Solution Structure of the Mature HIV-1 Protease Monomer
2003; Elsevier BV; Volume: 278; Issue: 44 Linguagem: Inglês
10.1074/jbc.m307549200
ISSN1083-351X
AutoresRieko Ishima, Dennis A. Torchia, Shannon M. Lynch, Angela M. Gronenborn, John M. Louis,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoWe present the first solution structure of the HIV-1 protease monomer spanning the region Phe1–Ala95 (PR1–95). Except for the terminal regions (residues 1–10 and 91–95) that are disordered, the tertiary fold of the remainder of the protease is essentially identical to that of the individual subunit of the dimer. In the monomer, the side chains of buried residues stabilizing the active site interface in the dimer, such as Asp25, Asp29, and Arg87, are now exposed to solvent. The flap dynamics in the monomer are similar to that of the free protease dimer. We also show that the protease domain of an optimized precursor flanked by 56 amino acids of the N-terminal transframe region is predominantly monomeric, exhibiting a tertiary fold that is quite similar to that of PR1–95 structure. This explains the very low catalytic activity observed for the protease prior to its maturation at its N terminus as compared with the mature protease, which is an active stable dimer under identical conditions. Adding as few as 2 amino acids to the N terminus of the mature protease significantly increases its dissociation into monomers. Knowledge of the protease monomer structure and critical features of its dimerization may aid in the screening and design of compounds that target the protease prior to its maturation from the Gag-Pol precursor. We present the first solution structure of the HIV-1 protease monomer spanning the region Phe1–Ala95 (PR1–95). Except for the terminal regions (residues 1–10 and 91–95) that are disordered, the tertiary fold of the remainder of the protease is essentially identical to that of the individual subunit of the dimer. In the monomer, the side chains of buried residues stabilizing the active site interface in the dimer, such as Asp25, Asp29, and Arg87, are now exposed to solvent. The flap dynamics in the monomer are similar to that of the free protease dimer. We also show that the protease domain of an optimized precursor flanked by 56 amino acids of the N-terminal transframe region is predominantly monomeric, exhibiting a tertiary fold that is quite similar to that of PR1–95 structure. This explains the very low catalytic activity observed for the protease prior to its maturation at its N terminus as compared with the mature protease, which is an active stable dimer under identical conditions. Adding as few as 2 amino acids to the N terminus of the mature protease significantly increases its dissociation into monomers. Knowledge of the protease monomer structure and critical features of its dimerization may aid in the screening and design of compounds that target the protease prior to its maturation from the Gag-Pol precursor. Catalytic activity of retroviral proteases requires dimer formation, unlike for cellular aspartic proteases that are monomeric. The active site of HIV-1 1The abbreviations used are: HIV-1, human immunodeficiency virus type 1; PR, mature HIV-1 protease bearing the 5 mutations, Q7K, L33I, L63I, C67A, C95A; TFP, transframe octapeptide; TFR, 56 amino acids of the transframe region comprising TFP and 48 amino acids of the p6pol domain; HSQC, heteronuclear single quantum coherence spectroscopy.1The abbreviations used are: HIV-1, human immunodeficiency virus type 1; PR, mature HIV-1 protease bearing the 5 mutations, Q7K, L33I, L63I, C67A, C95A; TFP, transframe octapeptide; TFR, 56 amino acids of the transframe region comprising TFP and 48 amino acids of the p6pol domain; HSQC, heteronuclear single quantum coherence spectroscopy. protease, similar to that of the Rous Sarcoma virus (RSV), Avian Myeloblastosis virus (AMV), and other retroviral proteases, is formed along the dimer interface. In HIV-1, a single copy of the protease, composed of 99 amino acids, is synthesized as part of the Gag-Pol polyprotein (Fig. 1) (1Oroszlan S. Luftig R.B. Curr. Top. Microbiol. Immunol. 1990; 157: 153-185PubMed Google Scholar). Thus, the initial critical step in the maturation of the protease involves the folding and dimerization of the protease domain in the form of a Gag-Pol precursor in order to catalyze the hydrolysis of the peptide bonds at its termini. The released active mature protease is expected to process the remainder of the Gag-Pol precursor and the Gag precursor at specific sites into the necessary mature functional and structural proteins required for viral maturation (1Oroszlan S. Luftig R.B. Curr. Top. Microbiol. Immunol. 1990; 157: 153-185PubMed Google Scholar, 2Louis J.M. Clore G.M. Gronenborn A.M. Nat. Struct. Biol. 1999; 6: 868-875Crossref PubMed Scopus (161) Google Scholar).Stage-specific regulation of the protease is crucial in the viral replication cycle, evident from studies showing that premature activation or partial inhibition of protease activity lead to impaired maturation of the virus (3Kaplan A.H. Zack J.A. Knigge M. Paul D.A. Kempf D.J. Norbeck D.W. Swanstrom R. J. Virol. 1993; 67: 4050-4055Crossref PubMed Google Scholar, 4Rose J.R. Babe L.M. Craik C.S. J. Virol. 1995; 69: 2751-2758Crossref PubMed Google Scholar, 5Karacostas V. Wolffe E.J. Nagashima K. Gonda M.A. Moss B. Virology. 1993; 193: 661-671Crossref PubMed Scopus (182) Google Scholar, 6Krausslich H.G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3213-3217Crossref PubMed Scopus (174) Google Scholar). Kinetics of the protease maturation from a model precursor containing only the two native cleavage sites, p6pol/PR at the N terminus and PR/RT at the C terminus (Fig. 1), showed that the reaction takes place in two independent sequential steps (7Louis J.M. Nashed N.T. Parris K.D. Kimmel A.R. Jerina D.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7970-7974Crossref PubMed Scopus (91) Google Scholar). The first step involves an intramolecular cleavage at the N terminus of the protease domain concomitant with a large increase in mature-like enzymatic activity and the appearance of the transient protease intermediate containing the flanking C-terminal polypeptide. The transient protease intermediate that exhibits similar kinetic parameters and dissociation constant to that of the mature protease is converted in a second step to release the mature protease via an intermolecular cleavage (8Wondrak E.M. Nashed N.T. Haber M.T. Jerina D.M. Louis J.M. J. Biol. Chem. 1996; 271: 4477-4481Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar).The native transframe region that flanks the N terminus of the protease in the Gag-Pol polyprotein, comprises two domains, the transframe octapeptide (TFP) followed by the 48 amino acid p6pol, both separated by protease cleavage sites (9Candotti D. Chappey C. Rosenheim M. M'Pele P. Huraux J.M. Agut H. C. R. Acad. Sci. III. 1994; 317: 183-189PubMed Google Scholar, 10Louis J.M. Dyda F. Nashed N.T. Kimmel A.R. Davies D.R. Biochemistry. 1998; 37: 2105-2110Crossref PubMed Scopus (83) Google Scholar). Reactions using the full-length TFP-P6pol-PR (Fig. 1) at pH 5.0, optimal for catalytic activity of the mature protease and the autocatalytic maturation reaction, showed the release of the protease to occur in two distinct steps (2Louis J.M. Clore G.M. Gronenborn A.M. Nat. Struct. Biol. 1999; 6: 868-875Crossref PubMed Scopus (161) Google Scholar, 11Louis J.M. Wondrak E.M. Kimmel A.R. Wingfield P.T. Nashed N.T. J. Biol. Chem. 1999; 274: 23437-23442Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The first cleavage occurs at the TFP-P6pol site to generate the intermediate precursor P6pol-PR. In the second step, P6pol-PR is converted to the mature protease concomitant with a large increase in catalytic activity. Thus, the two proteins, TFP-P6pol-PR and P6pol-PR, exhibit nearly the same low catalytic activity and the rate-limiting intramolecular cleavage at the p6pol-PR site is indeed concomitant with the appearance of mature-like enzymatic activity and stable tertiary structure formation characteristic of a protease dimer (2Louis J.M. Clore G.M. Gronenborn A.M. Nat. Struct. Biol. 1999; 6: 868-875Crossref PubMed Scopus (161) Google Scholar, 11Louis J.M. Wondrak E.M. Kimmel A.R. Wingfield P.T. Nashed N.T. J. Biol. Chem. 1999; 274: 23437-23442Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). These results are consistent with studies showing that HIV-1 particles of four different strains obtained from different cell lines contained only the 11-kDa mature protease and no p6pol-PR precursor (12Tessmer U. Krausslich H.G. J. Virol. 1998; 72: 3459-3463Crossref PubMed Google Scholar). Importantly, a mutation of the N-terminal protease cleavage site p6pol/PR leading to the production of an N-terminally extended 17-kDa protease species caused a severe defect in Gag polyprotein processing and a complete loss of viral infectivity (12Tessmer U. Krausslich H.G. J. Virol. 1998; 72: 3459-3463Crossref PubMed Google Scholar).The mature protease has been the target of drug development and hundreds of crystal structures of the protease dimer bound to various inhibitors have been solved (13Erickson J.W. Burt S.K. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 545-571Crossref PubMed Scopus (164) Google Scholar, 14Vondrasek J. van Buskirk C.P. Wlodawer A. Nat. Struct. Biol. 1997; 4: 8Crossref PubMed Scopus (51) Google Scholar). This has contributed to a large database for improved design of drugs that bind to the active site. However, treatment of HIV-1 infection on a longer term without the emergence of drug-resistance against protease inhibitors has been a challenge for the past decade (15Condra J.H. Haemophilia. 1998; 4: 610-615Crossref PubMed Scopus (62) Google Scholar). Although second generation active-site inhibitors are being developed in an effort to overcome the problem of drug-resistance, strategies to define an alternative mode of inhibition such as disrupting or preventing dimer formation may provide another avenue toward inhibitor design (16Weber I.T. J. Biol. Chem. 1990; 265: 10492-10496Abstract Full Text PDF PubMed Google Scholar). These latter inhibitors may have a greater success in curbing the emergence of drug resistance. There have been published reports of dimerization inhibitors of the protease (17Schramm H.J. Billich A. Jaeger E. Rucknagel K.P. Arnold G. Schramm W. Biochem. Biophys. Res. Commun. 1993; 194: 595-600Crossref PubMed Scopus (50) Google Scholar, 18Zutshi R. Franciskovich J. Shultz M. Schweitzer B. Bishop P. Wilson M. Chmielewski J. J. Am. Chem. Soc. 1997; 119: 4841-4845Crossref Scopus (111) Google Scholar); however, to date, no structural information of such monomer-inhibitor complexes is available. These studies have been hampered by the fact that monomeric protease is difficult to obtain since the mature protease is predominantly dimeric in solution with a dissociation constant < 5 nm (2Louis J.M. Clore G.M. Gronenborn A.M. Nat. Struct. Biol. 1999; 6: 868-875Crossref PubMed Scopus (161) Google Scholar, 19Wondrak E.M. Louis J.M. Biochemistry. 1996; 35: 12957-12962Crossref PubMed Scopus (47) Google Scholar, 20Todd M.J. Semo N. Freire E. J. Mol. Biol. 1998; 283: 475-488Crossref PubMed Scopus (161) Google Scholar).A network of interactions around the active site and termini of the mature protease are critical for its dimerization (21Lapatto R. Blundell T. Hemmings A. Overington J. Wilderspin A. Wood S. Merson J.R. Whittle P.J. Danley D.E. Geoghegan K.F. Hawrylik S.J. Lee S.E. Scheld K.G. Hobart P.M. Nature. 1989; 342: 299-302Crossref PubMed Scopus (409) Google Scholar, 22Spinelli S. Liu Q.Z. Alzari P.M. Hirel P.H. Poljak R.J. Biochimie (Paris). 1991; 73: 1391-1396Crossref PubMed Scopus (182) Google Scholar, 23Wlodawer A. Miller M. Jaskolski M. Sathyanarayana B.K. Baldwin E. Weber I.T. Selk L.M. Clawson L. Schneider J. Kent S.B. Science. 1989; 245: 616-621Crossref PubMed Scopus (1042) Google Scholar). To our knowledge there is no evidence for the existence of a folded monomer species during folding of the wild-type mature protease or structural data to support the dissociation of the dimer into a folded monomer. The existence of a folded monomer was recently observed only when unique dimer interface contacts were disrupted via mutations. These mutations increased the dissociation constant of the protease dramatically such that a monomer could be studied by solution NMR at a high protein concentration of up to 1 mm (in monomer) (24Ishima R. Ghirlando R. Tozser J. Gronenborn A.M. Torchia D.A. Louis J.M. J. Biol. Chem. 2001; 276: 49110-49116Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 25Louis J.M. Ishima R. Nesheiwat I. Pannell L.K. Lynch S.M. Torchia D.A. Gronenborn A.M. J. Biol. Chem. 2003; 278: 6085-6092Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). A series of mutants PRR87K, PRD29N, PRT26A, PR5–99, and PR1–95 has been described to exhibit a monomer fold in the absence of inhibitor. Of these, only PR1–95 did not form a ternary complex with DMP323, an inhibitor that binds tightly (K I < 10–9m) (26Lam P.Y. Ru Y. Jadhav P.K. Aldrich P.E. DeLucca G.V. Eyermann C.J. Chang C.H. Emmett G. Holler E.R. Daneker W.F. Li L. Confalone P.N. McHugh R.J. Han Q. Li R. Markwalder J.A. Seitz S.P. Sharpe T.R. Bacheler L.T. Rayner M.M. Klabe R.M. Shum L. Winslow D.L. Kornhauser D.M. Hodge C.N. J. Med. Chem. 1996; 39: 3514-3525Crossref PubMed Scopus (207) Google Scholar) to the protease dimer, up to protease concentrations of 1 mm.As a prelude to future structural studies of protease monomer-inhibitor complexes, here we present the first structure of the HIV-1 protease monomer, PR1–95 as determined by solution NMR. The structure of the monomer together with the NMR relaxation results has allowed comparison of the structure of the monomer with the subunit of the uninhibited dimer. In addition, using an inactive precursor bearing the active site D25N mutation, termed TFP-P6pol-PRD25N (Fig. 1), we show that the protease domain of the uninhibited precursor is mainly monomeric, adopting a tertiary fold very similar to that of the PR1–95 monomer. Systematic analyses of NMR and kinetic data of protease constructs, flanked either by 4 residues or 1 residue of the p6pol sequence, suggest that local interaction and packing of the terminal Pro1 and Phe99 residues in the mature protease are critical to achieve native-like dimer stability. Finally, the above results are discussed in the context of a model for the regulation of the HIV-1 protease in the viral replication cycle (2Louis J.M. Clore G.M. Gronenborn A.M. Nat. Struct. Biol. 1999; 6: 868-875Crossref PubMed Scopus (161) Google Scholar).EXPERIMENTAL PROCEDURESProtease Constructs—The protease (PR) domain in all constructs, optimized for NMR and kinetic studies, bears 5 mutations, Q7K, L33I, L63I to minimize the autoproteolysis of the protease and C67A and C95A to prevent cysteine-thiol oxidation (2Louis J.M. Clore G.M. Gronenborn A.M. Nat. Struct. Biol. 1999; 6: 868-875Crossref PubMed Scopus (161) Google Scholar). Plasmid DNA (pET11a, Novagen, Madison, WI) encoding TFP-p6pol-PR and PR (2Louis J.M. Clore G.M. Gronenborn A.M. Nat. Struct. Biol. 1999; 6: 868-875Crossref PubMed Scopus (161) Google Scholar) were used with the appropriate oligonucleotide primers to generate the constructs TFP-p6pol-PRD25N and PRD25N. Similarly, a stop codon was introduced to produce TFP-p6pol-PR1–95 and PR1–95. The PR-encoding plasmid was sequentially extended one codon at a time to produce SFNFPR. SFNFPR template was then used to introduce a D25N mutation. MIPR, MFPR and MGPR constructs were derived from PR. The initiator Met residue when not excised upon their expression in Escherichia coli is indicated as in the case of MIPR, MFPR and MGPR. All constructs were generated using the QuickChange mutagenesis protocol (Stratagene, La Jolla, CA) and verified by DNA sequencing and mass spectrometry. E. coli BL21(DE3) were grown in minimal media containing 15N ammonium chloride with or without 13C glucose as the sole nitrogen and carbon sources, respectively, at 37 °C and induced for expression. Proteins were prepared using an established protocol as described previously (24Ishima R. Ghirlando R. Tozser J. Gronenborn A.M. Torchia D.A. Louis J.M. J. Biol. Chem. 2001; 276: 49110-49116Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Specific cleavages giving rise to products due to the autoprocessing of active precursor proteins were accessed both by SDS-PAGE and mass spectrometry.NMR Spectroscopy and Structure Determination—All 1H-15N correlation spectra were recorded using ∼0.5 mm protein in monomer (unless noted otherwise) in 20 mm phosphate buffer at pH 5.8. NMR experiments for structure determination of PR1–95 were carried out using 0.4–0.5 mm protein in 20 mm phosphate buffer at pH 4.5 in 95% H2O/5% D2O and a sample volume of ∼280 μl in a 5-mm Shigemi tube (Shigemi, Inc., Allison Park, PA). Spectra were acquired on DMX500 spectrometers with or without a cryoprobe (Bruker Instruments, Billerica, MA) at 20 °C. Backbone and side chain resonance assignments, and heteronuclear seperated proton NOESY spectra, for structure determination, were obtained using standard triple-resonance three-dimensional NMR experiments (27Ferentz A.E. Wagner G. Q. Rev. Biophys. 2000; 33: 29-65Crossref PubMed Scopus (208) Google Scholar). Backbone dihedral angle restraints were obtained from 3JHNHα coupling constants, and χ1 angle restraints were determined from 3JNHβ coupling constants and NOESY data (28Clore G.M. Gronenborn A.M. Curr. Opin. Chem. Biol. 1998; 2: 564-570Crossref PubMed Scopus (103) Google Scholar). Residual 1DNH dipolar couplings were measured in 6% gel medium (29Tycko R. Blanco F.J. Ishii Y. J. Am. Chem. Soc. 2000; 122: 9340-9341Crossref Scopus (296) Google Scholar, 30Chou J.J. Gaemers S. Howder B. Louis J.M. Bax A. J. Biomol. NMR. 2001; 21: 377-382Crossref PubMed Scopus (218) Google Scholar). Gels were prepared from a stock solution of 36% w/v acrylamide and 0.92% w/v N,N′-methylenebisacrylamide yielding a acrylamide/bisacrylamide ratio of 39:1. Gels (280 μl) were cast to a diameter of 5.4 mm, rinsed in water overnight, dehydrated to about one-sixth the original size over a period of 5–6 h at 37 °C, soaked in protein solution (desired buffer and pH) for 16–20 h at room temperature and gently pushed into a Wilmad open-ended NMR tube (4.24 ± 0.012 ID) as described (29Tycko R. Blanco F.J. Ishii Y. J. Am. Chem. Soc. 2000; 122: 9340-9341Crossref Scopus (296) Google Scholar, 30Chou J.J. Gaemers S. Howder B. Louis J.M. Bax A. J. Biomol. NMR. 2001; 21: 377-382Crossref PubMed Scopus (218) Google Scholar). The bottom end of the tube was sealed with a susceptibility-matched plug and the top with a regular, susceptibility matched Shigemi (Allison Park, PA) microcell plunger.NMR data were processed and analyzed using the nmrPipe, nmrDraw, and PIPP software (31Garrett D.S. Powers R. Gronenborn A.M. Clore G.M. J. Magn. Reson. 1991; 95: 214-220Crossref Scopus (800) Google Scholar, 32Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11379) Google Scholar). Experimentally determined distance, dihedral angles, residual dipolar coupling constraints (Table I) were applied in a simulated annealing protocol using Xplor-NIH with conformational database torsion angle potentials (33Schwieters C.D. Kuszewski J.J. Tjandra N. Clore G.M. J. Magn. Reson. 2003; 160: 65-73Crossref PubMed Scopus (1841) Google Scholar). Simulated annealing and minimization calculations were carried out in Cartesian coordinate space with a final Powell minimization as the last step. Structures were analyzed using PROCHECK-NMR (34Laskowski R.A. Rullmann J.A.C. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4329) Google Scholar) and structure figures were generated using Insight II (MSI) and GRASP (35Nicholls A. Sharp K.A. Honig B. Proteins. 1991; 11: 281-296Crossref PubMed Scopus (5313) Google Scholar). Accessible surface area was calculated using NACCESS (36Hubbard S.J. Thornton J.M. Computer Program. University College, London1993Google Scholar)Table IExperimental constraints and structural statisticsNMR-derived constraintsInterproton distance constraints752Intraresidues (i— j = 0)224Sequential (i— j = 1)233Short range (1 < i— j < 5)56Long range (i— j > 4)205Dihedral angle constraints47Residual dipolar coupling (N-H) constraints63Total number of constraints862Total constraints per residue (excluding mobile N-terminal residues 1-10)10.1Structural quality20-Conformer ensembleRegularized mean structureRoot mean square deviations from experimental constraintsDistance constraints0.0175 ± 0.002580.01259Dihedral constraints (degree)0.1619 ± 0.14250.13731NH residues dipolar couplings (Hz)0.5135 ± 0.037360.45377Root mean square deviations from idealized covalent geometryBonds (angstrom)0.00144 ± 0.000090.0014562Angles (degree)0.42045 ± 0.008760.398953Improper (degree)0.33080 ± 0.036170.2811318ELI (kcal/mol)-135.098 ± 11.92-173.607Ramachandran statisticsaCalculated by PROCHECK-NMR. (%)Most favored region85.5 ± 1.385.3Additional allowed region10.8 ± 1.88.0Generously allowed region1.9 ± 2.25.3Disallowed region1.8 ± 1.11.3Coordinate precision (ordered regions of the structure)bCalculated for residues 11-15, 18-24, 32-34, 43-47 54-66, 69-78, and 83-90.Backbone atoms0.59 ± 0.104All nonhydrogen atoms1.11 ± 0.103a Calculated by PROCHECK-NMR.b Calculated for residues 11-15, 18-24, 32-34, 43-47 54-66, 69-78, and 83-90. Open table in a new tab Enzyme Kinetics—Kinetic parameters were measured using the substrate, Lys-Ala-Arg-Val-Nle-(4-nitrophenylalanine)-Glu-Ala-Nle-NH2 (California Peptide Research, Napa, CA) (37Richards A.D. Phylip L.H. Farmerie W.G. Scarborough P.E. Alvarez A. Dunn B.M. Hirel P.H. Konvalinka J. Strop P. Pavlickova L. J. Biol. Chem. 1990; 265: 7733-7736Abstract Full Text PDF PubMed Google Scholar) as described previously (7Louis J.M. Nashed N.T. Parris K.D. Kimmel A.R. Jerina D.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7970-7974Crossref PubMed Scopus (91) Google Scholar, 8Wondrak E.M. Nashed N.T. Haber M.T. Jerina D.M. Louis J.M. J. Biol. Chem. 1996; 271: 4477-4481Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 19Wondrak E.M. Louis J.M. Biochemistry. 1996; 35: 12957-12962Crossref PubMed Scopus (47) Google Scholar) in 50 mm sodium acetate buffer, pH 5 and 0.25 m NaCl at 25 °C. In a typical assay, 4 μl of enzyme was added to 96 μl of buffer in a 100 μl spectrophotometer cell. Reaction was initiated by the addition of 10 μl of substrate in water and monitored by following the decrease in absorption at 310 nm (Δϵ = 1800). In all cases, data were collected at substrate concentrations (10–460 μm) above and below Km at a final enzyme concentration of 250 nmMGPR and MIPR and 80 nm PR. The kinetic parameters, Km and k cat were obtained by fitting the Michaelis-Menten equation to initial rates. Assays to determine the Kd were performed under the same conditions without NaCl. Kd values were derived from plots of specific activity versus dimeric enzyme concentration as described previously (2Louis J.M. Clore G.M. Gronenborn A.M. Nat. Struct. Biol. 1999; 6: 868-875Crossref PubMed Scopus (161) Google Scholar, 19Wondrak E.M. Louis J.M. Biochemistry. 1996; 35: 12957-12962Crossref PubMed Scopus (47) Google Scholar) in a final substrate concentration of 390 μm. Enzyme concentrations were determined both spectrophotometrically (absorbance at 280 nm) and by Bio-Rad assay (Bio-Rad Laboratories, Hercules, CA).RESULTS AND DISCUSSIONUpon autocatalytic maturation at its N and C termini, the HIV-1 protease forms a stable homodimer exhibiting a dissociation constant in the subnanomolar range (2Louis J.M. Clore G.M. Gronenborn A.M. Nat. Struct. Biol. 1999; 6: 868-875Crossref PubMed Scopus (161) Google Scholar, 19Wondrak E.M. Louis J.M. Biochemistry. 1996; 35: 12957-12962Crossref PubMed Scopus (47) Google Scholar, 20Todd M.J. Semo N. Freire E. J. Mol. Biol. 1998; 283: 475-488Crossref PubMed Scopus (161) Google Scholar). Studies characterizing the HIV protease monomer were not feasible until we recently demonstrated that mutations of the interface residues, such as D29N, R87K, or deletion mutants of the terminal residues 1–4 or 96–99, destabilize the dimer (24Ishima R. Ghirlando R. Tozser J. Gronenborn A.M. Torchia D.A. Louis J.M. J. Biol. Chem. 2001; 276: 49110-49116Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 25Louis J.M. Ishima R. Nesheiwat I. Pannell L.K. Lynch S.M. Torchia D.A. Gronenborn A.M. J. Biol. Chem. 2003; 278: 6085-6092Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). While it is apparent that deletion of the terminal residues precludes the formation of the terminal β-sheet and disrupts dimerization, these studies also revealed that subtle intermonomer contacts formed by the conserved Asp29 and Arg87 residues are essential to the dimerization of the mature protease. NMR and equilibrium sedimentation analyses show that these mutants exhibit a monomer fold and a range of dimer dissociation constants. We chose to determine the monomer structure of PR1–95 because it is predominantly monomeric with no observable dimer formation up to a concentration of 1 mm, even in the presence of the high affinity inhibitor DMP323 (24Ishima R. Ghirlando R. Tozser J. Gronenborn A.M. Torchia D.A. Louis J.M. J. Biol. Chem. 2001; 276: 49110-49116Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar).Description of PR1–95 Monomer Structure—The three-dimensional structure of PR1–95 was determined using heteronuclear multidimensional NMR spectroscopy. Although PR1–95 undergoes aggregation that limited the number of experiments performed as well as the acquisition time of individual experiments, all necessary data for a high resolution structure were obtained. 40 of 100 calculated structures converged without angle or NOE violations greater than 5° or 0.5 Å, respectively. PR1–95 is a β-rich protein, composed of seven β-strands and one α-helix. A superposition of 10 conformers depicted in Fig. 2 shows that the structure is well defined except for disordered terminal and loop segments.Fig. 2Overall stereoview of the PR1–95 structure showing the final ensemble of 10 NMR conformers.View Large Image Figure ViewerDownload Hi-res image Download (PPT)A backbone superposition of the average NMR structure with the monomer subunit of two different crystal structures of the free mature protease dimer (21Lapatto R. Blundell T. Hemmings A. Overington J. Wilderspin A. Wood S. Merson J.R. Whittle P.J. Danley D.E. Geoghegan K.F. Hawrylik S.J. Lee S.E. Scheld K.G. Hobart P.M. Nature. 1989; 342: 299-302Crossref PubMed Scopus (409) Google Scholar, 38Rick S.W. Erickson J.W. Burt S.K. Proteins. 1998; 32: 7-16Crossref PubMed Scopus (65) Google Scholar) is shown in Fig. 3A. As is apparent, residues 10–90 of the PR1–95 monomer exhibit a nearly identical fold to that of one subunit of the protease dimer. This similarity in structures is consistent with the backbone chemical shifts of the PR1–95 monomer compared with those of the wild-type dimer (Fig. 4A). Characteristics that distinguish the PR1–95 structure from the monomer subunit of mature protease dimer are (I) disorder of the: (a) N-terminal residues 1–10, (b) flap residues 48–54, and (c) residues 91–95 at the C terminus of the α-helix and (II) solvent-exposed active site residues, comprising mainly polar amino acids. Interesting aspects of these regions of the monomer structure are discussed below.Fig. 3Structural details of the protease monomer. A, comparison of the average NMR structure of PR1–95 (blue) with one subunit of two free protease dimer crystal structures shown in green (21Lapatto R. Blundell T. Hemmings A. Overington J. Wilderspin A. Wood S. Merson J.R. Whittle P.J. Danley D.E. Geoghegan K.F. Hawrylik S.J. Lee S.E. Scheld K.G. Hobart P.M. Nature. 1989; 342: 299-302Crossref PubMed Scopus (409) Google Scholar) and yellow (38Rick S.W. Erickson J.W. Burt S.K. Proteins. 1998; 32: 7-16Crossref PubMed Scopus (65) Google Scholar). B, GRASP electrostatic surface potential of PR1–95 (excludes residues 1–10). Note: the crystal structure shown in green has a flap conformation that is more open than the crystal structure shown in yellow.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4Comparison of PR1–95 and PR chemical shifts and PR1–95 relaxation parameters. A, plot of difference of Cα chemical shifts of PR1–95 and PR. B, 15N-{H} heteronuclear NOE; C, 15N transverse relaxation time, T2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Terminal β-Sheet Interface—In the mature protease dimer, the terminal residues 1–4 and 96–99 of the two subunits form a well-ordered interfacial four-stranded anti-parallel β-sheet (16Weber I.T. J. Biol. Chem. 1990; 265: 10492-10496Abstract Full Text PDF PubMed Google Scholar). The lack of secondary structure of the N-terminal residues in the PR1–95 monomer is not surprising given that deletion of residues 96–99 precludes formation of the terminal interface β-sheet. The loss of the terminal β-sheet and chain flexibility observed for PR1–95 (Figs. 2 and 4) also occurs in monomer constructs such as PRT26A and PRR87K that contain intact terminal sequences. Thus, specific interactions distant from the terminal region clearly influence the stability of the termina
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