than the reverse. A
factor that is different in RT(Glu138Lys) compared
with RT(Val106Ala) is that the former mutation
slightly expands the NNRTI pocket in a way that is
not possible for resistance mutations at the hydropho-
bic core of the pocket which are more structurally con-
strained. For RT(Lys101Glu), where the H-bonding
network involving nevirapine is less disrupted (the link
to Glu138 being maintained) there is a smaller shift in
the nevirapine position and therefore suggesting there
is less opportunity to optimize interactions, resulting in

a greater overall loss of binding affinity. A corollary to
this is that the interactions of nevirapine with wild-
type RT would seem non-optimal. Indeed, there is
evidence of this for the mutation Pro236Leu which,
although it gives resistance to delavirdine [38], causes
hypersensitivity to nevirapine. There thus appears to
be an incompatibility between the competing require-
ments of nevirapine hydrogen bonding via water mole-
cules to side-chains at the outer edge of the pocket
(Lys101, Glu138) and aromatic ring stacking to tyro-
sines in the inner region. This would also explain why
nevirapine, although a much bulkier molecule than ef-
avirenz, and capable of making more interactions with
RT, is actually of much lower potency (30-fold) than
the latter drug. A further implication is that, as nevi-
rapine does not appear to optimally fit the NNRTI
pocket, there are possibilities for designing analogues
that better meet both hydrophobic and hydrogen
bonding requirements which might therefore have
greater potency. As an example, it may be possible to
introduce additional substituents into a nevirapine-like
inhibitor in order to displace the bound waters, allow-
ing direct interactions with the protein. Comparison
of the crystal structures of wild-type RT and
RT(Glu138Lys) with bound PETT-2 shows that there
is a loss of a van der Waals contact between the carb-
oxyl of Glu138 and the inhibitor. It would thus be
expected that there is an accompanying reduction in
binding affinity for PETT-2 for the Glu138Lys muta-
tion. In fact the EC

50
and IC
50
data both from the
mutant HIV in tissue culture and recombinant RT
enzyme assays clearly indicate essentially equal potency
for PETT-2 against WT and mutant forms. It is poss-
ible that the observed PETT-2 contact with the Glu138
carboxyl may be induced as a result of the crystals
being grown at pH 5.0, which approaches the range
of pKa values for glutamic acid side-chains (4.4).
Glu138 may therefore be protonated and as a conse-
quence induce a contact which would not be found
under conditions for assay of RT inhibition (pH 7.4).
It should also be born in mind that since the resolution
of the RT complexes with PETT-2 (both wild-type and
for RT(Glu138Lys)) are limited to 3.0 A
˚
, it is not
possible to fully define all aspects of the structure, par-
ticularly bound water molecules. It is thus not known
whether, for example, changes in hydrogen bonding
and water networks may occur as is the case in the
work reported here on the RT(Lys101Glu) and
RT(Glu138Lys) mutants in complexes with nevirapine.
Such changes might compensate for the loss of the
van der Waals interactions between PETT-2 and
RT(Glu138Lys) compared with wild-type. This limita-
tion in structural detail emphasizes the need to exercise
caution in the interpretation of molecular details of

inhibitor binding when using lower resolution X-ray
structures of HIV-1 RT.
For the Glu138Lys mutation, resistance to the
TSAO series of NNRTIs is thought to be due to the
loss of interaction of the side-chain carboxyl group
with a key amino group of the compounds [28]. Unfor-
tunately it has not been possible to obtain crystal
structures of TSAO bound to RT as inhibitors of
this series cause the protein to precipitate prior to co-
crystallization set-ups or induce disorder in pre-grown
crystals following soaking attempts. Such properties
may relate to the known disrupting effects of TSAO
on the RT dimer structure.
The work described here shows how the charge
inversion introduced by the Lys101Glu and Glu138Lys
mutations in RT results in a common structural
HIV-1 RT drug resistance mutations at 101 and 138 J. Ren et al.
3856 FEBS Journal 273 (2006) 3850–3860 ª 2006 The Authors Journal compilation ª 2006 FEBS
feature, the significant movement of mutated side-
chains away from the NNRTI pocket, due to the
repulsion of like charges. The structural consequences
of the mutant side-chain movement include partially
disrupting the hydrogen bond network involving nevi-
rapine, water and side-chain ⁄ main-chain interactions in
wild-type RT. Compensatory changes must occur for
the Glu138Lys mutation as this has little effect on
nevirapine binding which we suggest to be optimiza-
tion of inhibitor ring stacking interactions with
Tyr181. Lys101Glu provides an example of an indirect
mechanism of inducing drug resistance in RT but via

a different means to other reported examples viz
Lys103Asn, which apparently stabilizes the unliganded
RT conformation [7,35] whilst Val108Ile perturbs the
interaction of Tyr188 and Tyr181 with nevirapine [37].
Interestingly, the Lys101Glu mutation in RT also
appears to be capable of a direct mechanism of drug
resistance. For the carboxanilide, UC-38, and for ef-
avirenz there are van der Waals contacts between the
side-chain of Lys101 and the inhibitors [24]. Movement
of the Lys101 side-chain away from the NNRTI
pocket on mutation to glutamic acid, as observed for
the nevirapine complex, would thus cause loss of
contacts and a consequent weakening in binding for
UC-38 and efavirenz.
The selection of drug-resistant virus remains a signi-
ficant problem in controlling HIV infection and AIDS.
The work reported here is part of ongoing studies
aimed at understanding mechanisms of drug resistance
in HIV RT by the use of X-ray crystal structure analy-
sis. Combining such data with biochemical and virolo-
gical studies will be of value in contributing to the
development of further generations of NNRTIs with
improved resilience to existing drug resistance muta-
tions present in HIV. Such drugs are needed as an
important priority for continued efforts to treat HIV.
Experimental procedures
Crystallization and data collection
Expression vectors for HIV-1 RT (HXB-2 isolate) contain-
ing either Lys101Glu or Glu138Lys mutations were made
using site-directed mutagenesis as previously reported [34].

Purification of mutant RTs from recombinant Escherichia
coli was based on the ion-exchange procedures as described
in an earlier report [39]. A wide range of NNRTIs were
screened for crystallization with the mutant RTs, producing
crystals of three complexes suitable for structure determin-
ation. Crystals of complexes of RTs with NNRTIs were
grown and soaked in 50% PEG 3400 prior to data collec-
tion as described previously [40,41]. All the crystals of
mutant RT inhibitor complexes used in this work were
obtained by co-crystallization. X-ray data were either col-
lected at SRS, Daresbury, UK and APS, Chicago, IL,
USA, using the oscillation method or at the Photon Fac-
tory Tsukuba, Japan, using a Weissenberg camera [42].
Data collected for each mutant–inhibitor complex were
from crystals flash-cooled in liquid propane and maintained
at 100 K in a nitrogen gas stream. Indexing and integration
of data images were carried out with denzo, and data were
merged with scalepack [43]. Details of the X-ray data sta-
tistics are given in Table 1.
Structure solution and refinement
The molecular orientation and position of HIV-1 RT het-
erodimers in each unit cell were determined using rigid-
body refinement with cns [44]. The initial model used as
the starting point for the current structure determinations
was chosen from our collection of RT–NNRTI complexes
on the basis of closeness of unit cell parameters
[4,24,25,34,45–48]. All structures were refined with cns [44]
using positional, simulated annealing and individual B-fac-
tor refinement with bulk solvent correction and anisotropic
B-factor scaling. Model rebuilding was carried out using ‘o’

[49]. Table 1 gives the refinement statistics for the three
structures.
Structures of wild-type and mutant RTs were overlapped
using the program SHP [50]. The wild-type RT complexes
used for these comparisons were 1rtd (for nevirapine) [4]
and 1dtt (for PETT-2) [31].
Data deposition
Coordinates and structure factors for all of the HIV-1 RT
mutant structures reported here have been deposited in the
Protein Data Bank for immediate release on publication.
The codes are as follows: RT(Lys101Glu)-nevirapine,
2HND; RT(Glu138Lys)-nevirapine, 2HNY; RT(Glu138Lys)-
PETT-2, 2HNZ.
Potency of PETT-2 against HIV RT(Glu138Lys) in
tissue culture and enzyme assays
The assay to determine EC
50
values for PETT-2 used a
modified HeLa MAGI system in which HIV-1 infection is
detected due to the activation of the LTR-driven b-galac-
tosidase reporter in HeLa CD4-LTR-b-gal cells as des-
cribed previously [51]. Quantitation of the b-galactosidase
was carried out by measurement of the activation of the
chemoluminescent substrate, tropix, after 3 days infection.
The value of the EC
50
was the mean of three separate
experiments. PETT-2 inhibition of recombinant wild-type
HIV-1 RT and RT(Glu138Lys) were determined in an
enzyme assay using rC-dG as template-primer and measur-

J. Ren et al. HIV-1 RT drug resistance mutations at 101 and 138
FEBS Journal 273 (2006) 3850–3860 ª 2006 The Authors Journal compilation ª 2006 FEBS 3857
ing the incorporation of radiolabelled dGTP [39]. Curve fit-
ting of enzyme inhibition data and IC
50
determination were
carried out using origin (Microcal).
Acknowledgements
We thank the staff of the following synchrotrons for
their assistance in data collection: SRS, Daresbury,
UK; The Photon Factory, Tsukuba, Japan and
Advanced Photon Source, Chicago, IL, USA. Support-
ing grants from the Medical Research Council, the
Biotechnology and Biological Sciences Research Coun-
cil and the EC (QLKT-2000–00291, QLKT2-CT-2002–
01311) to DKS are acknowledged.
References
1 Richman DD (2001) HIV chemotherapy. Nature 410,
995–1001.
2 Brettle RP, Wilson A, Povey S, Morris S, Morgan R,
Leen CL, Hutchinson S, Lewis S & Gore S (1998) Com-
bination therapy for HIV: the effect on inpatient activ-
ity, morbidity and mortality of a cohort of patients. Int
J STD AIDS 9, 80–87.
3 Kohlstaedt LA, Wang J, Friedman JM, Rice PA &
Steitz TA (1992) Crystal structure at 3.5 A
˚
resolution of
HIV-1 reverse transcriptase complexed with an inhibi-
tor. Science 256, 1783–1790.

4 Ren J, Esnouf R, Garman E, Somers D, Ross C, Kirby
I, Keeling J, Darby G, Jones Y, Stuart D et al. (1995)
High resolution structures of HIV-1 RT from four RT-
inhibitor complexes. Nature Struct Biol 2, 293–302.
5 Goody RS, Muller B & Restle T (1991) Factors contri-
buting to the inhibition of HIV reverse transcriptase
by chain-terminating nucleotides in vivo. FEBS Lett 291,
1–5.
6 De Clercq E (1993) HIV-1-specific RT inhibitors: highly
selective inhibitors of human immunodeficiency virus
type 1 that are specifically targeted at the viral reverse
transcriptase. Med Res Rev 13, 229–258.
7 Esnouf R, Ren J, Ross C, Jones Y, Stammers D &
Stuart D (1995) Mechanism of inhibition of HIV-1
reverse transcriptase by non-nucleoside inhibitors.
Nature Struct Biol 2, 303–308.
8 Spence RA, Kati WM, Anderson KS & Johnson KA
(1995) Mechanism of inhibition of HIV-1 reverse
transcriptase by nonnucleoside inhibitors. Science 267,
988–993.
9 Schinazi RF, Larder BA & Mellors JW (2000) Muta-
tions in retroviral genes associated with drug resistance.
Int Antiviral News 8, 65–71.
10 Young SD, Britcher SF, Tran LO, Payne LS, Lumma
WC, Lyle TA, Huff JR, Anderson PS, Olsen DB,
Carroll SS et al. (1995) L-743, 726 (DMP-266): a novel,
highly potent nonnucleoside inhibitor of the human
immunodeficiency virus type 1 reverse transcriptase.
Antimicrob Agents Chemother 39, 2602–2605.
11 De Clercq E (2001) New developments in anti-HIV che-

motherapy. Curr Medical Chem 8, 1543–1572.
12 Jacobo-Molina A, Ding JP, Nanni RG, Clark ADJ,
Lu X, Tantillo C, Williams RL, Kamer G, Ferris AL,
Clark P et al. (1993) Crystal structure of human immu-
nodeficiency virus type 1 reverse transcriptase com-
plexed with double-stranded DNA at 3.0 A
˚
resolution
shows bent DNA. Proc Natl Acad Sci USA 90, 6320–
6324.
13 Huang H, Chopra R, Verdine GL & Harrison SC
(1998) Structure of a covalently trapped catalytic com-
plex of HIV-1 reverse transcriptase: implications for
drug resistance. Science 282, 1669–1675.
14 Byrnes VW, Sardana VV, Schleif WA, Condra JH,
Waterbury JA, Wolfgang JA, Long WJ, Schneider CL,
Schlabach AJ, Wolanski BS et al. (1993) Comprehensive
mutant enzyme and viral variant assessment of human
immunodeficiency virus type 1 reverse transcriptase
resistance to nonnucleoside inhibitors. Antimicrob
Agents Chemother 37, 1576–1579.
15 Buckheit RW, Snow MJ, Fliakas-Boltz V, Kinjerski TL,
Russell JD, Pallansch LA, Brouwer WG & Yang SS
(1997) Highly potent oxathiin carboxanilide derivatives
with efficacy against nonnucleoside reverse transcriptase
inhibitor-resistant human immunodeficiency virus iso-
lates. Antimicrob Agents Chemother 41, 831–837.
16 Petropoulos CJ, Parkin NT, Limoli KL, Lie YS, Wrin
T, Huang W, Tian H, Smith D, Winslow GA, Capon
DJ et al. (2000) A novel phenotypic drug susceptibility

assay for human immunodeficiency virus type 1.
Antimicrob Agents Chemother 44, 920–928.
17 Pelemans H, Aertsen A, Van Laethem K, Vandamme
AM, De Clercq E, Perez-Perez MJ, San-Felix A,
Velazquez S, Camarasa MJ & Balzarini J (2001) Site-
directed mutagenesis of human immunodeficiency virus
type 1 reverse transcriptase at amino acid position 138.
Virology 280, 97–106.
18 Chan JH, Freeman GA, Tidwell JH, Romines KR,
Schaller LT, Cowan JR, Gonzales SS, Lowell GS,
Andrews CW 3rd, Reynolds DJ, et al. (2004) Novel
benzophenones as non-nucleoside reverse transcriptase
inhibitors of HIV-1. J Med Chem 47, 1175–1182.
19 Schweighardt B, Ortiz GM, Grant RM, Wellons M,
Miralles GD, Kostrikis LG, Bartlett JA & Nixon DF
(2002) Emergence of drug-resistant HIV-1 variants in
patients undergoing structured treatment interruptions.
AIDS 16, 2342–2344.
20 Hanna GJ, Johnson VA, Kuritzkes DR, Richman DD,
Brown AJ, Savara AV, Hazelwood JD & D’Aquila RT
(2000) Patterns of resistance mutations selected by treat-
ment of human immunodeficiency virus type 1 infection
HIV-1 RT drug resistance mutations at 101 and 138 J. Ren et al.
3858 FEBS Journal 273 (2006) 3850–3860 ª 2006 The Authors Journal compilation ª 2006 FEBS
with zidovudine, didanosine, and nevirapine. J Infect
Dis 181, 904–911.
21 Bacheler LT, Anton ED, Kudish P, Baker D, Bunville
J, Krakowski K, Bolling L, Aujay M, Wang XV, Ellis
D et al. (2000) Human immunodeficiency virus type 1
mutations selected in patients failing efavirenz combina-

tion therapy. Antimicrob Agents Chemother 44, 2475–
2484.
22 Balzarini J, Brouwer WG, Felauer EE, De Clercq E &
Karlsson A (1995) Activity of various thiocarboxanilide
derivatives against wild-type and several mutant human
immunodeficiency virus type 1 strains. Antiviral Res 27,
219–236.
23 Buckheit RW, Kinjerski TL, Fliakas-Boltz V, Russell
JD, Stup TL, Pallansch LA, Brouwer WG, Dao DC,
Harrison WA, Schultz RJ et al. (1995) Structure-activity
and cross-resistance evaluations of a series of human
immunodeficiency virus type-1-specific compounds rela-
ted to oxathiin carboxanilide. Antimicrob Agents Chemo-
ther 39, 2718–2727.
24 Ren J, Esnouf RM, Hopkins AL, Warren J, Balzarini J,
Stuart DI & Stammers DK (1998) Crystal structures of
HIV-1 reverse transcriptase in complex with carboxani-
lide derivatives. Biochemistry 37, 14394–14403.
25 Ren J, Milton J, Weaver KL, Short SA, Stuart DI &
Stammers DK (2000) Structural basis for the resilience
of efavirenz (DMP-266) to drug resistance mutations in
HIV-1 reverse transcriptase. Structure Fold Des 8, 1089–
1094.
26 Balzarini J, Velazquez S, San-Felix A, Karlsson A,
Perez-Perez MJ, Camarasa MJ & De Clercq E (1993)
Human immunodeficiency virus type 1-specific [2¢,5¢-bis-
O-(tert- butyldimethylsilyl)-beta-d-ribofuranosyl]-3¢-
spiro-5¢-(4¢-amino-1¢,2¢-oxathiole-2¢,2¢-dioxide)-purine
analogues show a resistance spectrum that is different
from that of the human immunodeficiency virus type 1-

specific non-nucleoside analogues. Mol Pharmacol 43,
109–114.
27 Sluis-Cremer N, Arion D & Parniak MA (2002) Desta-
bilization of the HIV-1 reverse transcriptase dimer upon
interaction with N-acyl hydrazone inhibitors. Mol
Pharmacol 62, 398–405.
28 Rodriguez-Barrios F, Perez C, Lobaton E, Velazquez S,
Chamorro C, San-Felix A, Perez-Perez MJ, Camarasa
MJ, Pelemans H, Balzarini J et al. (2001) Identification
of a putative binding site for [2¢,5¢-bis-O-(tert-butyldi-
methylsilyl)-beta-d-ribofuranosyl]-3¢-spiro-5¢¢-(4¢¢-amino-
1¢¢,2¢¢
-oxathiole-2¢¢,2¢¢-dioxide) thymine (TSAO)
derivatives at the p51–p55 interface of HIV-1 reverse
transcriptase. J Med Chem 44, 1853–1865.
29 Zhang H, Vrang L, Backbro K, Lind P, Sahlberg C,
Unge T & Oberg B (1995) Inhibition of human immu-
nodeficiency virus type 1 wild-type and mutant reverse
transcriptases by the phenyl ethyl thiazolyl thiourea
derivatives trovirdine and MSC-127. Antiviral Res 28,
331–342.
30 Balzarini J, Kleim JP, Riess G, Camarasa MJ, De
Clercq E & Karlsson A (1994) Sensitivity of (138
Glu?Lys) mutated human immunodeficiency virus type
1 (HIV-1) reverse transcriptase (RT) to HIV-1-specific
RT inhibitors. Biochem Biophys Res Commun 201,
1305–1312.
31 Ren J, Diprose J, Warren J, Esnouf RM, Bird LE,
Ikemizu S, Slater M, Milton J, Balzarini J, Stuart DI
et al. (2000) Phenylethylthiazolylthiourea (PETT) non-

nucleoside inhibitors of HIV-1 and HIV-2 reverse tran-
scriptases: structural and biochemical analyses. J Biol
Chem 275, 5633–5639.
32 Das K, Ding J, Hsiou Y, Clark AD, Moereels H,
Koymans L, Andries K, Pauwels R, Janssen PA, Boyer
PL et al. (1996) 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. J Mol Biol 264, 1085–1100.
33 Hsiou Y, Das K, Ding J, Clark AD, Kleim J-P, Rosner
M, Winkler I, Riess G, Hughes SH & Arnold E (1998)
HIV-1 reverse transcriptase complexed with the non-
nucleoside inhibitor HBY 097: inhibitor flexibility is a
useful design feature for reducing drug resistance. J Mol
Biol 284, 313–323.
34 Ren J, Nichols C, Bird L, Chamberlain P, Weaver K,
Short S, Stuart DI & Stammers DK (2001) Structural
mechanisms of drug resistance for mutations at codons
181 and 188 in HIV-1 reverse transcriptase and the
improved resilience of second generation non-nucleoside
inhibitors. J Mol Biol 312, 795–805.
35 Hsiou Y, Ding J, Das K, Clark AD Jr, Boyer PL, Lewi
P, Janssen PA, Kleim JP, Rosner M, Hughes SH et al.
(2001) The Lys103Asn mutation of HIV-1 RT: a novel
mechanism of drug resistance. J Mol Biol 309, 437–445.
36 Lindberg J, Sigurosson S, Lowgren S, Andersson HO,
Sahlberg C, Noreen R, Fridborg K, Zhang H & Unge
T (2002) Structural basis for the inhibitory efficacy of
efavirenz (DMP-266), MSC194 and PNU142721
towards the HIV-1 RT K103N mutant. Eur J Biochem

269, 1670–1677.
37 Ren J, Nichols CE, Chamberlain PP, Weaver KL, Short
SA & Stammers DK (2004) Crystal structures of HIV-1
reverse transcriptase mutated at codons 100, 106 and
108 and mechanisms of resistance to non-nucleoside
inhibitors. J Mol Biol 336, 569–578.
38 Dueweke TJ, Pushkarskaya T, Poppe SM, Swaney SM,
Zhao JQ, Chen ISY, Stevenson M & Tarpley WG
(1993) A mutation in reverse transcriptase of bis(heter-
oaryl)piperazine-resistant human immunodeficiency
virus type 1 that confers increased sensitivity to other
nonnucleoside inhibitors. Proc Natl Acad Sci USA 90,
4713–4717.
J. Ren et al. HIV-1 RT drug resistance mutations at 101 and 138
FEBS Journal 273 (2006) 3850–3860 ª 2006 The Authors Journal compilation ª 2006 FEBS 3859
39 Wilson JE, Wright LL, Martin JL, Haire SE, Ray PH,
Painter GR & Furman PA (1996) Recombinant human
immunodeficiency virus type 1 reverse transcriptase is
heterogeneous. J Acquir Immune Defic Syndr Hum
Retrovirol 11, 20–30.
40 Stammers DK, Somers DON, Ross CK, Kirby I, Ray
PH, Wilson JE, Norman M, Ren JS, Esnouf RM,
Garman EF et al. (1994) Crystals of HIV-1 reverse
transcriptase diffracting to 2.2 A
˚
resolution. J Mol Biol
242, 586–588.
41 Esnouf RM, Ren J, Garman EF, Somers DON, Ross
CK, Jones EY, Stammers DK & Stuart DI (1998) Con-
tinuous and discontinuous changes in the unit cell of

HIV-1 reverse transcriptase crystals on dehydration.
Acta Crystallogr D54, 938–954.
42 Sakabe N (1991) X-ray diffraction data collection sys-
tem for modern protein crystallography with a Weissen-
berg camera and an imaging plate using synchrotron
radiation. Nucl Instr Methods Phys Res 303, 448–463.
43 Otwinowski Z & Minor W (1996) Processing of X-ray
diffraction data collected in oscillation mode. Methods
Enzymol 276, 307–326.
44 Brunger AT, Adams PD, Clore GM, Delano WL, Gros
P, Grosse KRW, Jiang JS, Kuszewski J, Nilges M,
Pannu NS et al. (1998) Crystallography and NMR sys-
tem: a new software suite for macromolecular structure
determination. Acta Crystallogr D54, 905–921.
45 Ren J, Esnouf R, Hopkins A, Ross C, Jones Y,
Stammers D & Stuart D (1995) The structure of HIV-1
reverse transcriptase complexed with 9-chloro-TIBO:
lessons for inhibitor design. Structure 3, 915–926.
46 Ren J, Esnouf RM, Hopkins AL, Stuart DI &
Stammers DK (1999) Crystallographic analysis of the
binding modes of thiozolo-isoindolinone non-nucleo-
side inhibitors to HIV-1 reverse transcriptase and
comparison with modelling studies. J Med Chem 42,
3845–3851.
47 Hopkins AL, Ren J, Esnouf RM, Willcox BE, Jones
EY, Ross C, Miyasaka T, Walker RT, Tanaka H,
Stammers DK et al. (1996) Complexes of HIV-1
reverse transcriptase with inhibitors of the HEPT series
reveal conformational changes relevant to the design of
potent non-nucleoside inhibitors. J Med Chem 39,

1589–1600.
48 Hopkins AL, Ren J, Tanaka H, Baba M, Okamato M,
Stuart DI & Stammers DK (1999) Design of MKC-442
(Emivirine) analogues with improved activity against
drug resistant HIV mutants. J Med Chem 42, 4500–
4505.
49 Jones TA, Zou JY, Cowan SW & Kjeldgaard M (1991)
Improved methods for building protein models in elec-
tron density maps and the location of errors in these
models. Acta Crystallogr A47, 110–119.
50 Stuart DI, Levine M, Muirhead H & Stammers DK
(1979) The crystal structure of cat pyruvate kinase at a
resolution of 2.6 A
˚
. J Mol Biol 134, 109–142.
51 Chan JH, Hong JS, Hunter RN 3rd, Orr GF, Cowan
JR, Sherman DB, Sparks SM, Reitter BE, Andrews CW
3rd, Hazen RJ et al. (2001) 2-Amino-6-aryl-
sulfonylbenzonitriles as non-nucleoside reverse tran-
scriptase inhibitors of HIV-1. J Med Chem 44, 1866–
1882.
HIV-1 RT drug resistance mutations at 101 and 138 J. Ren et al.
3860 FEBS Journal 273 (2006) 3850–3860 ª 2006 The Authors Journal compilation ª 2006 FEBS