Transient HIV-1 Gag–protease interactions revealed byparamagnetic NMR suggest origins of compensatorydrug resistance mutationsLalit Deshmukha, John M. Louisa, Rodolfo Ghirlandob, and G. Marius Clorea,1

aLaboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892;and bLaboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892

Contributed by G. Marius Clore, September 13, 2016 (sent for review September 1, 2016; reviewed by Hashim M. Al-Hashimi, Lewis E. Kay, and MichaelF. Summers)

Cleavage of the group-specific antigen (Gag) polyprotein by HIV-1protease represents the critical first step in the conversion ofimmature noninfectious viral particles to mature infectious virions.Selective pressure exerted by HIV-1 protease inhibitors, a mainstayof current anti–HIV-1 therapies, results in the accumulation of drugresistance mutations in both protease and Gag. Surprisingly, alarge number of these mutations (known as secondary or compen-satory mutations) occur outside the active site of protease or thecleavage sites of Gag (located within intrinsically disordered link-ers connecting the globular domains of Gag to one another), sug-gesting that transient encounter complexes involving the globulardomains of Gag may play a role in guiding and facilitating access ofthe protease to the Gag cleavage sites. Here, using large fragmentsof Gag, as well as catalytically inactive and active variants of pro-tease, we probe the nature of such rare encounter complexes usingintermolecular paramagnetic relaxation enhancement, a highly sen-sitive technique for detecting sparsely populated states. We showthat Gag-protease encounter complexes are primarily mediated byinteractions between protease and the globular domains of Gagand that the sites of transient interactions are correlated with sur-face exposed regions that exhibit a high propensity to mutate in thepresence of HIV-1 protease inhibitors.

HIV-1 Gag | HIV-1 protease | drug resistance mutations | invisible states |paramagnetic relaxation enhancement

The transformation of noninfectious viral particles into matureinfectious virions is a hallmark of the retroviral replication

cycle. In HIV type 1 (HIV-1), morphological remodeling is initi-ated on sequential hydrolysis of the group-specific antigen (Gag)polyprotein by the viral homodimeric aspartyl protease, whichgenerates a set of structural proteins (1). Not surprisingly, there-fore, HIV-1 protease inhibitors are major components of currentanti–HIV-1 therapies (2). The organization of HIV-1 Gag is asfollows: matrix (MA)–capsid (CA)–spacer peptide 1 (SP1)–nucleocapsid (NC)–spacer peptide 2 (SP2)–p6, with an HIV-1protease cleavage site located at each junction (Fig. 1A). MA, CA,and NC are globular domains, whereas SP1, SP2, and p6 are in-trinsically disordered in solution (3). At neutral pH in vitro, thefive cleavage sites in Gag are hydrolyzed by protease in the fol-lowing order: SP1jNC > SP2jp6 ∼ MAjCA > CAjSP1 ∼ NCjSP2(4). The corresponding in vivo cleavage order, deduced from vi-rions with mutated Gag cleavage sites, is consistent with in vitroobservations (5). The underlying mechanism governing orderedproteolysis is far from clear. Current understanding of Gag–pro-tease interactions is derived from crystal structures of activeprotease complexed to nonhydrolysable peptide analogs (6) and ofinactive protease (in which the active site Asp25 is replaced byAsn) bound to synthetic peptides comprising the Gag cleavagesites (7). Because all protease-bound peptides are in an extended,asymmetric β-strand conformation, it has been hypothesized thatan effective protease substrate comprises six to eight residues ofGag (8) and that protease recognizes the shape of Gag substrates

rather than their primary sequence (7). This “substrate envelope”hypothesis predicts variable interactions between side chains ofGag cleavage sites and protease (9), resulting in a unique pro-cessing rate for each site. Synthetic peptides, however, are apoor substitute for the Gag polyprotein in terms of long-rangeintermolecular interactions and conformational changes thatcan alter the accessibility of Gag cleavage junctions. Addition-ally both protease and Gag mutate and coevolve in response toprotease inhibitors in current clinical use (10–17). Primarymutations (i.e., mutations at the catalytic site of protease) andmutations within the Gag cleavage sites are readily amenable toinvestigation using peptide analogs. The effects of secondary orcompensatory mutations (i.e., mutations outside the proteasesubstrate binding cleft or far away from the Gag cleavage sites),however, cannot be unraveled using synthetic peptides, andtheir existence hints at a larger role played by the globular Gagdomains in Gag–protease interactions.Here, using state-of-the-art solution NMR methods, and in

particular intermolecular paramagnetic relaxation enhancement(PRE) measurements (18), we investigate Gag–protease inter-actions. We show that globular Gag domains transiently interactwith protease to form sparsely populated encounter complexes,and that the patches of surface-exposed residues on the Gagdomains and on protease that come into short-lived close contactwith one another exhibit a high propensity to mutate in thepresence of protease inhibitors.

Significance

Hydrolysis of group-specific antigen (Gag) polyprotein by pro-tease is essential for the formation of infectious HIV-1 virions. Inresponse to treatment with protease inhibitors, drug resistancemutations coevolve in protease and Gag, a significant number ofwhich (known as compensatory mutations) lie outside the activesite of protease and the Gag cleavage sites. We show, usingparamagnetic NMR spectroscopy, that transient, sparsely popu-lated encounter complexes are predominantly formed betweenthe globular domains of Gag and protease at sites that correlatewith the location of secondary drug resistance mutations. Theseresults provide a structural basis for the origins of secondarymutations and suggest that transient encounter complexes play asignificant role in guiding protease to the Gag cleavage sites.

Author contributions: L.D. and G.M.C. designed research; L.D. and R.G. performed research;J.M.L. contributed new reagents/analytic tools; L.D. and G.M.C. analyzed data; J.M.L. opti-mized and contributed new protease O samples used in the study; R.G. collected andanalyzed sedimentation velocity data; and L.D. and G.M.C. wrote the paper.

Reviewers: H.M.A.-H., Duke University Medical Center; L.E.K., University of Toronto; andM.F.S., Howard Hughes Medical Institute, University of Maryland Baltimore County.

The authors declare no conflict of interest.1To whom correspondence should be addressed. Email: mariusc@mail.nih.gov.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1615342113/-/DCSupplemental.

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ResultsGag and Protease Constructs. To elucidate the role of Gag domainsin Gag–protease interactions, we made use of a large Gag frag-ment containing the MA-CA-SP1-NC domains (group M, residues1–432, hereafter referred to as ΔGag; Fig. 1A). ΔGag exhibits amonomer-dimer equilibrium at high ionic strength (Kdimer ∼35 μMat ≥300 mM NaCl), but at low salt (≤100 mM NaCl) forms im-mature virus-like particles (3). We also made use of the construct,ΔGagW316A

M317A (Fig. S1), which carries a double mutation at theCA dimerization interface and is monomeric at high (300 mMNaCl) ionic strength (Fig. S1A), and the shorter constructCA-SP1-NCW316A

M317A, which is monomeric at low (50 mM NaCl) ionicstrength (Fig. S1B). Protease from HIV-1 group O (PR-O, pI ∼5.2)was used instead of the more common M group (PR-M, pI ∼9.5,70% sequence identity with PR-O), owing to its much better sol-ubility properties at high ionic strength.

Ordered Proteolytic Cleavage of Gag. In vitro cleavage of ΔGag byPR-M proceeds in the following order: NCjSP1 > MAjCA >CAjSP1 (3). Nearly identical cleavage patterns, time courses,and proteolysis rates are observed using PR-O for both ΔGag andΔGagW316A

M317A (Fig. 1B, Fig. S2, and Table S1), indicating that theorder of Gag proteolysis is conserved across HIV-1 groupsand is independent of Gag oligomerization state and protease

isoelectric point. The order of cleavage is also not dependent ondifferences in amino acid sequence of the cleavage sites, as asimilar sequential order of processing is observed for the constructΔGagMAjCA=SP1jNC

CAjSP1=SP1jNC, in which the MAjCA and CAjSP1 cleavagejunctions (128VSQNYjPIVQN137 and 359KARVLjAEAMS368, re-spectively) were mutated to the same sequence as that of theSP1jNC cleavage site (373SATIMjMQRGN383), although the cleav-age rate at the CAjSP1 junction is now enhanced and comparableto that at the MAjCA junction (Fig. 1C). These observations leadone to conclude that the globular Gag domains likely play a pivotalrole in Gag–protease interactions.

Intermolecular PRE Measurements. To probe transient interactionsbetween Gag and protease, we made use of NMR spectroscopyand specifically intermolecular PRE measurements (18). Theintermolecular PRE arises from dipolar interactions between anunpaired electron located on a paramagnetic tag covalently at-tached to one protein and the protons of the second protein: inour case, backbone amide protons that can be selectively moni-tored by isotopically labeling the second protein with 15N. Whenexchange between the unbound major species and the sparselypopulated transient encounter complex is fast on the PRE timescale (lifetime < 250–500 μs), the intermolecular PRE observedon the protons of the major species will simply be given by the“true” intermolecular PRE within the complex scaled by thepopulation of the complex (18). Owing to the large magneticmoment of the electron, the PRE effect (which is proportional tothe <r−6> separation between the unpaired electron and theproton) is very large, and hence transient complexes with occu-pancies as low as 0.5–1% can be detected (18).We first carried out PRE experiments with the paramagnetic tag

(maleimido-DOTA-Gd3+; see Fig. S3A) conjugated to two alter-native sites, V82C (PR-OD25N

V82C) and L72C (PR-OD25NL72C ), located

within the catalytic cleft and on the surface, respectively, of aninactive PR-O variant (D25N; Fig. 2A). Although the catalyticactivity of the V82C variant of PR-O is reduced compared with thewild-type (WT) version, the order of Gag cleavage is preserved(Fig. S4), and the structure of PR-OD25N

V82C, determined from back-bone amide residual dipolar couplings (RDCs) and backbonechemical shifts using CS-ROSETTA (19, 20), is the same as thatof PR-M with the flaps predominantly in the closed conformation(Fig. S5). The intermolecular PRE profiles observed on monomeric15N/13C/2H-labeled ΔGagW316A

M317A (high salt) and 15N/2H-labeledCA-SP1-NCW316A

M317A (low salt) are shown in Fig. 2 B and C. Verysimilar intermolecular PRE profiles are also observed for WTΔGag, although line broadening owing to monomer-dimerexchange precludes the measurement of PREs for much of thecapsid domain (Fig. S6). Although the magnitude of the PREsmay differ, the general pattern of intermolecular PREs obtainedwith the two paramagnetically tagged PR-O constructs is rathersimilar, with significant intermolecular PREs above background(1HN-Γ2 > 10 s−1) largely confined to specific surface-exposedregions within the three globular domains of Gag, namely MA,CA, and NC (Figs. 2 B–D). These intermolecular PREs are spe-cific to the Gag–protease system, as essentially no intermolecularPREs above background are observed in control experiments usingeither free DOTA-Gd3+ or DOTA-Gd3+ tagged maltose bindingprotein (MBP) (Fig. S7). Interestingly, two regions display signifi-cant differences in intermolecular PREs between the ΔGagW316A

M317Aand CA-SP1-NCW316A

M317A constructs. Specifically, larger intermolec-ular PREs are observed for CA-SP1-NCW316A

M317A at the N terminus ofCA with both paramagnetic PR-O constructs and in the NC do-main with PR-OD25N

L72C . The former is likely due to the fact that theN terminus of CA undergoes a conformational change from anintrinsically disordered linker to a β-hairpin subsequent to cleavageat the MAjCA junction of Gag (3), while the latter is primarilydriven by electrostatic interactions between the positively charged

Gag + PR-O (50:1) GagW316AM317A + PR-O (50:1)

MA-CA-SP1-NCMA-CA-SP1

CA-SP1

NC

0 1 3 5 10 15 20 25 30 35 40 50 60 120180

CA-SP1-NC

min0 1 3 5 10 15 20 25 30 35 40 50 60 120 180

MA-CA CAMA

B

GagCA|SP1 = SP1|NC + PR-O (100:1)

MA-CA-SP1-NCMA-CA-SP1

CA-SP1-NC

MA-CA

CA-SP1

NC

CAMA

min0 1 3 5 10 15 20 25 30 35 40 50 60120180

kSP1|NC6.5 ± 0.4

C

kMA|CA2.8 ± 0.2

kCA|SP10.2 ± 0.02

> >>>

MA|CA = SP1|NC

kSP1|NC6.5 ± 0.4

kMA|CA2.6 ± 0.3

kCA|SP12.5 ± 0.3

> ~pmoles/min

pmoles/min

A

CA NTD133-277

CACTD282-363

NCN-Zinc378-407

NCC-Zinc413-432SP1364-377linker278-281

MA1-132

KARVL AEAMSQVTNSATIM MQRGN359 382

VSQNY PIVQN137128

Fig. 1. Cleavage kinetics of ΔGag by PR-O. (A) Schematic of ΔGag organiza-tion. Sequences of the cleavage sites are shown with the MAjCA, CAjSP1, andSP1jNC junctions indicated by dashed lines and scissors. Note that in full-lengthGag, the intrinsically disordered SP2 and p6 domains are located C-terminal tothe NC domain, and there are cleavage sites at the NCjSP2 and SP2jp6 junctions.NTD and CTD refer to the N- and C-terminal domains, respectively of CA.Cleavage time course of (B) WT ΔGag (Left) and the monomeric ΔGagW316A

M317A

mutant (Right) and of (C) the ΔGag construct, ΔGagMAjCA=SP1jNCCAjSP1=SP1jNC, in which the

MAjCA and the CAjSP1 cleavage sites (highlighted in bold in A) have beenmutated to the same sequence as that of the SP1jNC cleavage site (SAT-IMjMQRGN). In all cases, Gag processing by PR-O occurs in the same sequentialorder. Cleavage rates obtained from analysis of the time courses of the SDS/PAGE band intensities (see Fig. S2 and Table S1) are indicated. Aliquots of thereaction mixture at room temperature were taken at regular time intervals andvisualized by PageBlue staining [18% (wt/vol) Tris-glycine gel]. The concentra-tion of PR-O was 1 μM (in subunits), and the ratio of Gag to protease (in sub-units) was 50:1 in B and 100:1 in C. Buffer conditions were as follows: 20 mMsodium phosphate, pH 6.5, 300 mM NaCl, 0.1 mM ZnCl2, and 1 mM TCEP.

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NC domain and the negatively charged protease, which are mag-nified at low salt.The unexpected absence of intermolecular PREs in the vi-

cinity of the Gag cleavage sites can be attributed to two factors:(i) the occupancy of transient encounter complexes (lifetimes ≤250–500 μs probed by PRE) (18) involving the cleavage sites ismuch lower than that involving the globular domains of Gag; and(ii) the slow formation of weak (KD ∼300 μM) productive Gag–protease complexes are rate limited by protease flap opening(21, 22). The flaps of free protease are predominantly closed(23), blocking access to substrate; flap opening is a rare pro-cess resulting in slow productive complex formation (lifetime∼100 ms) (22), which is manifested by a significant reduction in1HN/

15N cross-peak intensity for residues at the SP1jNC junctionof ΔGagW316A

M317A on addition of a 3:1 excess of unlabeled protease(Fig. S8A). [Note that 1HN/

15N cross-peaks for residues at theMAjCA and CAjSP1 junctions are not affected at the concen-trations of protease used, indicating that the SP1jNC junction

serves as the primary point of association between Gag andprotease, consistent with the observed order and rates of pro-teolytic cleavage (see Fig. 1B).] The converse experiment ti-trating a threefold excess of unlabeled ΔGagW316A

M317A into 15N/2H-labeled PR-OD25N

V82C results in a decrease in intensity of 1HN/15N

cross-peaks within the flaps and catalytic site of protease with nochanges in chemical shifts (Fig. S8B), as expected for a system onthe slow side of intermediate exchange on the chemical shifttime scale.To map the interaction sites for transient encounter complexes on

the protease, we carried out intermolecular PRE experiments using15N/2H-labeled PR-OD25N

V82C and two different paramagnetically(Gd3+)tagged Gag constructs. Site-specific incorporation of a paramagneticprobe on ΔGag presents a challenge owing to the presence of 10native cysteine residues. For one construct, we therefore used atruncated Gag comprising only MA and the N-terminal domain ofCA (MA-CANTD), in which one of the two native cysteines wasmutated to Thr (C57T), whereas the other, Cys87, located close to

Residues150 200 250 300 350 400

0

20

40

60

50 100 150 200 250 300 350 400

0

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40

60

MA1-132 CA133-277NTD CA282-363

CTD NC378-432

SP1364-377

H33 A196 K272

G178 D235

L243G357

R286

M423

12 3

W414Q195

S148

K30E12

S77T81

G412

E312T186 A220

Q139S148

Gag

M31

7AW

316A

+ P

R-O

L72C

/ V

82C

D25

ND

25N

CA

-SP

1-N

C M

317A

W

316A

+ P

R-O

L72C

/ V

82C

D25

ND

25N

MA

CANTD

P133

NC

T81

Zn2+ M378

A364

Q195, A196

E312

SP1

W414 M423

R286CACTD

D

L72C

V82C

D25N

DOTA-Gd3+

A

B

C

FlapsH219

R409

M200

E12

H33

T401I389, V390

D235

Core

Active SiteK30

G178

G357

K272

L243

G412

L75, R76

T186Y79

Zn2+

Fig. 2. PRE mapping of transient interactions of Gag with PR-O. (A) Ribbon diagram of PR-O with sites of paramagnetic tagging with DOTA-Gd3+ indicated(L72C, blue; V82C, red). Intermolecular PRE profiles observed on (B) 15N/13C/2H-labeled ΔGagW316A

M317A and (C) 15N/2H-labeled CA-SP1-NCW316AM317A monomeric Gag

constructs arising from Gd3+-tagged PR-OD25NL72C (blue) and PR-OD25N

V82C (red). Regions with intermolecular PREs above background (dashed lines) are indicated bythe gray bars; residues exhibiting large PREs and associated with drug resistance (14–16, 28, 29) are indicated by the bold italic labels. The concentration of PR-O(in subunits) is 100 μM with a Gag to PR-O ratio of 3:1 and 2:1 for ΔGag and CA-SP1-NC constructs, respectively. (D) Ribbon representation of ΔGag (note therelative orientation of theMA, CA, and NC domains is arbitrary as the globular domains reorient semi-independently of one another in solution) (3). Gag cleavagejunctions are highlighted with black circles and scissors. Regions of ΔGagW316A

M317A exhibiting PREs above background in the presence of paramagnetically taggedPR-OD25N

V82C are colored in blue; residues that exhibit large PREs and are associated with drug resistance are depicted as dark blue spheres; those exhibiting large PREsonly are shown as light blue spheres. Three residues (Thr186, Met200, and His219) in CA also associated with drug resistance (15, 16) are depicted as orangespheres; two of these (Thr186 and His219) give large PREs at low ionic strength (see C ), whereas Met200 is close to the patch of CA residues (residues 192–198) that exhibit large PREs at both high and low ionic strengths.

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the MAjCA cleavage junction, was conjugated with DOTA-Gd3+

(MA-CAC57T,C87NTD ) (Fig. 3A). In the second, monomeric ΔGagW316A

M317Awas complexed to a single-stranded (ss)DNA 8mer, d(TG)4 con-taining a single DOTA-Gd3+ derivatized deoxythymidine (Fig. 3Aand Fig. S3B). A slight molar excess of ΔGagW316A

M317A was used, en-suring that all ssDNA was bound to NC, as the interaction of MAwith ssDNA is very weak in comparison (3, 24). With para-magnetically tagged MA-CAC57T,C87

NTD , regions of protease exhibitingPREs above background (1HN-Γ2 > 4 s−1) comprise residues near orin the catalytic site (residues 6–8 and 26–31), residues in the flaps(residues 46–53 and 82–83), and a small stretch of core residues(residues 88–94; Fig. 3B, Upper). With paramagnetically taggedΔGagW316A

M317A-d(TG)4, regions of protease exhibiting significant PREsinclude residues near or in the catalytic site (residues 5–10 and

23–29), the flaps (residues 30–43, 46–59, 71–77 and 80–83), and partof the core (residues 64–66 and 87–97; Fig. 3B, Lower). Essentially nointermolecular PREs above background are observed on pro-tease in control experiments using DOTA-Gd3+ or Gd3+-taggedMBP (Fig. S9).

DiscussionRole of Gag–Protease Encounter Complexes. The observed patternof intermolecular PRE profiles are indicative of the formation oftransient encounter complexes involving the globular domains ofGag, which serve to guide protease in the vicinity of the Gagcleavage sites such that productive complex formation can occurefficiently on rare protease flap opening (Fig. 4). These en-counter complexes are likely to have a significant impact on the

A364

P133

M378

dT-DOTA-Gd3+

C87-DOTA-Gd3+

Zn2+

d(TG)4

MA

CANTD

CACTD

NC

SP1

0

5

10

N25

Flaps

C57T

20 40 60 800

10

20

W6T26

D7

T74

G94

Residues

A B

CTip of flaps

D25N

10

93

4336

535048

4746

8890

73

74

71

82833334

54

64

7677

24

Flaps

L19

G40T80L72

T26M46

G51Q61

D29N88

Subunit B Subunit A

catalyticsite

32

F53

Fig. 3. PRE mapping of transient PR-O interactions with Gag. (A) Ribbon diagram of ΔGag with the location of the Gd3+ paramagnetically tagged sitesindicated. Site-specific incorporation of a paramagnetic probe on ΔGag using traditional cysteine chemistry is difficult due to the presence of 10 nativecysteine residues. Two constructs were therefore used. The first comprises MA-CAC57T,C87

NTD in which Cys57 is mutated to Thr and Cys87 is paramagneticallytagged. The second comprises ΔGagW316A

M317A complexed via the NC domain to the single stranded oligonucleotide d(TGT*GTGTG), where T* is DOTA-Gd3+

derivatized deoxythymidine. (B) Intermolecular PRE profiles observed on 15N/2H-labeled PR-OD25NV82C in the presence of the paramagnetically tagged Gag var-

iants MA-CAC57T,C87NTD (blue; Upper) and ΔGagW316A

M317A-d(TG)4 (red; Lower). Regions with intermolecular PREs above background (dashed lines) are indicated by thegray bars. Residues exhibiting large PREs and associated with drug resistance (17) are labeled in bold italics. The concentration of PR-O was ∼150 μM(in subunits), with PR-O to Gag molar ratios of 5:1 for MA-CAC57T,C87

NTD and 3:1.2 for ΔGagW316AM317A with 50 μM ssDNA. (C) Ribbon representation of the PR-O dimer.

Residues exhibiting large intermolecular PREs from paramagnetically tagged MA-CAC57T,C87NTD and ΔGagW316A

M317A-d(TG)4 are shown in blue (on subunit A) and red(on subunit B), respectively. Residues that show large PREs and are associated with drug resistance (17) are depicted as spheres.

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cleavage rates at the different Gag junctions. (It should be noted,however, that functional testing of the role of encounter com-plexes by in vitro mutagenesis is likely to be very difficult as theinterfaces are relatively large and the contribution of any indi-vidual intermolecular interaction is likely to be very small.Hence, a large number of cumulative mutations would likely berequired.) Other lines of biochemical evidence support this pic-ture: the presence of single-stranded nucleic acids significantlyaccelerates hydrolysis at the SP1jNC junction while leaving thecleavage rate at the MAjCA and CAjSP1 unaltered (3). The twozinc knuckles of NC sample a large region of conformationalspace relative to one another in the absence of nucleic acids butbehave as a single globular entity when bound to nucleic acids(24, 25), which would be predicted to enhance the formation oftransient encounter complexes with protease. The two slowestcleavage sites (CAjSP1 and NCjSP2) occur at junctions that are notflanked by globular domains, suggesting that transient confinementof protease between Gag domains is important in sequential pro-cessing. A recent report (26) suggested that the six-helix bundleformed by SP1 (27) in immature Gag assemblies may block accessto the CAjSP1 junction, thereby slowing down the proteolysis atthis site. However, SP1 and neighboring residues are intrinsicallydisordered and fully accessible in solution in the context of ΔGag,as evidenced by NMR chemical shifts, relaxation parameters, andbackbone amide RDCs (3, 24), and yet hydrolysis at the CAjSP1junction is remarkably slow in vitro (compare Fig. 1B), even whenmutated to the sequence of the SP1jNC junction (see Fig. 1C).Moreover, of the five Gag cleavage sites, only the CAjSP1 andSP1jNC junctions, located a mere 15 residues apart, are in-terdependent on one another such that blocking hydrolysis at theSP1jNC junction by site-directed mutagenesis accelerates hydro-lysis of the upstream CAjSP1 junction (4). These observationssuggest that protease, guided by the CA and NC domains, cancompetitively interact with both cleavage junctions on either side ofSP1, with an intrinsic preference for the SP1jNC junction. Basedon these observations, we conclude that sequential in vitro hy-drolysis of Gag is governed by several factors: transient interactionof Gag domains with protease, accessibility of Gag cleavage junc-tions, and the primary sequence of Gag cleavage sites. Moreover,because the sequential order of proteolytic processing is the samein vitro and in vivo, we speculate that these factors also dictate Gaghydrolysis in vivo. Additionally, steric hindrance due to the as-sembled Gag lattice and the protonation states of cleavage siteresidues are likely to also contribute to Gag processing in vivo.

Correlation of Sites of Encounter Complexes with Compensatory DrugResistance Mutations. A comparison of regions involved in tran-sient Gag–protease encounter complexes with residues associ-ated with drug-resistance mutations (14–17, 28) in Gag andprotease is provided in Figs. 2D and 3C, respectively (also see

Table S2 for additional details). The emergence of mutationsresistant to HIV-1 protease inhibitors usually involves a stepwiseprocess whereby primary mutations in protease alter the sub-strate-binding pocket leading to a reduction in inhibitor bindingand concomitantly productive Gag–protease complex formation,whereas secondary or compensatory mutations in both prote-ase and Gag may help partially restore the loss of viral fitnesscaused by the primary mutations (10, 15, 16). The regions of Gagand protease that undergo compensatory mutations are strik-ingly similar to those exhibiting significant intermolecular PREs,thereby providing a plausible underlying structural basis behindthese mutations. For example, conservative noncleavage sitemutations in MA, specifically K30R, R76K, Y79F, and T81A,restore the fitness deficit arising from drug-resistant protease byimproving replication capacity and generating a significant re-duction in susceptibility to protease inhibitors in vivo (14, 29).These residues along with two other drug-resistance mutations,E12K and L75R, reside in regions of MA that give large in-termolecular PREs. Similarly, among the few compensatorymutations in CA that have been reported, T186M and H219Qdisplay large intermolecular PREs, whereas the third, M200I,is close to a region of CA that exhibits large PREs (residues192–198). Further, several residues of NC mutate on exposureto protease inhibitors, and every single one displays large in-termolecular PREs. It should also be noted that some of theseresidues are involved in other well-established functions [e.g.,Lys30 and Arg76 of MA are involved in Gag–membrane inter-actions (30, 31), and His219 is located in an exposed loop withinthe N-terminal domain of CA loop that binds to cyclophilin-A(32)], implying multiple functions for these residues. The pri-mary and secondary mutations in protease also show a closecorrelation with residues that exhibit significant intermolecularPREs, including residues in the core (residues 10, 71, 73, 88, 90,and 93) and flaps (residues 32–34, 36, 46, 47, 48, 50, 53, 54, and82). Based on the above correlations, we conclude that HIV-1Gag–protease interactions have evolved a conserved mechanismwhereby the globular domains of Gag act as guideposts forthe formation of transient encounter complexes that facilitateaccess of the protease to the cleavage junctions, thereby en-hancing cleavage rates and modulating the sequential order ofGag cleavage.

Materials and MethodsProtein Expression and Purification. Full details on cloning, expression, site-directed mutagenesis, isotope (2H/15N/13C and 2H/15N) labeling, purification,paramagnetic tagging with Gd3+, and analytical ultracentrifugation exper-iments are provided in SI Materials and Methods. Samples for NMR wereprepared in a buffer containing 20 mM sodium phosphate, pH 6.5, 0.1 mMZnCl2, and 1 mm Tris(2-carboxyethyl)phosphine (TCEP); additionally, the

Gag

Protease Encountercomplexes

fast slow

Slow conformational transitions

(flaps: closed - open)

hydrolysis

PREs Chemical ShiftsPeak Intensity

Real-time NMR

Fig. 4. Schematic of the interactions of HIV-1 protease and Gag. Each illustrated step occurs at successively slower time scales that are accessible to differenttypes of NMR measurements (as indicated in green). Transient encounter complexes, in fast exchange with the unbound species, are detected by PREmeasurements (see Figs. 2 and 3). These ultra-weak complexes, involving quite extensive interactions surfaces, are initially formed and serve to guide theformation of productive complexes, thereby dictating the sequential order of Gag cleavage (i.e., we speculate that these transient encounter complexesaccelerate the formation of productive complexes). The formation of productive complexes at the Gag cleavage sites are associated with rare conformationaltransitions involving flap opening (22, 23) that are detected by changes in cross-peak intensities and chemical shifts (intermediate exchange; see Fig. S8).Finally, cleavage/liberation of individual MA, CA, and NC domains is a slow process that can be monitored by real-time NMR by observing site specific build-upof cross-peaks arising from the cleavage products (3).

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buffer contained 300 and 50 mM NaCl in the case of ΔGag and CA-SP1-NCconstructs, respectively.

NMR Spectroscopy. All heteronuclear NMR experiments were carried out at30 °C on Bruker 500-, 600-, and 800-MHz spectrometers equipped withz-gradient triple resonance cryoprobes. Full details of the sequential assign-ment, PRE and RDC experiments, and NMR spectral processing and analysis areprovided in SI Materials and Methods.

ACKNOWLEDGMENTS. We thank M. Bayro, J. Werner-Allen, C. D. Schwieters,D. Appella, and V. Tugarinov for useful discussions; Y. Shen for help withCS-ROSETTA calculations; Y. Kim for help with DOTA-Gd3+ purification;A. Aniana for technical assistance; and J. Lloyd [National Institute of Diabe-tes and Digestive and Kidney Diseases (NIDDK) Advanced Mass SpectrometryCore] for technical support. This work was supported by the Intramural Pro-gram of NIDDK/NIH and the AIDS Targeted Antiviral Program of the Officeof the NIH Director (to G.M.C.).

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