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

Structural insights into mechanisms of non-nucleoside

drug resistance for HIV-1 reverse transcriptases mutated

at codons 101 or 138

Jingshan Ren

, Charles E. Nichols

, Anna Stamp

, Phillip P. Chamberlain

, Robert Ferris

Kurt L. Weaver

, Steven A. Short

and David K. Stammers

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: daves@strubi.ox.ac.uk

(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 ª2006 The Authors Journal compilation ª2006 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-ribofuranosyl]-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

Nevirapine

PETT-2

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 ª2006 The Authors Journal compilation ª2006 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

˚)

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

mergeb

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

work

⁄R

free

) 0.198 ⁄0.270 0.216 ⁄0.288 0.226 ⁄0.277

R-factor

(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

)

59 ⁄65 ⁄37 ⁄44 62 ⁄68 ⁄39 ⁄48 73 ⁄81 ⁄60 ⁄–

Rms backbone B-factor deviation (A

) 4.4 4.2 3.4

All crystals belong to space group P2

[40,41];

merge

¼S|I–<I>|⁄S <I>;

R factor ¼S|F

-F

|⁄SF

;

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 ª2006 The Authors Journal compilation ª2006 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 3.5rshowing 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 ª2006 The Authors Journal compilation ª2006 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 ª2006 The Authors Journal compilation ª2006 FEBS

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

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