Revisiting Monomeric HIV-1 Protease

CHARACTERIZATION AND REDESIGN FOR IMPROVED PROPERTIES*

John M. LouisDagger §, Rieko Ishima§||**, Issa NesheiwatDagger , Lewis K. PannellDagger Dagger , Shannon M. LynchDagger , Dennis A. Torchia||, and Angela M. GronenbornDagger

From the Laboratories of Dagger  Chemical Physics and Dagger Dagger  Bioorganic Chemistry, NIDDK and the || Molecular Structural Biology Unit, NIDCR, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, September 22, 2002, and in revised form, December 2, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Interactions between the C-terminal interface residues (96-99) of the mature HIV-1 protease were shown to be essential for dimerization, whereas the N-terminal residues (1-4) and Arg87 contribute to dimer stability (Ishima, R., Ghirlando, R., Tozser, J., Gronenborn, A. M., Torchia, D. A., and Louis, J. M. (2001) J. Biol. Chem. 276, 49110-49116). Here we show that the intramonomer interaction between the side chains of Asp29 and Arg87 influences dimerization significantly more than the intermonomer interaction between Asp29 and Arg8'. Several mutants, including T26A, destablize the dimer, exhibit a monomer fold, and are prone to aggregation. To alleviate this undesirable property, we designed proteins in which the N- and C-terminal regions can be linked intramolecularly by disulfide bonds. In particular, cysteine residues were introduced at positions 2 and 97 or 98. A procedure for the efficient preparation of intrachain-linked polypeptides is presented, and it is demonstrated that the Q2C/L97C variant exhibits a native-like single subunit fold. It is anticipated that monomeric proteases of this kind will aid in the discovery of novel inhibitors aimed at binding to the monomer at the dimerization interface. This extends the target area of current inhibitors, all of which bind across the active site formed by both subunits in the active dimer.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The mature HIV-11 protease, a homodimer composed of two 99-amino acid polypeptides, plays a critical role in the life cycle of retroviruses by processing the precursor proteins Gag and Gag-Pol into the essential mature structural and functional proteins (1). Dimerization of the protease is indispensable for catalytic activity because each subunit contributes one of the two catalytic aspartic acid residues (Asp25) that form the active site (1). The mature protease catalyzes its own release from the Gag-Pol precursor and forms a highly stable dimer (2, 3). The two major areas that constitute the dimer interface are 1) the active site region 24-29 comprising the triplet Asp25-Thr26-Gly27 (DTG) forming a hydrogen bond network, called the "fireman's grip" (4, 5) and 2) the four-stranded anti-parallel beta -sheet comprising residues 1-4 and 96-99 (Fig. 1) (4, 6). These two interfaces are adjacent in the three-dimensional structure and form a nearly continuous region. Inhibitor or substrate binding to the protease further enhances the dimer stability (7, 8).


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Fig. 1.   A, tube drawing of the polypeptide backbone of the HIV-1 protease (Protein Data Bank code 1A30) with one protease monomer in green and the other in orange. The residues Gly86-Arg87-Asn88 are colored gray. Residues Asp29 and Arg87 are represented as a ball and stick model; the dotted yellow lines indicate hydrogen bonds between the side chains. Residues Gln2, Leu97, and Asn98 that were mutated to cysteine residues are yellow. B, side chain orientation of residues Gln2, Leu97, and Asn98 in one of the monomers (depicted in A) shown in ball and stick representation. C and D, schematic drawing depicting the four-stranded terminal beta -sheet of the native protease dimer and comparison with a possible orientation of the terminal strands linked by a single, intramonomer disulfide bridge. Residues in the second monomer of the dimer are labeled by prime symbols. Note that the beta -strands in D merely represent the orientation, and there is no evidence that they form beta -sheets.

The active site of the mature protease has been the predominant target for drug development in the fight against AIDS (4, 9). However, long term treatment of any HIV-infected individual is complicated by genetic variation in viral strains resulting in the selection of drug-resistant variants during therapy (10). Based on the three-dimensional structure of the protease, it has been suggested that interfering with the terminal beta -sheet, particularly disrupting the interaction between the two C-terminal strands, may provide an alternative mechanism for protease inhibition and drug design (6). This mode of inhibition may carry the added advantage of reducing the emergence of drug-resistant strains. Several reports indicate that peptides derived from the terminal regions of the protease inhibit enzymatic activity by blocking dimer formation (11, 12). However, to date this has not been confirmed structurally, either by x-ray crystallography or NMR. In addition, potential leads of this kind have not been developed further for possible clinical use, to our knowledge.

We are particularly interested in understanding the structural role of conserved regions in the protease sequence for monomer folding and dimerization. Residues in the dimer interface are well conserved both from the point of genetic variation and drug resistance (13). In addition, the unique triad Gly86-Arg87-Asn/Asp88, is also invariant among retroviral proteases (Figs. 1 and 2) (14). These residues are located in the helical stretch of the structure, connecting the main body of the protein to the last beta -strand of the terminal beta -sheet (6). This motif appears to constitute a crucial structural element, because even a conservative mutation, R87K, disrupted dimerization of the mature protease (15, 16). Interestingly, substrates with micromolar affinity to protease did not restore the dimeric form of PRR87K, as evidenced by its poor catalytic activity. In contrast, inhibitors with nanomolar affinity do induce dimer formation. We reasoned that the R87K mutation disturbed the interfacial beta -sheet and therefore carefully assessed deletion mutants in which either the N- or C-terminal residues were absent (Fig. 2). Studies on PR-(1-95), PR-(5-99), and PR-(5-95) demonstrated that even though both the N- and C-terminal strands contributed to dimer stability, interactions between the C-terminal residues 96-99 that form the inner strands of the sheet were absolutely essential for dimer formation at protease concentrations below 1 mM (16).

Here we analyze the influence of two conserved active site interface residues on monomer folding and dimer formation. NMR studies indicated that both mutants, PRD29N and PRT26A, are monomers with a tertiary fold similar to that of the PRR87K monomer. In the three-dimensional structure of the protease dimer, Asp29 is involved in two pivotal interactions; the first is an hydrogen bond between one of the carboxylate oxygens and the guanidinium group of Arg87 within the monomer, whereas the second is an intermolecular hydrogen bond across the dimer interface between the second Asp29 carboxylate oxygen and guanidine group of Arg8' of the other monomer (Fig. 1; Protein Data Bank code 1A30) (6).2 Our present studies of PRD29N suggest that the interaction between the Asp29 and Arg87 is more critical for dimerization than that between residues Asp29 and Arg8'.

We propose that the folded monomer of protease will aid in the design of dissociative inhibitors of the protease by targeting the exposed interface. To that end, detailed structural information on the monomer and candidate complexes either by x-ray crystallography or NMR need to be obtained. We therefore designed protease constructs in which the terminal strands can be linked by disulfide bonds. This allows for 1) destabilization of the native-like terminal beta -sheet dimer interface and 2) stabilization of the monomer by an intramolecular staple, i.e. the S-S bond. Three related constructs, PRQ2C/L97C, PRD25N/Q2C/L97C, and PRQ2C/N98C, all with only one intrachain disulfide bond, were prepared and characterized.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Construction of Protease Mutants-- The DNA template, PR (bearing five mutations Q7K, L33I, L63I, C67A, and C95A) (17) was used to construct PRT26A and PRD29N, PRD25N, PRQ2C/L97C, and PRQ2C/N98C. The PRD25N template was subsequently employed to make PRD25N/Q2C/L97C. All of the mutations were introduced using the QuikChange mutagenesis protocol (Stratagene, La Jolla, CA), the nucleotide sequences of all of the constructs were verified by DNA sequencing, and the proteins were verified by mass spectrometry. The intrachain disulfide-linked forms of PRQ2C/L97C, PRD25N/Q2C/L97C, and PRQ2C/N98C are termed PRC2-S-S-C97, PRD25N/C2-S-S-C97, and PRC2-S-S-C98, respectively.

Expression and Isolation of Inclusion Bodies-- The cells were grown at 37 °C either in Luria-Bertani medium or in a modified minimal medium for uniform (>99%) 15N labeling with 15NH4Cl as the sole nitrogen source and induced with 2 mM isopropyl-beta -D-thiogalactopyranoside for 4 h. Cells derived from 1 liter of culture were suspended in 20 volumes of buffer A (50 mM Tris-HCl, pH 8.2, 10 mM EDTA, and 10 mM dithiothreitol) and lysed by sonication at 4 °C in the presence of 100 µg/ml lysozyme. The insoluble fraction was washed by resuspension in buffer A containing 2 M urea and 0.5% Triton X-100 and subsequently in buffer A. In both cases, the insoluble fraction (inclusion bodies) was pelleted by centrifugation at 20,000 × g for 30 min at 4 °C. The final pellet of inclusion bodies was solubilized in 50 mM Tris-HCl, pH 8.0, 7.5 M guanidine HCl, 5 mM EDTA, 10 mM dithiothreitol to yield a protein concentration not exceeding 20 mg/ml estimated by the Bio-Rad protein assay.

Purification and Folding of PRT26A and PRD29N Constructs-- PRT26A and PRD29N were isolated using an established protocol comprising isolation of inclusion bodies followed by fractionation of the protease by size exclusion chromatography in buffer B (50 mM Tris-HCl, pH 8, 4 M guanidine HCl, 5 mM EDTA, 2 mM dithiothreitol) and reversephase high pressure liquid chromatography (see below). The proteins were folded according to the procedure previously described (16). The concentrations of purified proteins were determined spectrophotometrically.

Purification and Folding of Mutant PRs Containing Cysteine Residues with Intrachain Disulfide Bond Formation-- For the PRQ2C/L97C, PRD25N/Q2C/L97C, and PRQ2C/N98C constructs, 20 mg of the solubilized protein (inclusion bodies) at a concentration of 12.5 mg/ml was gradually diluted into 100 ml of buffer B without dithiothreitol (buffer C) and dialyzed against 2 liters of buffer C at room temperature overnight. The protein was concentrated to about 2 ml and applied to a Superdex-75 column (HiLoad 2.6 × 60 cm; Amersham Biosciences) equilibrated in buffer C at a flow rate of 3 ml/min at ambient temperature. Peak fractions corresponding to the monomer were combined and subjected to reverse-phase HPLC on POROS 20 R2 resin (Perceptive Biosystems, Framingham, MA) using a linear gradient of 0 to 60% acetonitrile, 0.05% trifluoroacetic acid. The peak fractions were combined and stored at -80 °C. The protein (2 mg) was diluted to about 0.33 mg/ml in 35% acetonitrile, water, 0.05% trifluoroacetic acid or in 0.1 M formic acid and dialyzed (Slide-A-Lyzer, 10K dialysis cassettes; Pierce) against 2 liters of 30 mM formic acid, pH 2.8, for 1-1.5 h. The sample was drawn out of the dialysis cassette, diluted with 5 volumes of 10 mM sodium acetate, pH 6.0, and dialyzed further against 4 liters of 20 mM sodium phosphate, pH 5.8, for 1.5-2 h. Finally the protein was concentrated to ~8 mg/ml using Centriprep YM-10 and Centricon YM-10 concentrators (Millipore Corporation, Bedford, MA) and stored at 4 °C or used for the experiments described below.

Protease Digests and Mass Spectroscopy-- PRD25N/Q2C/L97C or PRQ2C/N98C was digested overnight either with Glu C in 25 mM ammonium acetate, pH 4.0, at room temperature or with trypsin in 25 mM ammonium bicarbonate, pH 7.8, at 37 °C. The protein to enzyme ratio was 50:1 for Glu C and 10:1 for trypsin. 50-100 pmol of the digest was injected onto a Zorbax C3 column (2.1 × 150 mm; Agilent, San Jose, CA) fitted to an HP1100 integrated high pressure liquid chromatography/electrospray mass spectrometer (Agilent) and equilibrated in 5% acetic acid. The peptides were eluted using a linear gradient of 0 to 100% acetonitrile over a period of 25 min at a flow rate of 200 µl/min. The peptides that eluted into the mass spectrometer were scanned from m/z 300 to 1700 every 4 s. The spectra were deconvoluted using the Agilent software to yield the mass of the peptides.

Sedimentation Equilibrium-- Sedimentation equilibrium experiments were conducted at 20 °C and three different rotor speeds (16,000, 20,000 and 24,000) on a Beckman Optima XL-A analytical ultracentrifuge. The protein samples were prepared and loaded into the ultracentrifuge cells at nominal loading concentrations of ~0.7 absorbance at 280 nm (~60 µM in monomer). The data were analyzed in terms of a single ideal solute to obtain the buoyant molecular mass, M(1 - v), using the Optima XL-A data analysis software (Beckman). The value for the experimental molecular mass M was determined using the calculated values for the density (determined at 20 °C using standard tables) and partial specific volume v (calculated on the basis of amino acid composition; see Ref. 18).

Protease Assays-- The mutant enzymes were assayed using a spectrophotometric substrate (19), Lys-Ala-Arg-Val-Nle-(4-nitrophenyl-alanine)-Glu-Ala-Nle-NH2, in 100 mM sodium acetate buffer, pH 5.0, at 25 °C at final enzyme concentrations of 29 µM PRD29N, 22.8 µM PRC2-S-S-C97, or 1 µM PRC2-S-S-C98 and 460 µM substrate.

NMR Experiments-- All of the NMR experiments were carried out at protein concentrations in the range of 0.6-1.0 mM in monomer (unless noted otherwise) in 20 mM phosphate buffer at pH 5.8, 95% H2O, 5% D2O at 20 °C with a sample volume of ~280 µl in a 5-mm Shigemi tube (Shigemi, Inc., Allison Park, PA). NMR spectra were acquired on a DMX500 spectrometer (Bruker Instruments, Billerica, MA). The data were processed and analyzed using nmrPipe, nmrDraw, and PIPP software (20, 21). Protein aggregation was followed at 20 °C by measuring amide signal intensities in 1H-15N HSQC spectra recorded over a period of 4 days. The initial protein concentration used in these latter experiments was ~1 mM.

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Disrupting the D29/R87 Interaction Destabilizes Dimer Formation-- We previously demonstrated that a protease construct bearing the conservative mutation R87K does not exhibit dimer formation, even at concentrations up to ~1 mM (16). This suggested that an interaction involving the side chain of Arg87, most likely the one between Arg87 and Asp29, stabilized the dimer, possibly by anchoring the alpha -helix and thereby correctly positioning the two C-terminal strands of the beta -sheet. Here we constructed and investigated PRD29N, a construct containing the complementary change to that of PRR87K in the Arg87/Asp29 pair. PRR87K and PRD29N were ~4600- and 920-fold less active than PR (Fig. 2). The 1H-15N HSQC spectrum of PRD29N recorded on a 0.9 mM sample is displayed in Fig. 3A. Characteristic peaks resulting from the monomeric protein (squares) as well as dimer peaks (dashed squares) are apparent. This demonstrated that PRD29N is also a dimerization-deficient mutant.


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Fig. 2.   Amino acid sequences of mature HIV-1 protease variants. The two highly conserved regions in all retroviral proteases, the active site (DTG) and C-terminal (GR(N/D)) triads, are highlighted in gray. All of the variants bear five mutations: three mutations (Q7K, L33I, and L63I) that abrogate autoproteolysis and two mutations (C65A and C95A) to prevent nonspecific thiol cross-linking (17). The mutations and their designations are indicated. PR exhibits essentially the same kinetic parameters and stability as the wild-type mature HIV-1 protease (17).


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Fig. 3.   500 MHz 1H-15N HSQC spectra of PRD29N, PRT26A, and PRD25N in 20 mM sodium phosphate buffer, pH 5.8, and 20 °C. Freshly prepared PRD29N (A), PRT26A in the absence (B) and presence of DMP323 (C), and PRD25N (D). The inset in A shows duplicate lanes of PRD29N sample in the absence of inhibitor analyzed by SDS-PAGE under reducing conditions after the NMR experiment.

In the structure of the protease dimer, an extensive network of backbone and side chain interactions exist between residues Arg8', Asp29, Arg87, and Asn88 (6). Noteworthy among these interactions are the hydrogen bonds formed between the Asp29 carboxylates and the Arg87 and Arg8' side chains. Each oxygen of the Asp29 side chain accepts a hydrogen bond from one of the terminal amino groups of each arginine (Fig. 1A). Previous results indicated that the intermonomer interaction between Asp29 and Arg8' does not significantly influence the maturation, stability, or enzymatic activity of the protease (17). For example, the R8Q mutant of the mature PR (PRR8Q) does not exhibit any changes in the dimer dissociation constant (17). Furthermore, the kinetic parameters for mature PRR8Q-catalyzed hydrolysis of a peptide substrate and the inhibition constant for the hydrolytic reaction with an inhibitor are comparable with those of PR.

This contrasts results obtained for PRD29N and PRR87K mutants. Drastic effects on dimer stability and catalytic activity were noted. It is also known that the conserved Asp29 residue is involved in substrate/inhibitor binding (22, 23). Thus, the poor catalytic activity of PRD29N arises both from the destabilization of the dimer (increase in dissociation constant, Kd) as well as changes in the active site environment. Separation of the kinetic parameters was precluded by the very low catalytic activity of PRD29N reaching the sensitivity limit of the spectrophotometric assay. Consistent with its poor catalytic activity, autoproteolysis of PRD29N is significantly impaired (Fig. 3A, inset), although a significant fraction of PRD29N is dimeric at 0.9 mM concentration. Slow aggregation of this mutant was observed, similar to results for other monomers investigated previously.

The Active Site Mutant PRT26A Is a Folded Monomer-- The dimer interface of the free mature protease is formed by residues contributing to the active site and those located at the N and C termini. Of these, terminal residues constitute about 50% of the total interface interactions (6, 24). Complementing our mutational studies of the terminal beta -sheet dimer interface, earlier studies have shown that a T26A mutation of the active site interface significantly affects dimer formation as compared with the mutations T26C or T26S (5). To compare the effect of these two distinct interfaces (active site residues versus terminal beta -sheet) on monomer folding, we investigated the T26A mutant further. The T26A substitution mutation was introduced into our idealized PR construct, and the protein was purified and assessed by NMR. The 1H-15N HSQC spectrum of PRT26A is similar to those of PR-(1-95) and PRR87K monomers (Fig. 3B) (16), demonstrating that PRT26A is also a folded monomer. 15N NMR relaxation measurements on PRR87K in the absence of inhibitor DMP323 revealed significant motions on the subnanosecond time scale for the N- and C-terminal residues 2-10 and 93-99 unique to the monomer (16). These motions were attributed in part to the loss of the interfacial beta -sheet in PRR87K. This notion is supported by the random coil chemical shifts of the C-terminal residues of PRR87K. As illustrated in Fig. 3, residues Thr96 and Phe99 of PRT26A exhibit nearly identical shifts to those of PRR87K, suggesting that the terminal strands of PRT26A are also disordered and flexible, resembling those of PRR87K. The disorder of the terminal residues observed for these monomers is most likely due to the loss of the formation of a terminal beta -sheet arrangement and not a direct effect of the mutation. Thus, in the absence of a bound inhibitor, the highly conserved Thr26 residue of the active site DTG triad is as critical to dimer stability as Arg87, Asp29, or the N-terminal residues 1-4.

Unlike PR-(1-95), which does not show evidence of dimer formation even in the presence of the potent inhibitor DMP323, PRT26A, like PRR87K and PR-(5-99), forms dimers in the presence of DMP323, as evidenced by its 1H-15N HSQC spectrum (Fig. 3C). Thus, interactions between the inhibitor and the active site/flap residues for these mutants offset the effect of the mutation on the dimerization constant. 15N and 1H chemical shifts of the terminal residues, e.g. Ile3 and Ala95, of PRT26A/DMP323 are nearly identical to those of the PR/DMP323, implying very similar terminal beta -sheet arrangements of PR and PRT26A.

Comparison of the Aggregation Properties of the Protease Monomers-- Like other protease monomers studied previously, PRT26A slowly aggregates. The decay in signal intensity of selected peaks in the 1H-15N HSQC spectra for PRT26A and PR-(1-95) at 1 mM protein concentration indicates the loss of monomer species at approximately similar rates. The time course for the Gly68 resonances is plotted for both proteins in Fig. 4. Note that a comparison with PRR87K at pH 5.8 and PR-(5-99) could not be carried out because the former aggregates considerably faster and the latter experiences substantial autoproteolysis (17).


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Fig. 4.   Time dependence of signal intensity for the amide resonance Gly68 (tentative assignment) in the 1H-15N HSQC spectra of PRD25N/C2-S-S-C97 (A), PRT26A (B), and PR-(1-95) (C) in 20 mM sodium phosphate buffer, pH 5.8, at 20 °C. Ten other peaks also showed similar patterns of decay to that of Gly68 with an average error of 5%.

It is generally assumed that partially or misfolded forms of proteins are prone to aggregation (25). In the case of the protease monomers investigated here, we propose that the terminal beta -sheet residues, when not properly engaged, and those at the active site present a substantial solvent-exposed hydrophobic surface that would naturally cause nonspecific aggregation. Our 1H-15N HSQC spectra (Fig. 3) (16) indicate that the folds of the PRT26A and PR-(1-95) monomers are very similar to the monomeric unit in the PR dimer. The fact that PRT26A aggregates at a rate comparable with PR-(1-95), although different regions of the chain are involved, suggests that both the active site region and the termini can be responsible for aggregation.

Disulfide Bridge Formation between the N- and C-terminal Strands of Protease: a Strategy to Overcome Monomer Aggregation-- As illustrated in Fig. 1, the terminal residues of the monomer subunits in the protease dimer form a four-stranded anti-parallel beta -sheet, thereby interleaving strands from the two subunits. We previously demonstrated that interfering with the formation of this sheet by deleting either the N- or C-terminal 4 residues disrupts dimerization of the free protease. Except for the conservative substitutions L97V, no other major drug-resistant mutations of protease terminal residues have been reported (23). This suggests that hydrophobic packing of the terminal side chains may be critical for dimer stability. Based on the crystal structure of the protease dimer, we reasoned that substitution of cysteines for one amino acid in each of the terminal regions (1-4 and 96-99) that can form intrachain disulfide bonds may prevent the formation of the four-stranded beta -sheet. In addition, we anticipated that linking the terminal segments of the monomer would reduce the rate of monomer aggregation by reducing the probability of random interaction of exposed terminal hydrophobic residues.

Although PR carries three mutations (Q7K, L33I, and L63I) to suppress autoproteolysis, it is prone to slow cleavage in the absence of inhibitor at protein concentrations of ~0.5 mM required for NMR experiments. In addition, protease constructs containing mutations that significantly reduce dimer stability, such as PR5-99, are prone to autoproteolysis at NMR concentrations (16). We therefore chose the protease mutant PRD25N as the template to create an intrachain disulfide bridge between the N- and C-terminal strands. This active site mutant PRD25N forms a stable but inactive dimer at 0.5 mM (Fig. 3D), which makes it ideal for studies of the mature PR dimer either free or bound to a native substrate (22). The mutation D25N may also influence the dimer stability of the mature protease (KD = ~10-6 M),3 albeit to a much lesser extent than the mutants that form monomers discussed above (also see Table I). The construct PRD25N/Q2C/L97C was used to optimize the protocol for disulfide bond formation (see "Experimental Procedures" for details). We opted for oxidation prior to the final size exclusion column chromatography because this allows for efficient separation of monomer, intermolecularly disulfide-linked dimer, and multimeric forms of the protein. In addition, this scheme always provided higher yields of the intramolecularly disulfide-linked form of PRD25N/Q2C/L97C, now termed PRD25N/C2-S-S-C97 compared with the intermolecular linked species. After reverse-phase HPLC purification and prior to refolding, the oligomeric state of the protein was assessed by SDS-PAGE under nonreducing conditions. In general, a single band corresponding to the molecular mass of the protease monomer revealed that the majority of the protein contained an intrachain disulfide bond (Fig. 5A, inset).

                              
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Table I
Summary of folded forms of protease variants
1H-15N correlation spectra were acquired on approx 0.6 mM samples (in monomer) in 20 mM sodium phosphate buffer, pH 5.8, either in the absence or approx 5-fold excess of potent inhibitor DMP323 (KI = approx 1 nM) (16). Except for PRT26A, PRD29N, and PRD25N analyzed by NMR, the major folded forms of the protease were determined both by NMR and sedimentation equilibrium studies. PRD25N/C2-S-S-C97 as determined by sedimentation equilibrium studies is a monomer in the presence of DMP323 yielding a molecular mass of 10690 ± 640. In addition to the monomer, there is evidence for a polydisperse aggregate with a mass of approx 20 times that of the monomer in the PRD25N/C2-S-S-C97, PRC2-S-S-C97, and PRC2-S-S-C98 samples. This is present in quantities of less than 0.5% in terms of concentration. It is possible that this small fraction of aggregate may account for the variation in the experimental mass. The results for PR, PRR87K, PR-(5-99), PR-(1-95), and PR-(5-95) were published previously (16) and are shown here for comparison only.


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Fig. 5.   1H-15N HSQC spectra of oxidized (A) and oxidized and reduced (B) PRD25N/2C/L97C. Signals arising from a small fraction of protease containing the free cysteine sulfhydrals are indicated by arrows in B. The inset shows duplicate lanes of PRD25N/C2-S-S-C97 analyzed by SDS-PAGE under nonreducing conditions. No intermolecularly disulfide-linked dimers or multimeric forms of the protein are observed (sensitivity <=  5%).

In addition, the proportion of intrachain disulfide-linked (circular chain) versus linear (free cysteine side chains) monomer forms in the preparation of purified, uniformly 15N-labeled mutant PRD25N/Q2C/L97C was assessed by digestion with endopeptidase Glu C and liquid chromatography/electrospray mass spectrometry. A species of molecular mass 5944 Da (calculated mass = 5953 Da) corresponding to the combined mass of residues 1-21 and 66-99 was observed, whereas the individual 1-21 and 66-99 peptides were not detected. This demonstrated that essentially 100% intrachain disulfide bond formation between residues Cys2 and Cys97 occurred. Similar analyses for the PRQ2C/N98C construct after digestion with trypsin yielded a fragment of molecular mass 2173.1 Da corresponding to the combined mass of residues 1-7 and 88-99 (calculated mass = 2175 Da). Again the individual 1-7 and 88-99 peptides were not detected. The presence of the individual peptide masses was confirmed by incubating an aliquot of the digest with a reducing agent prior to a second liquid chromatography/electrospray mass spectrometer run.

The Intrachain Disulfide-linked Protease Is a Folded Monomer-- As evidenced from the 1H-15N HSQC spectrum of PRD25N/C2-S-S-C97 (Fig. 5A), the chemical shifts are similar to those of other protease monomers, such as PRR87K and PR-(1-95). No characteristic dimer peaks were observed (dotted squares), indicating that the proportion of the dimer was less than 5%. Sedimentation equilibrium experiments (detailed under "Experimental Procedures") yielded molecular masses of 11,680 ± 120 and 13,320 ± 600 Da for PRC2-S-S-C97, lacking the active site mutation D25N, and PRD25N/C2-S-S-C97 proteins consistent with a monomeric mass (the expected mass for the monomer was 10,800 Da; Table I). Therefore S-S bond formation between residues 2 and 97 does not interfere with the folding of the monomeric protein.

To investigate the effect of the free sulfhydrals on protease dimerization and stability, PRD25N/Q2C/L97C was purified under reducing conditions. Disulfide bond formation between Cys2 and Cys97 occurs during folding of the protein upon increasing the pH from 2.8 to 5.8 and concentration (see "Experimental Procedures"). Fig. 5B displays the 1H-15N HSQC spectrum of PRD25NQ2C/L97C immediately after its preparation. Clearly, under ambient conditions, oxidation cannot be avoided, and peaks that correspond to the oxidized form (identical positions to those in Fig. 5A) are observed. These constitute the majority of the signals. In addition, other small intensity signals were noted, and although not assigned yet, a tentative comparison of the chemical shifts with those of PRR87K suggests that the major differences are observed in the region corresponding to the terminal beta -strand residues. We therefore believe that these additional signals arise from a small portion of protein containing free cysteine sulfhydrals. These signals can only be observed transiently, and after a few hours of incubation at 20 °C only signals of the oxidized species remain. It is most likely that any monomer with free cysteine side chains cross-links rapidly via intermolecular disulfide bonds, leading to signal loss in the spectrum.

The behavior of the oxidized PRD25N/C2-S-S-/C97 with respect to aggregation was assessed by 1H-15N HSQC spectroscopy. The relative signal intensity of at least 10 individual peaks was followed over time at 20 °C. These data are illustrated for the Gly68 peak in Fig. 4. The decrease in signal intensity is clearly less than observed for the PRT26A and PR-(1-95) monomers. This indicates that the folded monomer, at least for PRD25N/C2-S-S-/C97 containing the intrachain disulfide bond, exhibits superior attributes with respect to long term stability to permit detailed biophysical characterization. PRC2-S-S-/C97 and PRC2-S-S-/C98 were excluded from these analyses because they form active dimers in the presence of substrate with significant catalytic activity (Table I and see below). Because PRD25N/C2-S-S-/C97 is monomeric even in the presence of DMP323 (Table I), it appears that the intrachain disulfide bond in conjunction with the D25N mutation in PRD25N/C2-S-S-C97 decreases dimer stability to a far greater extent than the D25N mutation alone.

Inhibitor Binding and Enzymatic Activity of Intrachain Disulfide-linked Protease-- Inhibitor-mediated interactions enhance dimer formation by the protease (7, 8). In the absence of inhibitor PRD25N/C2-S-S-C97 is clearly a monomer, and we used the equivalent PRC2-S-S-C97 protein to assess whether inhibitor binding promotes dimerization. A comparison of the HSQC spectrum of PRC2-S-S-C97 with that of PRD25N/C2-S-S-C97 clearly demonstrated that PRC2-S-S-C97 is also mainly monomeric with a tertiary fold nearly identical to that of PRD25N/C2-S-S-C97. Interestingly, in the presence of the potent inhibitor DMP323, peaks characteristic of both the monomer and the dimer are observed (Table I), indicating that DMP323 binding by PRC2-S-S-C97 promotes dimer formation. Consistent with this observation, PRC2-S-S-C97 (at 22 µM) is enzymatically active, albeit about 200-fold less than PR, indicating that ligand binding promotes the active dimeric PRC2-S-S-C97 complex.

Previous studies of protease terminal deletion mutants indicated that the interaction between the two C-terminal strands is crucial for dimer formation even in the presence of inhibitor. Thus, it is highly likely that the two C-terminal strands of the PRC2-S-S-C97 may form favorite contacts upon DMP323 binding. In the wild-type mature HIV-1 protease dimer the C-terminal strands flank the inside of the four-stranded anti-parallel beta -sheet with a 1-99'-99-1' configuration (Fig. 1). In the dimeric form of PRC2-S-S-C97, the intramolecular disulfide link between the N- and C-terminal strands will position the N and C termini from the same monomeric unit next to each other. Therefore, the most likely configuration for these mutants will be based on a 1-99-99'-1' association.

In addition to the Cys2-Cys97 cross-linked constructs described above, we also analyzed a construct, PRC2-S-S-C98, in which Asn98 was substituted by a Cys to form an intrachain disulfide bridge with Cys2. NMR analyses indicated that this construct was also a folded monomer at ~0.6 mM concentration. Interestingly, PRC2-S-S-C98 displays only a 15-fold decrease in kcat/Km when compared with PR under identical conditions (17). For comparison, this value for PRC2-S-S-C98 is ~18 times higher than that of PRC2-S-S-C97. The kinetic parameters kcat and Km for PRC2-S-S-C98-catalyzed hydrolysis of the substrate were 0.87 ± 0.07 s-1 and 0.427 ± 0.06 mM, respectively, representing approximately a 6.3- and 2.4-fold decrease in kcat and Km, respectively, when compared with PR (17). In the free and inhibitor/substrate bound three-dimensional structures of the mature protease dimer, the Gln2 and Asn98 side chains are both solvent-exposed, whereas the Leu97 side chain points inward into the protein core (Fig. 1, A and B). Based on these side chain orientations, we reason that an intrachain disulfide link between Cys2 and Cys98 may not introduce significant conformational distortion, whereas the one between Cys2 and Cys97 most likely causes a strained and somewhat distorted terminal strand.

The deletion construct PR-(5-99) exhibits 3- and 50-fold lower kcat/Km values as compared with those of PRC2-S-S-C98 and PR. Despite a lower catalytic activity than PRC2-S-S-C98, PR-(5-99) clearly undergoes autoproteolysis (see spectrum in Ref. 17). Surprisingly, we did not observe peaks in the 1H-15N HSQC spectrum that correspond to products of autoproteolysis for PRC2-S-S-C98 even at NMR concentrations. In fact, PRC2-S-S-C98 at a concentration of 1.5 mg/ml is stable for up to 6 months stored at 4 °C with no loss in catalytic activity. Therefore, PRC2-S-S-C98 monomer is unique from the point that it is stable, forms a dimer in the presence of substrate with nearly native-like enzymatic activity, and is apparently resistant to autoproteolysis at a protein concentration up to 0.6 mM. It is possible that either the mutations Q2C or L98C or the intramolecular disulfide bond between these residues may restrict autoproteolysis particularly at the major Leu5-Trp6 site.

Concluding Remarks-- Mature wild-type HIV-1 protease upon its maturation from the Gag-Pol precursor forms a stable dimer exhibiting a subnanomolar dissociation constant (<5 nM) (3, 17). A structure-assisted mutagenesis approach interfaced with experimental assessment of the designed variants by NMR allowed us to define key interactions that influence the monomer-dimer equilibrium (Table I). At ~0.6 mM, all of the mutant proteins are mainly monomeric, except for the constructs PRD29N and PR-(5-99), which show an approximately equal proportion of monomer and dimer. Based on the similarities of chemical shifts for PRR87K and the other variants, we think that the folds of these monomers are very similar to that of an individual subunit of the dimer, except for the N- and C-terminal regions. All of the variants showed inhibitor-promoted dimerization at NMR concentrations, except PR-(1-95), which carries a 4-residue deletion at the C terminus, indicating that pivotal interactions between the C-terminal residues exist. Intermonomer interactions involving Thr26 and residues 1-4 as well as a critical intramonomer hydrogen bond between the side chain carboxylate of Asp29 and the amino group of Arg87 stabilize the dimer. Future inhibitors may be designed to interfere with these interactions and incorporate features that complement the unique surface of the folded monomer. We anticipate that these inhibitors may address problems related to multi-drug resistance inevitably observed with current HIV-1 protease drugs.

Structural studies by NMR require stable monomeric protease at a reasonably high protein concentration (~1 mM). Because all described linear chain monomers tend to aggregate within a day or two at 1 mM concentration, we designed PRD25N/C2-S-S-C97, PRC2-S-S-C97, and PRC2-S-S-C98, in which the terminal beta -strands were linked through a disulfide bridge. Most notably we demonstrate that PRD25N/C2-S-S-C97 is less prone to aggregation, even at a relatively high protein concentration of ~1 mM. It is anticipated that these monomer forms may facilitate future NMR studies of monomer-ligand complexes.

    ACKNOWLEDGEMENTS

We thank F. Delaglio and D. Garrett for data processing software and R. Ghirlando for equilibrium sedimentation studies. We are grateful to S. Oroszlan for many thoughtful and inspiring discussions.

    FOOTNOTES

* This work was supported by the Intramural AIDS Targeted Anti-Viral Program of the Office of the Director of the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These authors contributed equally to this work.

To whom correspondence may be addressed: Laboratory of Chemical Physics, Bldg. 5, Rm. 411, NIDDK, NIH, Bethesda, MD 20892. Tel.: 301-594-3122; Fax: 301-480-4001; E-mail: jmlouis@helix.nih.gov.

** To whom correspondence may be addressed: Molecular Structural Biology Unit, Bldg. 30, Rm. 109, NIDCR, NIH, Bethesda, MD 20892-4307. Tel.: 301-402-3160; Fax: 301-402-5321; E-mail: rishima@dir. nidcr.nih.gov.

Published, JBC Papers in Press, December 4, 2002, DOI 10.1074/jbc.M209726200

2 The prime symbol with the position number indicates a residue in the second monomer of the dimer.

3 D. A. Torchia, R. Ishima, and J. M. Louis, unpublished data.

    ABBREVIATIONS

The abbreviations used are: HIV-1, human immunodeficiency virus, type 1; PR, wild-type mature HIV-1 protease bearing the mutations Q7K, L33I, L63I, C67A, and C95A; HSQC, heteronuclear single quantum coherence spectroscopy.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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