Letter

Nature 455, 693-696 (2 October 2008) | doi:10.1038/nature07342; Received 7 July 2008; Accepted 12 August 2008; Corrected 14 November 2008

Visualizing transient events in amino-terminal autoprocessing of HIV-1 protease

Chun Tang1,2, John M. Louis1, Annie Aniana1, Jeong-Yong Suh1 & G. Marius Clore1

  1. Laboratory of Chemical Physics, Building 5, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, USA
  2. Present address: Department of Biochemistry, University of Missouri, Columbia, Missouri 65211, USA.

Correspondence to: G. Marius Clore1 Correspondence and requests for materials should be addressed to G.M.C. (Email: mariusc@mail.nih.gov).

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HIV-1 protease processes the Gag and Gag-Pol polyproteins into mature structural and functional proteins, including itself, and is therefore indispensable for viral maturation1, 2. The mature protease is active only as a dimer3, 4, 5 with each subunit contributing catalytic residues6. The full-length transframe region protease precursor appears to be monomeric yet undergoes maturation via intramolecular cleavage of a putative precursor dimer5, 7, 8, 9, 10, 11, concomitant with the appearance of mature-like catalytic activity7, 9. How such intramolecular cleavage can occur when the amino and carboxy termini of the mature protease are part of an intersubunit beta-sheet located distal from the active site is unclear. Here we visualize the early events in N-terminal autoprocessing using an inactive mini-precursor with a four-residue N-terminal extension that mimics the transframe region protease precursor5, 12. Using paramagnetic relaxation enhancement, a technique that is exquisitely sensitive to the presence of minor species13, 14, 15, 16, we show that the mini-precursor forms highly transient, lowly populated (3–5%) dimeric encounter complexes that involve the mature dimer interface but occupy a wide range of subunit orientations relative to the mature dimer. Furthermore, the occupancy of the mature dimer configuration constitutes a very small fraction of the self-associated species (accounting for the very low enzymatic activity of the protease precursor), and the N-terminal extension makes transient intra- and intersubunit contacts with the substrate binding site and is therefore available for autocleavage when the correct dimer orientation is sampled within the encounter complex ensemble.

The regulation of HIV-1 protease autoprocessing is modulated by the N-terminal flanking transframe region (TFR) sequence (Fig. 1a)2. The catalytic activity of the monomeric protease precursor is approximately three orders of magnitude less than that of the mature protease dimer (which has a monomer–dimer equilibrium dissociation constant Kd < 10 nM)2, 5. The appearance of mature-like catalytic activity and stable dimer formation is directly correlated with a single rate-limiting step comprising intramolecular (first order) cleavage of a putative transient dimeric precursor species at the p6pol–protease (PR) junction7, 9, 10. Mutations within the latter that prevent cleavage lead to the production of an N-terminally extended 17-kDa protease precursor species, and cause a severe defect in Gag polyprotein processing and the complete loss of viral infectivity in vivo17, 18. Subsequent cleavage at the C terminus of protease at the PR–reverse transcriptase (RT) junction (Fig. 1a) occurs via an intermolecular (second order) reaction catalysed by a fully active protease dimer19. Mutations within the PR–RT junction that block C-terminal cleavage do not significantly affect either enzymatic activity and dimerization of the protease in vitro19, 20 or processing of HIV-1 precursor proteins, virus maturation, viability and morphology in vivo20, indicating that the presence of the C-terminal reverse transcriptase sequence has negligible influence on the protease precursor19, 20. Thus, only autoprocessing at the N terminus of protease at the p6pol–PR junction is an absolute prerequisite for stable protease dimer formation, the appearance of mature catalytic activity and complete processing of viral precursors. Before cleavage at the p6pol–PR junction, intermediate precursor forms may be liberated by intramolecular cleavage at competing sites (for example, p2–NC and TFP–p6pol; see Fig. 1a) that become available for productive binding and hydrolysis11, but these precursors will show the same low catalytic activity as that of the p6pol–PR precursor9, 10.

Figure 1: Intermolecular PRE profiles.
Figure 1 : Intermolecular PRE profiles. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com

a, Organization of the Gag-Pol polyprotein1, 2. b–d, Intermolecular PREs observed on U-[2H/13C/ 15N]-labelled SFNFPR(D25N) originating from a spin label conjugated to T12C (b), E34C (c) and V82C (d) of SFNFPR(D25N) at natural isotopic abundance. Residues broadened beyond detection are denoted by open bars. Error bars represent 1 s.d. Gamma2 rates back-calculated from the structure of the mature dimer (for the core residues 10–94) at populations of 1% and 2% are shown as blue and green lines, respectively. Average Gamma2 rates derived from the top 20 structures of the Ne = 4 simulated annealing calculations at a population of 5% heterodimer are shown as black lines. Grey shaded areas delineate residues that are buried at the dimer interface in the mature protease. e, f, Observed intermolecular PREs originating from the spin label attached to T12C (e) and V82C (f) colour-coded on a ribbon diagram of the mature dimer24 (spin label attached to the blue subunit). Atomic probability density maps25 (plotted at a threshold of 10% of maximum) showing the distribution of the spin-label oxygen radicals are shown as red meshes.

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As little as a four-residue extension at the N terminus of protease, corresponding to the C-terminal residues of p6pol, in conjunction with a D25N mutation result in an effectively monomeric species5, 12. Disruption of the native protease dimer by N-terminal extension is due to removal of the protons on the secondary amine of the N-terminal proline residue, disrupting the interstrand hydrogen bond between the amine of the N-terminal proline of one subunit and the C-terminal carbonyl oxygen of the second subunit6. C-terminal extension, however, does not have an impact on this interstrand hydrogen bond because the secondary amine of Pro 1 is preserved. Therefore, we made use of the mini-precursor, bearing only the N-terminal cleavage site, to visualize the early transient events involved in autoprocessing of the protease at the p6pol–PR junction that is required for the formation of a fully active, stable protease dimer.

The optimized mini-precursor protease construct SFNFPR(D25N) comprises a four-residue N-terminal extension (Ser-Phe-Asn-Phe) derived from the TFR (Fig. 1a), a D25N mutation to abolish all residual catalytic activity, and C67A and C95A mutations to remove surface cysteines (Supplementary Fig. 1a)9, 10, 12. The corresponding active SFNFPR(D25) mini-precursor construct undergoes autoprocessing during expression to release the mature protease (see Methods). NMR analysis of SFNFPR(D25N) shows that it is monomeric (with an upper limit of approx10% dimer from translational diffusion measurements); the secondary and tertiary structures of the mature protease are preserved with the exception of the N- and C-terminal strands which form an intersubunit four-stranded anti-parallel beta-sheet in the mature dimer; and residues -4 to 9 and 95–99 are disordered and highly mobile (see Methods and Supplementary Fig. 1b–e).

Because enzymatically active protease is dimeric, and the rate-limiting step in autoprocessing is unimolecular7, 9, transient self-association of the precursor must occur to initiate autoprocessing. To visualize this phenomenon we measured intermolecular paramagnetic relaxation enhancements (PREs) by introducing a spin label via conjugation to three engineered surface-exposed cysteine residues: T12C, E34C and V82C (one at a time). These sites are frequently mutated in viable HIV-1 variants2. T12C and V82C are located at the periphery of the substrate-binding cleft in the mature dimer, whereas E34C is relatively far removed from the dimer interface (Fig. 1e, f). In a rapidly exchanging system, the PRE 1HN-Gamma2 rates21 are population-weighted averages of the PRE rates of the species present13, 14. Because the PRE rate for a paramagnetic centre-proton pair is proportional to the < r-6 > average of the distance between them, and the PRE effect is large owing to the high magnetic moment of an unpaired electron, the PRE in the fast exchange regime is very sensitive to the presence of lowly populated (<5%), highly transient species in solution providing there are paramagnetic centre-proton distances in the minor species that are shorter than in the predominant species13, 14, 15.

PREs were measured on a 1:1 mixture of 0.2 mM U-[2H/13C/15N]-labelled SFNFPR(D25N) and spin-labelled SFNFPR(D25N) at natural isotopic abundance. Because 1HN-Gamma2 rates are measured using 1H–15N correlation-based experiments21, the observed 1HN-Gamma2 rates arise solely from intermolecular interactions between the spin-labelled protein and the isotopically labelled protein (Fig. 1b–d). For the E34C spin label, no 1HN-Gamma2 rates greater than 5 s-1 are observed (Fig. 1c); this sample therefore provides a negative control, excluding the existence of solvent PRE effects arising from diffusion and random elastic collisions, or from direct intermolecular interactions between the spin label and the U-[2H/13C/15N]-labelled protein. The PRE profiles for the T12C (Fig. 1b) and V82C (Fig. 1d) spin labels are similar but the magnitude for the latter is 4- to 8- fold greater than for the former. Within the ordered core of the precursor (residues 10–94), large intermolecular PREs are observed for residues 21–30, 46–55 and 80–85 located at or close to the dimer interface. Residues 21–30 encompass the catalytic triad, residues 46–66 correspond to the flap region which gates the active site, and residues 80–81 and 83–84 are located in the substrate binding cleft (Fig. 1e, f). In addition, the N-terminal region experiences sizeable PREs from the T12C (Fig. 1b) and V82C (Fig. 1d) spin labels. These data demonstrate that transient self-association of the precursor involves residues located at the dimer interface in the mature dimer. A similar intermolecular PRE profile is observed from V82C spin-labelled, full-length TFR–PR(D25N) precursor to U-[2H/13C/15N]–SFNFPR(D25N), indicating that the transient dimerization interface is preserved on further N-terminal extension of the protease precursor (Supplementary Fig. 2a).

Back-calculation of the PREs from the structure of the mature dimer shows that almost zero PRE values are expected for the T12C and E34C spin labels at a population of 1–2% mature heterodimer (Fig. 1b, c). For the V82C label, small PRE values at a population of 1–2% mature heterodimer are predicted for residues 27–30 and 48–50 (Fig. 1d, blue line). The mature dimer does not predict the large observed PRE values observed for residues 20–26, 30–35 and 80–83. Furthermore, in the mature dimer residues 80–83 of one subunit are located on the opposite side of the dimer interface from residues 80–83 of the other subunit, and thus the large intermolecular PREs observed from the V82C spin label to residues 80–83 would require a approx180° rotation of one subunit relative to its position in the mature dimer. Thus, the upper limit of the total population of mature dimer (heterodimer and homodimer) cannot exceed 2–4%.

Transient interactions between SFNFPR(D25N) precursor monomers were visualized semi-quantitatively using rigid-body simulated annealing calculations14, 16, 22 to optimize the agreement between observed and calculated Gamma2 rates arising from the T12C, E34C and V82C spin labels simultaneously (see Methods). The flexible N- and C-terminal regions (residues -4 to 9 and 95–99, respectively) were excluded from the calculations. A single conformer representation (Ne = 1) for the transient dimer does not account for the PRE data and even at a heterodimer population of 15% the PRE Q-factor23 (see Methods for definition) has a value of greater than 0.4 (Fig. 2a). Thus, the dimeric SFNFPR(D25N) precursor is an ensemble of multiple encounter complexes. For Ne greater than or equal to 2, the average PRE Q-factor decreases rapidly as the heterodimer population is increased above 1%, levelling off at a population of approx5% (Fig. 2a). The best results are obtained with Ne = 4, and larger ensemble sizes are unjustified and would result in over-fitting the data. For Ne = 4, the PRE Q-factors at a heterodimer population of 3–5% are close to the expected PRE Q-factor based on experimental error (Fig. 2a), consistent with translational diffusion data (Methods and Supplementary Fig. 1d). Given a total protein concentration of 0.4 mM, the apparent Kd for self-association is therefore 3–6 mM. A comparison of the calculated and observed PRE profiles and a correlation plot of observed versus calculated Gamma2 rates for Ne = 4 at a heterodimer population of 5% are shown in Fig. 1b–d and Fig. 2b, respectively.

Figure 2: Ensemble simulated annealing and the protease mini-precursor encounter complex ensemble.
Figure 2 : Ensemble simulated annealing and the protease mini-precursor encounter complex ensemble. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com

a, PRE Q-factor as a function of ensemble size and population of heterodimer. Dashed line denotes the expected Q-factor when agreement between observed and calculated Gamma2 rates is comparable to the experimental error in the measurements. b, Correlation between observed and calculated Gamma2 rates for Ne = 4 and a heterodimer population of 5%. Qee is the ensemble of ensembles average PRE Q-factor for the 20 calculated Ne = 4 ensembles and r the correlation coefficient. Error bars in a and b represent 1 s.d. c, Atomic probability density map25 (grey mesh, plotted at a threshold of 20% of maximum) showing the distribution of the spin-labelled subunit relative to the isotopically labelled subunit (red ribbon) in the SFNFPR(D25N) encounter complexes. The location of the second subunit in the mature dimer is shown as a blue ribbon. d, Orientations in spherical coordinates of the vector joining the centre of masses of the two interacting molecules in the encounter complexes relative to the coordinate system shown in c with the z axis corresponding to the C2 symmetry axis of the mature dimer. The phi,theta angles for the mature dimer are located at the crosshair. e–g, Representative encounter complexes (labelled and denoted by red dots in d) corresponding to the structures with the closest spherical angles (e), the smallest d.r.m.s. (f) and the smallest atomic r.m.s. displacement (g) relative to the mature dimer. The Calpha atom of Gly 51 at the tip of the flap is shown as a sphere to guide the eye. The isotopically labelled and spin-labelled subunits are shown in red and grey, respectively; the blue subunit corresponds to the orientation relative to the red subunit seen in the mature dimer. h, Histogram of the d.r.m.s. metric for the Ne = 4 structures (total of 20 times 4 = 80 conformers) at a population of 5% heterodimer.

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The distribution of the spin-labelled monomer relative to the isotopically labelled monomer in the computed ensemble of SFNFPR(D25N) encounter complexes is shown in Fig. 2c. The predominant interactions between the two monomers involve the same residues that comprise the dimer interface in the mature dimer, and one subunit of the mature dimer is embedded within the ensemble distribution of the spin-labelled subunit. The orientation of the subunits in the encounter complex ensembles can be described by spherical angles describing the orientation of the vector joining the centre of masses of the two subunits to the coordinate axis frame. Many members within the calculated ensemble are clustered around the values corresponding to the mature dimer (Fig. 2d). This is reflected in the distribution of the distance root mean square (d.r.m.s.; see Methods) deviation metric where over one-half of the ensemble members have d.r.m.s. values less than 6 Å (Fig. 2h). However, the structures with spherical angles close to the mature dimer (indicated by arrows in Fig. 2d) and low d.r.m.s. values have a widespread range of relative self-rotations, as illustrated by three examples comprising the ensemble members with the closest spherical angles to the mature dimer (Fig. 2e), the smallest d.r.m.s. (Fig. 2f) and the smallest Calpha atomic r.m.s. displacement (Fig. 2g). The difference from the mature dimer in rotation angle about the axis joining the centre of masses of the two subunits ranges from 13° (Fig. 2g) to 135° (Fig. 2e), with an intermediate rotation angle of 70° for the structure in Fig. 2f (see Supplementary Fig. 3 for definitions). One can therefore conclude that the actual occupancy of a structure within the encounter complex ensemble corresponding to the mature dimer is very small.

To probe the conformational space sample by the disordered N-terminal flanking sequence of the SFNFPR(D25N) precursor we introduced a spin label on a Cys residue inserted immediately after the N-terminal serine (S(C)FNFPR(D25N)). PRE measurements were carried out on a 1:1 mixture of 0.2 mM U-[2H/13C/15N]–SFNFPRD25N precursor and 0.2 mM spin-labelled, natural isotopic abundance S(C)FNFPR(D25N) to detect intermolecular PREs, and on a sample of 0.2 mM spin-labelled, U-[2H/13C/15N]-labelled S(C)FNFPR(D25N) to observe both inter- and intramolecular PRE effects. Although the overall PRE profiles for the two samples are similar (although differences in detail are apparent), the magnitude of the PREs for the second sample is much larger than for the first, reflecting the contribution from intramolecular PREs (Fig. 3a). The N-terminal residues -4 to 9, and residues comprising the active site, flap and substrate-binding cleft, display large inter- and intramolecular PREs (Fig. 3a, c). The intermolecular PREs involving residues 82–84 are fully consistent with the large intermolecular PREs observed on the N-terminal residues from spin-labelled V82C (Fig. 1d). These data indicate that the N-terminal tail can insert itself into the active site and make transient contact with both subunits in the encounter complex ensemble. The spin label is located four residues proximal to the scissile peptide bond, and the observation that large PREs are observed for both sides of the active site (see Fig. 3c) suggests that the tail shuttles back and forth within the substrate binding cleft formed by the two subunits in the context of a dimer. Such translational movement is a functional requirement, as the protease precursor cuts the N-terminal transframe region in two major locations before cleaving its C terminus (Fig. 1a)9, 10. This is confirmed by the observation of a very similar intermolecular PRE profile from full-length TFR–PR(D25N) spin-labelled at position -44, four residues downstream from the TFP–p6pol cleavage site at residues -48/-49, to U-[2H/13C/15N]–SFNFPR(D25N) (Supplementary Fig. 2b).

Figure 3: PRE profiles with spin labels attached at the N- and C termini of the SFNFPR(D25N) mini-precursor.
Figure 3 : PRE profiles with spin labels attached at the N- and C termini of the SFNFPR(D25N) mini-precursor. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com

a, Intermolecular PREs (red) observed for a 1:1 mixture (0.2 mM each) of N-terminal spin-labelled S(C)FNFPR(D25N) at natural isotopic abundance and U-[2H/13C/15N]–SFNFPR(D25N), and the sum of the inter- and intramolecular PREs (blue) observed for 0.2 mM N-terminal spin-labelled U-[2H/13C/15N]–S(C)FNFPR(D25N). Residues broadened beyond detection are denoted by open bars. b, Intermolecular PREs observed for a 1:1 mixture (0.2 mM each) of U-[2H/13C/15N]–SFNFPR(D25N) and C-terminal spin-labelled (at N98C) SFNFPR(D25N) at natural isotopic abundance. Grey shaded areas in a and b delineate residues that are buried at the dimer interface in the mature protease. Error bars in a and b represent 1 s.d. c, d, Inter- and intramolecular PREs with Gamma2 rates >10 s-1 colour-coded in red and blue, respectively, onto the molecular surface of the mature protease dimer originating from the N-terminal (c) and the C-terminal (d) spin labels. The intramolecular PRE rates are given by the difference in PRE rates between the blue and red profiles in a. Cartoons of modelled N-terminal (residues -4 to 9) and C-terminal (residues 95–99) regions bearing the spin labels are included in c and d, respectively.

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The C-terminal region of the SFNFPR(D25N) precursor was spin-labelled at N98C. The resulting intermolecular PREs are much smaller than those with the spin label at the N terminus, but the PRE profiles are similar (Fig. 3b, d). Thus, the C-terminal flexible region can also make intermolecular contacts with the active site and substrate-binding cleft in the context of the precursor encounter complex ensemble. Because the N- and C termini are highly mobile, intermolecular PREs between the N- and C termini will be significantly attenuated. Nevertheless, intermolecular PREs are observed on residues 95–97 from the spin label at the N terminus (Fig. 3a), and on residues 5–8 (Fig. 3b) from the spin label at the C terminus (Fig. 3b). Small intermolecular PREs are also observed from the N98C spin label to the C-terminal region (residues 95–99). These observations might suggest the existence of transient, loose interactions between the N- and C termini that may partially approximate a portion of the intersubunit beta-sheet in the mature dimer.

The PRE data presented here demonstrate that although the HIV-1 protease precursor is predominantly monomeric, transient encounter complex dimers are formed using the same interface as that of the mature dimer but with a wide range of relative subunit orientations. Only a very small fraction of the encounter complexes adopt the same subunit orientation as in the mature protease, accounting for the very low enzymatic activity of the precursor. This small subset, which may be partially stabilized by transient, loose interactions involving the N- and C-terminal regions, can accommodate transient insertion of the N-terminal region including the N-terminal cleavage site in the substrate binding cleft, thereby providing a structural model for autoprocessing at the N terminus of the protease leading to the formation of a stable dimer with mature catalytic activity.

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Methods Summary

Sample preparation and NMR spectroscopy

Protein expression, mutagenesis, purification and conjugation of engineered surface cysteine residues to 3-iodomethyl-(1-oxy-2,2,5,5-tetramethylpyrroline) are described in the Methods. Samples for NMR were in 20 mM sodium phosphate buffer, pH 5.8. NMR experiments were collected at 20 °C at a 1H spectrometer frequency of 600 MHz. 1HN PRE data were acquired using a two-dimensional 1H–15N correlation-based pulse scheme with an interleaved two time-point measurement21.

Simulated annealing calculations

Conjoined rigid-body/torsion angle dynamics simulated annealing calculations on the basis of the PRE data were carried out using Xplor-NIH22 as described14.

Full methods accompany this paper.

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Supplementary Information

Supplementary information accompanies this paper.

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Acknowledgements

We thank R. Ishima for providing initial backbone assignments for the SFNFPR(D25N) protease construct; C. Schwieters for many discussions; Y. Sheng for help with the CS-Rosetta calculations; Y. Kim for providing the code for structure clustering and d.r.m.s. calculations; and J. Sayer for MALDI measurements. This work was supported by funds from the Intramural Program of the NIH, NIDDK and the AIDS Targeted Antiviral program of the Office of the Director of the NIH (to G.M.C.).

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Online Methods

Vector construction and protein sample preparation for NMR studies

Mutations T12C, E34C, V82C and N98C within the 99-amino-acid-long HIV-1 protease sequence and the mutation to insert a Cys in the flanking SFNF sequence (C-terminal residues of p6pol within the transframe region; see Fig. 1a and Supplementary Fig. 1) to generate S(C)FNFPR(D25N) were introduced in the SFNFPR(D25N) template5 using the appropriate forward and reverse primers and the QuikChange kit and protocol (Stratagene). The S(C)FNFPR(D25N) construct was used because we were unable to obtain efficient spin-labelling of a precursor protein bearing an N-terminal cysteine. Mutations A(-44)C (fifth residue of p6pol) and V82C (in the protease sequence) were also introduced in the full-length TFR–PR(D25N) construct (that is, TFP–p6pol–PR(D25N); see Fig. 1a and Supplementary Fig. 1a) using the same protocol. (The TFR is 56 residues in length and adopts a random coil conformation.) The newly introduced mutations were verified both by DNA sequencing and mass spectrometry. (Note that the SFNFPR(D25N) template, in addition to the D25N mutation which eliminates all traces of catalytic activity, and the C67A and C95A mutations which remove all additional surface cysteine residues other than that to which the spin label is going to be attached, also contains three other mutations, Q7K, L33I and L63I; the latter three mutations restrict autoproteolysis of the mature protease dimer, and have been shown to have indiscernible effects on structure, stability and catalytic activity of the mature dimer10.)

Escherichia coli BL21 (DE3) host cells bearing the appropriate vector were grown in Luria-Bertani medium or in D2O-based minimal medium containing 15N-NH4Cl and 13C6,2H7-glucose as the sole nitrogen and carbon sources, respectively, at 37 °C, and induced for expression. Proteins were purified from inclusion bodies using an established protocol as described previously involving size-exclusion chromatography under denaturing conditions followed by reverse-phase HPLC9, 26. Peak fractions (approx0.5 mg ml-1) were stored in aliquots at –70 °C. Alternatively, two aliquots (2.5 mg) of the proteins were lyophilized and stored at –20 °C.

A total of 2.5 mg of the lyophilized protein was dissolved in 1.2 ml of 4 M guanidinium-HCl, 1.7 mM HCl, pH 1.6. Spin-label conjugation was carried out by dissolving 0.5 mg of 3-iodomethyl-(1-oxy-2,2,5,5-tetramethylpyrroline) (catalogue number I709500; Toronto Research Chemicals) in 10 mul of ethanol, followed by the addition of 140 mul of 1 M Tris-HCl, pH 8, and adding the resulting mixture to the protein solution. After incubation for 1 h at room temperature, 30 mul of 1 M dithiothreitol was added and the incubation continued for another 1.5 h. The sample was loaded onto a Superdex-75 column (1.6 times 60 cm, GE HealthCare) equilibrated in 4 M guanidinium-HCl, 20 mM sodium formate, pH 2.6, at a flow rate of 1.4 ml min-1 at room temperature. Peak fractions were pooled and the concentration was estimated by measuring absorbance at 280 nm. The extent of labelling was 100% as determined by MALDI–TOF analysis on a Voyager-DE instrument (Perceptive Biosystems). Spin-labelling does not perturb the structure of the SFNFPR(D25N) mini-precursor as judged by NMR spectroscopy. The three mutations within the protein core, T12C, E34C and V82C, are frequently mutated in viable HIV-1 variants and are therefore not expected to alter significantly the catalytic properties of the protease2. It should be noted that V82C is located close to the substrate binding cleft comprising residues 80–81 and 83–85, but its side chain points outwards towards solvent. In the one instance where kinetic data are available for a mutation at position 82 (V82A), only a modest 10–15% decrease in kcat/Km relative to wild type is observed, and structural differences between wild-type protease and the V82A mutant are insignificant, with an r.m.s. deviation between the two crystal structures of only 0.12 Å for all main chain atoms27. Thus, the presence of a bulky spin label at position 82 would not be expected to result in a major perturbation in catalytic activity.

After extensive dialysis against 7 mM HCl, 1.4 mg each of the conjugated protein and the U-[2H/13C/15N]-labelled SFNFPR(D25N) protein were mixed and adjusted to a final concentration of 0.25 mg ml-1 protein, 35% acetonitrile and 0.05% trifluoroacetic acid. The solution was dialysed against 2 l of 7 mM HCl and 4 l of 20 mM sodium phosphate, pH 5.8, each for a period of 1.5–2 h and concentrated to approx400 muM using Amicon Ultra-4 (10,000 MWCO) devices. Protein concentration (mg ml-1) was determined spectrophotometrically using epsilon (0.1%) = 1.097 at 280 nm.

Control active SFNFPR(D25) mini-precursor protease construct

The SFNFPR(D25N) precursor construct does not undergo autoprocessing owing to the substitution of the active site Asp 25 by Asn. To verify that SFNFPR(D25N) represents a suitable model system we examined the autoprocessing activity of the corresponding SFNFPR(D25) precursor; that is, the precursor without the active site mutation. Most of the expressed protein undergoes maturation at the N terminus (between Phe-Pro) of the protease in the control SFNFPR(D25) precursor to produce the mature protease as expected. This was confirmed by subjecting an aliquot of the purified (dissolved) inclusion bodies to electrospray-mass spectrometry. The measured mass of 10,728 Da clearly corresponds to the PR(D25) mature protease (calculated mass of 10,728.3 Da). Under identical conditions of analysis for SFNFPR(D25N), which is devoid of catalytic activity, only the full-length protein corresponding to a mass of 11,222 Da (calculated mass of 11,222.8 Da) is observed consistent with previous observations from studies using the inactive full-length TFR–PR(D25N) precursor, which does not undergo maturation5, as compared to the active TFR–PR(D25) precursor, which exhibits time-dependent processing at the p6pol–protease junction to release the mature protease9, 10.

NMR experiments

All NMR data were acquired at 20 °C on a Bruker DRX600 spectrometer equipped with a z-gradient triple resonance cryoprobe.

Measurement of translational diffusion coefficients (Ds) by pulse field gradient NMR28 was carried out using the Watergate BPP-LED pulse scheme described previously29. The translational diffusion coefficient Ds is derived from a linear least-squares fit to a plot of ln[I(f)/I(f0)] versus (f2 - f02):

Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com

where I(f) and I(f0) are the intensities of the NMR signal at fractional gradient strengths of f and f0; f0 is the fractional gradient strength of the reference spectrum (0.1); f is the fractional gradient strength with values of 0.2, 0.3, 0.4, 0.5 and 0.6 times Gmax, the maximum gradient strength (70 times 10-4 T cm-1); italic gamma is the gyromagnetic ratio of 1H (2.6752 times 108 s-1 T-1); Delta = 15.4 ms; delta = 5 ms (gradient duration); and tau = 0.2 ms. The overall diffusion delay is 10 ms. The value of the scaling factor (italic gammadeltaGmax)2(Delta- delta/3 - tau/2) is 1.19 times 1010 s m-2. The values of Ds were 9.3(plusminus0.4) times 10-11 and 12.9(plusminus0.5) times 10-11 m2 s-1 for the mature PR(D25N) dimer and the SFNFPR(D25N) precursor, respectively, at the same (0.4 mM) subunit concentration (Supplementary Fig. 1d). The ratio of the two Ds values (0.72 plusminus 0.04) is fully consistent with the expected value of 0.75 for a Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com ratio28, placing an upper limit of about 10% for the population of dimeric species.

15N-{1H} heteronuclear NOE measurements were carried out using a flip-back scheme as described30. Residues -4 to 9 and 95–99 of SFNFPR(D25N) have heteronuclear 15N-{1H} NOE values ranging from -1 to 0.5 indicating that they are disordered and highly mobile. Backbone assignments were derived using the following three-dimensional triple resonance experiments: HNCO, HN(CO)CA and CBCA(CO)NH31, 32. The weighted mean backbone chemical shift difference between different constructs is given by [Deltadelta2HN + Deltadelta2N/25 + Deltadelta2Calpha/4]1/2 as described previously33. A comparison of 1H/15N/13Calpha chemical shifts reveals significant perturbations relative to the corresponding mature dimeric PR(D25N) for residues located at the dimer interface (Supplementary Fig. 1b), but only minor perturbations relative to the equivalent monomeric PR(1–95) construct obtained by deletion of the C-terminal four residues (Supplementary Fig. 1c)5. Analysis of the chemical shift index (based on 13Calpha, 13Cbeta and 13C' shifts)34 for SFNFPR(D25N) and PR(D25N) indicates that the secondary structure elements are preserved in the precursor with the exception of the N- and C-terminal strands which form an intersubunit four-stranded antiparallel beta-sheet in the mature dimer (Supplementary Fig. 1e).

PRE 1HN-Gamma2 rates are given by the difference in R2 relaxation rates between the paramagnetic (spin-labelled) and diamagnetic states of the protein. R2 rates were determined from a two-time-point interleaved two-dimensional 1H–15N correlation-based experiment, as described previously21. The time interval between the two time points was 32 ms for the intermolecular PRE measurements and 4 ms for the intramolecular PRE measurements. The short time interval for the latter is used to minimize any errors in Gamma2 rates introduced by any potential diamagnetic contamination (that is, spin-labelling less than 100%)21.

Tertiary structure of SFNFPR(D25N)

To verify that the tertiary structure of the ordered region of SFNFPR(D25N) (that is, residues 10–94) is the same as that of an individual subunit of the mature protease, we made use of the CS-Rosetta chemical shift structure determination algorithm which uses a hybrid approach of chemical-shift-based fragment selection and ROSETTA Monte Carlo driven fragment assembly35. The resulting ten lowest energy models are essentially identical to the corresponding region of the mature dimer with a backbone r.m.s. deviation of only 1.3 plusminus 0.2 Å (Supplementary Fig. 1e).

PRE calculations and ensemble refinement

Because the electron relaxation rate taus of the free radical is much longer than that of the protein rotational correlation time taur21, the PRE correlation time tauc [ = (taur-1 + taus-1)-1] for the calculation of intermolecular PRE rates was assumed to be the same as taur (12 ns) for the mature protease dimer36. To account for the flexibility of the linker between the spin label and the protein backbone, a ten-conformer randomized ensemble was used to represent the conformational space sampled by the spin label. The randomized ensemble was generated by high-temperature simulated annealing and slow cooling in Xplor-NIH22 subject to a target function comprising stereochemical terms, a quartic van der Waals repulsion term to prevent atomic overlap between the spin label and the protein, and a multidimensional conformational database potential of mean force37 describing the phi/psi/chi1 conformational space available to the surface cysteine residue to which the spin label was conjugated. Note that overlap between the members of the Cys spin-label ensemble is permitted as the ten-member ensemble represents a distribution of states. To ensure full sampling of the conformational space available to the spin label a different ten-conformer randomized ensemble was used for each structure calculation. Agreement between observed and calculated Gamma2 rates is given by the PRE Q-factor, QPRE:23

Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com

where Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com and Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com are the observed and ensemble average calculated transverse Gamma2 rates for residue i, respectively, and p is the overall population of the encounter complex species. All members of an ensemble of size Ne are weighted equally. For the average Q-factor < Q > for all calculated n ensembles, Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com is averaged over the members of each Ne ensemble. For the ensemble of ensembles average PRE Q-factor, Qee, Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com is averaged over all ensemble members and all ensembles14.

The coordinates used in the Xplor-NIH22 calculations were taken from the X-ray structure of the unliganded mature HIV-1 protease dimer (Protein Data Bank accession code 1HHP)24. Residues 10–94 were treated as a rigid body, and the flexible N- and C-terminal residues were not included in the calculations. The coordinates of the isotopically labelled subunit were held fixed, the initial positions of the spin-labelled subunit (at natural isotopic abundance) were randomized, and rigid-body simulated annealing was carried out against the PRE data sets for the spin label conjugated to the T12C, E34C and V82C sites simultaneously. The target function comprises a PRE restraint term23, a quartic van der Waals repulsion term to prevent atomic overlap between the spin-labelled and isotopically labelled subunits, and a very weak radius of gyration term38 to ensure that each member of the ensemble makes at least some intermolecular contacts14, 39. Note that atomic overlap between ensemble members of spin-labelled subunits is permitted as these represent separate but rapidly interconverting configurations of the encounter complex species14, 39. A grid search was performed varying the population of heterodimer and the ensemble size Ne used to represent the self-associated species14. For each ensemble size and population of encounter complex species, 100 calculations were carried out. Ensembles were ranked by PRE Q-factor and van der Waals repulsion energies, and the top 20 ensembles with the smallest PRE Q-factors were used for subsequent analysis39. Structures were rendered using PyMol (http://www.pymol.org) and re-weighted atomic probability density maps were generated using Xplor-NIH22 as described25.

d.r.m.s. metric

One metric we used to compare the precursor encounter complexes with the mature dimer was the distance root mean square (d.r.m.s.) metric defined by40:

Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com

where N is the number of distinct residue pairs (i, j), and Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com and Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com are the distance matrices in a calculated precursor encounter complex structure and the mature HIV-1 protease dimer structure, respectively.

Spherical coordinate systems used to describe relative subunit orientation in the encounter complexes

Two spherical coordinate systems are used to describe the relative orientation of the subunits in the precursor encounter complexes40. The first (polar angle phi and azimuth angle theta) describes the orientation of the vector joining the centre of masses of the two subunits (shown as grey spheres in Supplementary Fig. 3a) to an external axis system with the z axis corresponding to the C2 symmetry axis of the mature dimer. The second (polar angle alpha and azimuth angle beta) describes the orientation of a vector joining the centre of mass of the second subunit to an arbitrarily chosen atom of the same subunit (Calpha atom of Gly 51) relative to an axis system with the z' axis given by the vector joining the centre of masses of the two subunits (with the red subunit in Fig. 2 corresponding to the fixed reference subunit) (Supplementary Fig. 3a).

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