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Human Immunodeficiency Virus 1 Protease Expressed in Escherichia

Human Immunodeficiency Virus 1 Protease Expressed in Escherichia

Proc. Nati. Acad. Sci. USA Vol. 86, pp. 1841-1845, March 1989 Biochemistry Human 1 expressed in Escherichia coli behaves as a dimeric (pr55iag /oligopeptide / crosslinking/retroviral protease/ inactivation) THOMAS D. MEEK*t, BRIAN D. DAYTON*, BRIAN W. METCALF*, GEOFFREY B. DREYER*, JAMES E. STRICKLERt, JOSELINA G. GORNIAKt, MARTIN ROSENBERG§, MICHAEL L. MOORE¶, VICTORIA W. MAGAARD¶, AND CHRISTINE DEBOUCK11 Departments of *Medicinal Chemistry, tMacromolecular Sciences, §Biopharmaceutical Research and Development, VPeptide Chemistry, and I'Molecular Genetics, Smith Kline & French Laboratories, King of Prussia, PA 19406 Communicated by Brian W. Matthews, December 16, 1988 (receivedfor review October 20, 1988) ABSTRACT Recombinant human immunodeficiency vi- date (13-16) consist of <130 amino acids and, although they rus 1 (HIV-1) protease, purified from a bacterial expression exhibit limited sequence homology (25-35% identity), they system, processed a recombinant form of its natural substrate, contain two highly conserved regions [Leu-(Val/Leu)-Asp- Pr55sag, into protein fragments that possess molecular weights (Thr/Ser)-Gly and (Ile/Leu)-(Ile/Leu)-Gly-Arg-(Asp/Asn)]. commensurate with those of the virion gag . Molecular Sequences similar to both of these are present in the aspartic weights of the protease obtained under denaturing and non- pepsin, , and D. The active sites of denaturing conditions (11,000 and 22,000, respectively) and these aspartic proteases contain two aspartic residues, both chemical crosslinking studies were consistent with a dimeric of which occur in the sequence Asp-Thr-Gly. The retroviral structure for the active enzyme. The protease appropriately contain only one such Asp-Thr-Gly sequence and cleaved the nonapeptide Ac-Arg-Ala-Ser-Gln-Asn-Tyr-Pro- are less than halfthe size ofthe aspartic proteases. Computer Val-Val-NH2 between the and residues. HIV-1 modeling of retroviral protease structure has led to the protease was sensitive to inactivators of the aspartic proteases. proposal that if the enzymes exist as dimers of identical The aspartic protease inactivator 1,2-epoxy-3-(4-nitrophen- subunits, with each monomer contributing an Asp-Thr-Gly oxy)propane produced irreversible, time-dependent inactiva- sequence to the , then they more closely resemble tion of the protease. The pH-dependent kinetics of this inacti- the bilobal structure of the aspartic proteases (17). vator were consistent with the requirement of an unprotonated We (18) and others (10, 19-22) have subcloned and ex- carboxyl group in the active site of the enzyme, suggesting that pressed HIV-1 protease in Escherichia coli. Our studies HIV-1 protease is also an aspartic protease. demonstrated that (i) the protease activity resides entirely within the pol of HIV-1; (ii) the recombinant The of the type 1 human immunodeficiency virus enzyme can apparently catalyze its own cleavage from a fusion (HIV-1) possesses the same 5'-gag-pol--3' organization as protein precursor in vivo; (iii) the N terminus of the recom- other (1-4). The initial product of the binant protease, Pro-Gln-Ile-Thr-Leu, is consistent with pro- HIV-1 gag is a 55-kDa precursor (Pr55M) that is cessing at the proteolytic consensus sequence Ser-Phe-Asn- subsequently processed into the gag proteins p17, p24, and p15 Phe*Pro-Gln [residues 65-70 in pol (numbering as in ref. 1)]; (p7/p6) during virion formation (2, 5). The pol coding region, and (iv) in E. coli, the expressed protease can correctly which overlaps gag in the -1 reading frame, is expressed as process a Pr55S&j-like . One of the products of a fusion product of gag and pol by a translational frameshift this cleavage is a 24-kDa protein having an N-terminal se- upstream of the gag termination codon (6). This gag-pol quence identical to that ofthe authentic p249'5 protein from the precursor (Pr16099P0l) is processed into the gag proteins, a virion. We report here biochemical characterization of the protease, a , and an endonuclease. In all purified recombinant HIV-1 protease and address the model of retroviruses, the gag and gag-pol precursors are cleaved into the enzyme as a dimeric aspartic protease. their mature protein products by a specific, virally encoded protease (7). One conserved sequence recognized by EXPERIMENTAL PROCEDURES retroviral proteases (8) and found repeatedly within the trans- lated HIV-1 gag and pol is (Ser/Thr)-Xaa-Yaa-(Tyr/ Source and Preparation of Recombinant HIV-1 Protease. Phe)-Pro-Zaa. Proteolysis ofthis sequence occurs between the The constructs, derived from the pAS expression vector (23), aromatic amino and proline residues. Retroviruses made and methods for expression of recombinant HIV-1 protease deficient in protease by deletion or point are char- and Pr55sag in E. coli have been described (18). HIV-1 acterized by immature morphology and incompe- protease was obtained from the PRO4 expression vector in E. tence (9-11), suggesting that the retroviral proteases are coli strain AR58 by heat induction (24) or AR120 by nalidixic essential for the development ofinfectious virus. Accordingly, acid induction (25). Under these conditions, rapid autopro- inhibition of the active protease within infectious HIV or teolysis of the expressed 25-kDa fusion-protein precursor HIV-infected cells represents a new therapeutic opportunity resulted in the production of a small quantity of a soluble for the treatment of acquired immunodeficiency syndrome 11-kDa protein (denatured molecular mass) that reacted with (AIDS) and related disorders. a polyclonal raised against the PRO4 product (18). Protein sequence homologies suggest that the retroviral The purification of active protease from such bacterial cell proteases belong to the family of aspartic proteases (12). The lysates will be described in detail elsewhere (J.E.S., unpub- retroviral proteases that have been purified and sequenced to Abbreviations: Ac-RASQNYPVV-NH2, Ac-Arg-Ala-Ser-Gln-Asn- Tyr-Pro-Val-Val-NH2; DMS, dimethyl suberimidate; EPNP, 1,2- The publication costs of this article were defrayed in part by page charge epoxy-(4-nitrophenoxy)propane; HIV-1, human immunodeficiency payment. This article must therefore be hereby marked "advertisement" virus 1. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed.

1841 Downloaded by guest on September 30, 2021 1842 Biochemistry: Meek et al. Proc. Natl. Acad. Sci. USA 86 (1989) lished data). In brief, packed E. coli cells containing the compounds. Aliquots of 10 Al were then removed and induced PRO4 plasmid were lysed by sonication, and the assayed for protease activity as described above in a 20-/l protease was precipitated from the lysis supernatant with total volume containing 2 mM Ac-RASQNYPVV-NH2 in ammonium sulfate. Precipitated proteins were resuspended MENDT buffer (pH 6.0). Inactivation of protease by 1,2- and subjected to size-exclusion HPLC on a TSK G2000 SW epoxy-3-(4-nitrophenoxy)propane (EPNP; Sigma; recrystal- column. Purified HIV-1 protease was stored indefinitely at lized from methanol) was examined for pH and concentration -200C in 20 mM TrisHCI, pH 7.5/1 mM dithiothreitol/1 mM dependence in a similar manner. Reaction mixtures (50 Al) EDTA/200 mM NaCl/40% (vol/vol) glycerol at protein containing 50 mM Mes (pH 6.0), 0.1 mM EDTA, 0.2 M NaCl, concentrations of 1-200 gg/ml. 0.1% Triton X-100, 10%o dimethyl sulfoxide, 40 ng of prote- Peptidolytic Assay of HIV-1 Protease Activity and Proteol- ase, and 0.6-9.0 mM EPNP were preincubated at 220C. At ysis of p55. HIV-1 protease activity in crude extracts and in various times, 5-/l aliquots were removed to 50-/A assay purified preparations was measured by quantification of the mixtures (30 minm 370C) containing 3 mM Ac-RASQNYPVV- peptidolysis products of the synthetic nonapeptide Ac-Arg- NH2 in MENDT buffer. The pH dependence of EPNP Ala-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2 (Ac-RASQNYPVV- inactivation was determined similarly by preincubation of 40 NH2). A typical peptidolytic assay contained 2-3 mM Ac- ng of protease and 10 mM EPNP in a mixed buffer (50 mM RASQNYPVV-NH2 and crude PRO4 extract or 8-250 ng of each glycine, sodium acetate, Mes, and Tris; pH 3-8) purified protease in 10 ptl of 50 mM Mes, pH 6.0/1 mM containing 0.1 mM EDTA, 0.1% Triton X-100, 10% dimethyl EDTA/0.2 M NaCI/1 mM dithiothreitol/0.1% Triton X-100 sulfoxide, and NaCl added at levels sufficient to hold the (MENDT buffer) at 370C. Reaction was quenched after 2-15 ionic strength at 0.25. Following a 4-hr incubation at 22°C, min with an equal volume of 0.6 M trichloroacetic acid, and protease activity was determined at pH 6.0 as described peptidolytic products were analyzed by reverse-phase HPLC above. The chemical stability of EPNP at pH 3-6 was [Beckman Ultrasphere ODS column (4.5 mm x 25 cm); investigated in a control experiment in which EPNP was mobile phase, 5-20% acetonitrile (15 min) and 20% acetoni- pretreated over the same range of pH values for 4 hr in the trile (5 min) in 0.1% trifluoroacetic acid at 1.5 ml/min; absence of protease. Subsequently, the inactivation of pro- detection at 220 nm]. Proteolysis of p55 by purified fractions tease by this pretreated EPNP was determined at pH 6.0. of HIV-1 protease was performed at 37°C in 10-/Al reaction Other Methods. Ac-RASQNYPVV-NH2 was synthesized mixtures containing 1.0 ,g of p55 and 30-210 ng of the by standard solid-phase methods (28). Protein concentrations enzyme in MENDT buffer. Reaction was quenched after 30- were determined by the method of Bradford (29), and by 60 min by boiling the reaction mixtures in NaDodSO4/PAGE composition analysis for fractions of purified sample buffer. Samples were then subjected to NaDodSO4/ protease. Initial-rate data and the pH dependence ofprotease PAGE in 15% polyacrylamide minigels (4.5 cm x 8 cm x 1 inactivation (log kobs vs. pH) were fitted to Eqs. 1 and 2, mm) and proteins were visualized by silver staining. respectively, by using the Fortran program ofCleland (30), in Analytical Gel Filtration and Glycerol Density Gradient which v is the measured initial rate, A is the substrate Ultracentrifugation of HIV-1 Protease. Crude PRO4 extract concentration, Vmax is the maximal velocity, Km is the (0.75 ml, 32 mg ofprotein) was chromatographed on a column Michaelis constant, y is kobs, c is the pH-independent value (2.5 cm x 20 cm) of Sephacryl S-200 superfine (Pharmacia) of y, H is the proton concentration, and K is an acid equilibrated with 50 mM Tris-HCl, pH 7.0/1 mM EDTA/0.1 dissociation constant. M NaCl/1 mM dithiothreitol/0.15% Triton X-100 (TENDT buffer) at a flow rate of 8 ml/hr (4°C). Protease activity in v = VmaxA/(Km + A) [1] log y = c/(1 + H/K) [2] column fractions was determined by the peptidolytic assay. Ultracentrifugation of HIV-1 protease was performed in a glycerol density gradient. Purified protease (5 ,g, 0.24 ml) RESULTS AND DISCUSSION was layered onto a 4-ml 17-35% (vol/vol) glycerol density Proteolysis of p55 and Peptidolysis of a Nonapeptide Sub- gradient (26) prepared in TENDT buffer (pH 7.0; with and strate by HIV-1 Protease. The recombinant protein p55 is a without 0.15% Triton X-100). An identical sample contained fusion protein containing -75% of the coding sequence of 0.05 mg of each of the protein standards cytochrome c, HIV-1 Pr55Sag (18). This protein comprises most of the p17 chymotrypsinogen A, and ovalbumin. Samples were ultra- region expressed as a fusion product, all of p24, and about centrifuged for 40 hr (40C) at 55,000 rpm in a Beckman SW60 half of p15. NaDodSO4/PAGE analysis and detection by rotor. Aliquots (0.2 ml) were assayed for standard proteins by silver staining (Fig. 1) showed that increasing amounts of the spectral analysis (280 nm, 410 nm) and by NaDodSO4/ PAGE, and HIV-1 protease activity was assayed by the kDo 2 3 4 5 6 7 peptidolytic assay as described above. Crosslinking ofHIV-1 Protease. Purified HIV-1 protease was crosslinked with dimethyl suberimidate (DMS) according to 43 the method of Davies and Stark (27). Reaction mixtures containing 0.2 M bicine (pH 8.5) and purified HIV-1 protease 25- (1.4-69 ,ug/ml) with or without 1.7-8.7 mM DMS were 18- incubated at room temperature for 5-8 hr, then denatured with 15- NaDodSO4/PAGE sample buffer and subjected to NaDod- S04/PAGE in 15% polyacrylamide minigels as described 6- above. The protein bands were electrophoretically transferred onto nitrocellulose paper, and the resulting blot was probed with a polyclonal antibody (18) raised against the PRO4 FIG. 1. NaDodSO4/PAGE analysis of proteolysis of p55 by product. Protein-antibody complexes were visualized by au- HIV-1 protease. The indicated molecular masses are from prestained toradiography following treatment with 1251I-labeled protein A. low molecular weight standards obtained from Bethesda Research avail- Laboratories. In 10-1.d samples, p55 was digested by purified HIV-1 Inactivation Studies ofHIV-1 Protease. Commercially protease for 60 min (pH 6.0, 370C) as described in Experimental able protease inactivators (0.1 and 10 mM) were preincubated Procedures. One microgram of p55 was present in lanes 1-5. with 0.7,4g purified protease for 1 hr at 370C in 10 gl of50mM Protease amounts were 0 ng (lane 1), 30 ng (lane 2), 70 ng (lane 3), Mes, pH 6/0.2 M NaCl/0.1% Triton X-100. Dimethyl sul- 130 ng (lane 4), 340 ng (lane 5), 70 ng (lane 6), and 210 ng (lane 7). foxide at 5% final concentration was used for water-insoluble Proteins were visualized by silver staining. Downloaded by guest on September 30, 2021 Biochemistry: Meek et al. Proc. Natl. Acad. Sci. USA 86 (1989) 1843 purified protease (30-340 ng) effected progressive proteolysis constants Km = 5.5 + 0.3 mM and Vma,, = 1.59 ± 0.04 nmol/ of p55 (51 kDa) to 41-kDa, 32-kDa, 24-kDa, and 21-kDa min (8 ng of protease) were obtained (37°C, pH 6.0) by fitting protein fragments in 1 hr. The sizes ofthese protein products the data to Eq. 1. Ifit is assumed that HIV-1 protease contains are consistent with cleavage at the p24-p15junction (41 kDa), one active site per 22-kDa dimer (see below), the resulting kcat the p17-p24junction (32 kDa), or at bothjunctions to form the and kcat/Km values for this substrate and enzyme preparation p245ag (24 kDa) and the fusion protein pl7gag (21 kDa) (the p15 would be 70 sec-1 and 13 mM-'sec-1, respectively. product, predicted to be an 8-kDa protein, is not observed). Native Molecular Mass of the Protease Suggests a Dimeric Upon extended incubation times, the p55 and 41-kDa pro- Structure. As suggested by structural modeling of their teins were completely absent and the 32-kDa protein was primary sequences, the retroviral proteases would most diminished but no additional of the 24-kDa and resemble the structure of the aspartic proteases if they 21-kDa proteins was apparent. These findings suggest that existed as protein dimers (17). To investigate this model, we the recombinant HIV-1 protease properly processes a re- sought to compare the molecular mass of HIV-1 protease combinant form of its natural substrate, Pr55sag. obtained under nondenaturing conditions with the value of 11 The highly purified recombinant HIV-1 protease (Fig. 1, kDa found upon NaDodSO4/PAGE analysis of the purified lanes 6 and 7) appeared as a single protein band of 11 kDa enzyme. Analytical gel filtration of PRO4 extract on Sepha- (>90% of total protein by densitometric analysis). The cryl S-200 demonstrated that HIV-1 protease activity eluted autoprocessing in E. coli of a 25-kDa precursor construct of in a sharp peak (74.7-76.0 ml) prior to the protein standard the HIV-1 pol coding region (PRO4) to an active protease of myoglobin, consistent with an apparent native molecular 11 kDa (18) is consistent with the premise that the HIV-1 mass of 18-19 kDa (Fig. 3A). That the native molecular mass protease comprises a 99-residue sequence (codons 69-167 in obtained from gel filtration is nearly twice that of the the pol reading frame; numbering as in ref. 1) between two denatured protein has also been reported for the retroviral consensus cleavage sequences [(Thr/Ser)-Xaa-Yaa-Phe*Pro)] protease from Moloney murine virus (31). The at both the amino and carboxyl termini. Stokes radius of the protease (a = 2.0 + 0.2 nm) was In addition to the protein substrate, the protease hydrolyzes determined from these data by the method of Laurent and the nonapeptide Ac-RASQNYPVV-NH2, which bears a se- Killander (32). quence similar to the known p17-p24 junction in Pr55sag (2). From glycerol density gradient ultracentrifugation of the Analysis of the peptidolytic fragments of Ac-RASQNYPVV- purified protease, the enzyme activity sedimented at a NH2 by reverse-phase HPLC (Fig. 2) provided a routine, distance consistent with that of a 21-kDa protein (Fig. 3B), quantitative assay for preliminary studies of the enzymolog- yielding a sedimentation coefficient of2.4 ± 0.1 S (average of ical properties of HIV-1 protease. Carboxyl-terminal se- three determinations). This observed sedimentation coeffi- quence analysis of a preparative sample ofthe peptide eluted cient was invariant in the presence or absence of Triton at 10 min was consistent with the sequence Xaa-Ala-Ser-Gln- X-100. The molecular weight (M) of the protease was calcu- Asn-Tyr (J.E.S., unpublished data), demonstrating that as lated from the sedimentation coefficient (s - 2.4 x 10-13 sec), expected, HIV-1 protease cleaved the nonapeptide between the Stokes radius (a = 2.0 x 10-7 cm), and the calculated the tyrosine and proline residues. Under reaction conditions partial specific volume [v = 0.757 cm3/g (33)] by using the identical to those used with the protease, neither p55 nor Ac- equation of Siegel and Monty (34): M = 61rnasN/(1 - vp), in RASQNYPVV-NH2 gave the observed cleavage patterns which r1 and p are, respectively, the viscosity and density of upon incubation with identically prepared bacterial extracts water at 20°C and N is Avogadro's number. From this, the containing a pAS plasmid that lacked the PRO4 insert. These molecular weight of the native HIV-1 protease was deter- results make unlikely the possibility that the observed pro- mined to be 22,000 ± 3000, a value twice that expected from teolysis and peptidolysis by material from PRO4-containing the amino acid composition. These findings strongly suggest were due to bacterial proteases. that the active form of HIV-1 protease is a dimer of identical The time course of the peptidolytic activity obtained from 11-kDa subunits. integration of the Ac-RASQNY and Ac-RASQNYPVV-NH2 Oligomeric protein structures have also been detected by peaks was found to be linear for up to 20 min (37°C, <20o chemical crosslinking as evidenced by NaDodSO4/PAGE conversion to product), and initial rates were determined for (27). Purified HIV-1 protease was crosslinked at pH 8.5 by samples in which product formation had not exceeded 15%. the amine-specific reagent DMS in large excess. An immu- From a double reciprocal plot ofthe initial rates at various Ac- noreactive 22-kDa protein band approximately equal in RASQNYPVV-NH2 concentrations (1-15 mM), the kinetic intensity to the monomeric protease (11 kDa) was visible

AC-RASQNYPW-NH2' Ac-RASONY- FIG. 2. Reverse-phase HPLC of peptidolytic fragments of Ac- RASQNYPVV-NH2 digested with HIV-1 protease. Profile a: reten- tion times of authentic representing peptidolysis of Ac- b RASQNYPVV-NH2 at 20% com- pletion [PVV-NH2 (0.6 mM, 9.22 min), AcRASQNY (0.6 mM, 9.95 a min), and Ac-RASQNYPVV-NH2 (2.4 mM, 16.7 min)]. Profile b: 20-min digestion of 3 mM Ac- I I - RASQNYPVV-NH2 by purified 0 10 20 HIV-1 protease as described in Retention time, min Experimental Procedures. Downloaded by guest on September 30, 2021 1844 !3iochemistry: Meek et al. Proc. Natl. Acad. Sci. USA 86 (1989) 4.0

Thyroglobulin A e0 3.0 13.0 0 cm Gamma globulin x 0) 11.0 ~~\Oval~~~vabumin 0) ¢ 2.0 Is RMyoglobin cn Protes\ a-0 9.0 _ ~~~~~HIV 0 Vitamin . 612j 1.0 7.0 0.4 0.5 0.6 0.7 O..0 0.2 0.4 0.6 0.8 1.0 Fr Kav

FIG. 3. (A) Analytical gel filtration of HIV-1 protease on Sephacryl S-200. The protein calibration standards used and their respective elution volumes (Ve) were Blue dextran, V. = 38.0 ml; thyroglobulin (670 kDa), V, = 43.0 ml; bovine gamma globulin (158 kDa), Vr = 54.5 ml; ovalbumin (44 kDa), Ve = 67.1 ml; myoglobin (17 kDa), Ve = 76.0 ml; vitamin B-12 (1.4 kDa), Ve = 96.3 ml [Vt = 97 ml; Kav = (Ve - VO)/(Vt - YO)]. The peak of protease activity, measured by the peptidolytic assay, appeared to be in fractions 59-60 (74.7-76.0 ml; Ka, = 0.63). (B) Glycerol density gradient ultracentrifugation of HIV-1 protease. The protein standards used were cytochrome c (12.5 kDa, 1.9 S), chymotrypsinogen A (25 kDa, 2.7 S), and ovalbumin (44 kDa, 3.5 S). HIV-1 protease was assayed by the peptidolytic assay. Relative mobility (Fr) = peak fraction (from bottom of tube)/total fractions (from bottom of tube).

upon immunoblot analysis (Fig. 4). Although larger oligo- was not restored following extensive dialysis of the EPNP- meric forms were apparent at the highest concentrations of treated enzyme. From a double reciprocal plot ofinactivation protease used (10 pg), the observation of both 22-kDa and rates (kobs) vs. EPNP concentrations (Fig. 5A Inset; fitted to 11-kDa species over a 100-fold concentration range of the Eq. 1), the concentration of EPNP resulting in half-maximal protein is in accord with an "intramolecular" reaction inactivation, Kinact, was 11 ± 2 mM and the maximum between the subunits of a protein dimer. inactivation rate, Vinact, was 0.040 ± 0.005 min-'. The Inactivators of Aspartic Proteases Inactivate HIV-1 Prote- addition of a competitive, synthetic peptide inhibitor (G.B.D. ase., The activity of the enzyme was measured following and B.D.D., unpublished data) at 10 times its K, value pretreatment with a number of protease inactivators. At high afforded complete protection against inactivation by 10 mM concentrations (0.1 mM and 10 mM), none ofthe inactivators EPNP, suggesting thait protease inactivation results from of the serine proteases [phenylmethylsulfonyl fluoride, ben- epoxide attack on an active-site residue. zamidine, N-tosyl-L-phenylethyl chloromethyl ketone, soy- The pH dependence of EPNP inactivation of HIV-1 pro- bean inhibitor (0.2 mg/ml), leupeptin (2.2 mg/ml)] or tease was examined at 10 mM EPNP and plotted as log kob, of the metalloproteases (1,10-phenanthroline, EDTA, phos- vs. pH, with the data fitted to Eq. 2 (Fig. 5B). The rate of phoramidon) effected significant inactivation of the peptido- protease inactivation was diminished upon protonation of an lytic activity of HIV-1 protease. In contrast, the sulfhydryl- enzymic residue of pK = 3.8 ± 0.09, suggesting that the specific reagents N-ethylmaleimide and iodoacetamide inac- epoxide esterifies an unprotonated carboxylic residue. The tivated the enzyme to <10% of its initial activity at inactivation of the protease at pH 6.0 by samples of EPNP concentrations of 0.1 mM, suggesting that a cysteine residue pretreated at pH 3-6 was invariant (Fig. 5B), indicating that is important to its catalytic activity or its structure. Inacti- the decrease in inactivation observed at lower pH values is vators of the aspartic proteases each effected some or not due to acid-catalyzed hydrolysis of the epoxide. Further- Pretreatment of the significant loss of protease activity. more, kinetic plots of log Vmax and log Vmax/Km vs. pH (pH 10 mM EPNP, and 10 mM enzyme with 0.1 mM pepstatin A, 2-6; Ac-RASQNYPVV-NH2 as substrate) are both charac- ethyl ester (0.1 mM cupric ion) N-diazoacetylnorleucine values below pH 4 = 3.6 and 3.4, of protease activity. These terized by decreasing (pKa resulted in >50% inactivation respectively), which also implicates the involvement of an findings are similar to those reported for the retroviral virus and bovine unprotonated carboxylic residue in the catalytic mechanism. proteases from avian myeloblastosis (35) to the aspartic proteases, leukemia virus (36). In view of the sequence homology we propose from our mechanistic studies that the HIV-1 EPNP has been shown by and by x-ray This one or both of the active protease also belongs to this class ofproteases. proposal crystallographic analysis to esterify studies in which the site-specific residues of pepsin (37-39). In our hands, in- is supported by two recent site aspartic the residue within the conserved Asp- creasing concentrations of EPNP effected time-dependent mutation of aspartic inactivation of HIV-1 protease (Fig. SA). Protease activity Thr-Gly sequence of the HIV-1 protease (expressed in E. colt) to an asparagine (10) or an alanine (21) residue resulted kDa 2 3 4 in an inactive protease. *fA: :^¢:,4,!.r...illlil6kallw -... We have demonstrated that the active form of HIV-1 255- protease has biochemical properties similar to the aspartic 18- proteases and possesses a native molecular mass twice that of the denatured protein. These findings are in concert with 15- the proposal that the "symmetric" active-site structure 6- found in the aspartic proteases, resulting from two distinct can be achieved in the smaller retroviral FIG. 4. Immunoblot analysis of chemically crosslinked HIV-1 protein domains, 0.3 of proteases by dimerization of single subunits such that each protease. Lane 1, 0.3 ,ug of protease, no DMS; lane 2, ,ug to protease, 8.7 mM DMS; lane 3, 0.15 j&g of protease, no DMS; lane monomer contributes an identical Asp-Thr-Gly sequence 4, 0.15 yg of protease, 8.7 mM DMS. an active site formed at an interface of subunits (17). This Downloaded by guest on September 30, 2021 Biochemistry: Meek et al. Proc. Natl. Acad. Sci. USA 86 (1989) 1845

0.4 2. Sanchez-Pescador, R., Power, M. D., Barr, P. J., Steimer, AEPNP, K. S., Stempien, M. M., Brown-Shimer, S. L., Gee, W. W., mm Renard, A., Randolph, A., Levy, J. A., Dina, D. & Luciw, 01 P. A. (1985) 227, 484-492. 3. Wain-Hobson, S., Sonigo, P., Danos, O., Cole, S. & Alizon, M. -0N4 0.6 (1982) Cell 40, 9-17. 1.5 4. Muesing, M. A., Smith, D. H., Cabradilla, C. D., Benton, g-0.8 P C.\V.,l Lasky, L. A. & Capon, D. J. (1985) Nature (London) 3.0 450-458. 3.0 5. ~~~~~313,Veronese, F., Rahman, R., Copeland, T., Oroszlan, S., Gallo, - 1.2 R. C. & Sarngadharan, M. G. (1987) AIDS Res. Hum. Retro- 3, 253-264. -1.6-16400- \ \ 6. Jacks, T., Power, M. D., Masiarz, F. R., Luciw, P. A., Barr, I.? 200- w\ X 6.0s-\ P. J. & Varmus, H. E. (1988) Nature (London) 331, 280-283. - :,OO 7. Dickson, C., Eisenman, R., Fan, H., Hunter, E. & Teich, N. - 2.0 X _ _ < (1982) in Molecular Biology of Tumor Viruses, eds. Weiss, R., 0/[EPN6] (mM,2) > 9.0 Teich, N., Varmus, H. & Coffin, J. 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(1987) Nature (London) 329, 351-354. 3.4 4.8 6.2 7.6 9.0 18. Debouck, C., Gorniak, J. G., Strickler, J. E., Meek, T. D., Metcalf, B. W. & Rosenberg, M. (1987) Proc. Natl. Acad. Sci. USA 84, 8903-8906. FIG. 5. Inactivation of HIV-1 protease by EPNP. (A) Semilog- 19. Framerie, W. G., Loeb, D. D., Casavant, N. C., Hutchison, arithmic plot ofprotease activity remaining after preincubation at the C. A., III, Egell, M. H. & Swanstrom, R. (1987) Science 236, indicated times with 0.6-9.0 mM EPNP (pH 6.0). The v1/vo values 305-308. were normalized at each time point to control samples containing no 20. Graves, M. C., Lim, J. J., Heimer, E. P. & Kramer, R. A. EPNP. Pseudo-first-order inactivation rates (kobJ) at each concen- (1988) Proc. Natl. Acad. Sci. USA 85, 2449-2453. tration of EPNP were obtained from fitting the data to ln(v1/vo) = 21. Mous, J., Heimer, E. P. & LeGrice, S. F. J. (1988) J. Virol. 62, -kob~t. (Inset) Double reciprocal replot of 1/kob, vs. 1/[EPNP]; the 1433-1440. line drawn through the experimental points was obtained from fitting 22. Hutta, J., Billich, S., Schulze, T., Sukrow, S. & Moelling, K. the data to Eq. 1. (B) pH dependence of EPNP inactivation of HIV-1 (1988) EMBO J. 7, 1785-1791. protease (220C). Inactivation rates (kob,; min') were measured at pH 23. Shatzman, A. R. & Rosenberg, M. (1987) Methods Enzymol. 6.0 following treatment ofthe enzyme at the indicated pH with 10mM 152, 661-673. EPNP (e). The solid line drawn through the experimental points was 24. Devare, S. G., Shatzman, A., Robbins, K. C., Rosenberg, M. obtained by fitting the data to Eq. 2. Control values (o) are kobs values & Aaronson, S. A. (1984) Cell 36, 43-49. at pH 6.0 by 10 mM EPNP that had been pretreated (4 hr, 220C) at 25. Mott, J. E., Grant, R. A., Ho, Y. & Platt, T. (1985) Proc. NatI. the indicated pH values. Acad. Sci. USA 82, 88-92. 26. Ho, Y.-S., Lewis, M. & Rosenberg, M. (1982) J. 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