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• W2014734546 abstract "Autoprocessing of HIV-1 protease (PR) precursors is a crucial step in the generation of the mature protease. Very little is known regarding the molecular mechanism and regulation of this important process in the viral life cycle. In this context we report here the first and complete residue level investigations on the structural and folding characteristics of the 17-kDa precursor TFR-PR-Cnn (161 residues) of HIV-1 protease. The precursor shows autoprocessing activity indicating that the solution has a certain population of the folded active dimer. Removal of the 5-residue extension, Cnn at the C-terminal of PR enhanced the activity to some extent. However, NMR structural characterization of the precursor containing a mutation, D25N in the PR at pH 5.2 and 32 °C under different conditions of partial and complete denaturation by urea, indicate that the precursor has a high tendency to be unfolded. The major population in the ensemble displays some weak folding propensities in both the TFR and the PR regions, and many of these in the PR region are the non-native type. As both D25N mutant and wild-type PR are known to fold efficiently to the same native dimeric form, we infer that TFR cleavage enables removal of the non-native type of preferences in the PR domain to cause constructive folding of the protein. These results indicate that intrinsic structural and folding preferences in the precursor would have important regulatory roles in the autoprocessing reaction and generation of the mature enzyme. Autoprocessing of HIV-1 protease (PR) precursors is a crucial step in the generation of the mature protease. Very little is known regarding the molecular mechanism and regulation of this important process in the viral life cycle. In this context we report here the first and complete residue level investigations on the structural and folding characteristics of the 17-kDa precursor TFR-PR-Cnn (161 residues) of HIV-1 protease. The precursor shows autoprocessing activity indicating that the solution has a certain population of the folded active dimer. Removal of the 5-residue extension, Cnn at the C-terminal of PR enhanced the activity to some extent. However, NMR structural characterization of the precursor containing a mutation, D25N in the PR at pH 5.2 and 32 °C under different conditions of partial and complete denaturation by urea, indicate that the precursor has a high tendency to be unfolded. The major population in the ensemble displays some weak folding propensities in both the TFR and the PR regions, and many of these in the PR region are the non-native type. As both D25N mutant and wild-type PR are known to fold efficiently to the same native dimeric form, we infer that TFR cleavage enables removal of the non-native type of preferences in the PR domain to cause constructive folding of the protein. These results indicate that intrinsic structural and folding preferences in the precursor would have important regulatory roles in the autoprocessing reaction and generation of the mature enzyme. Retroviruses including human immunodeficiency virus (HIV), 1The abbreviations used are: HIV, human immunodeficiency virus; RT, reverse transcriptase; PR, HIV-1 protease; MBP, maltose-binding protein; TFR, N-terminal transframe region; AIDS, acquired immuno-deficiency syndrome; MALDI-TOF, matrix-associated laser desorption ionization-time of flight; HSQC, heteronuclear single quantum coherence; TOCSY, total correlated spectroscopy; RT, reverse transcriptase; TFP, transframe octapeptide. use their minimal genetic information by encoding their structural proteins and enzymes as two polyprotein precursors Gag and Gag-Pol (1Vaishnav Y.N. Wong-Staal F. Annu. Rev. Biochem. 1991; 60: 577-630Crossref PubMed Scopus (281) Google Scholar). Autoprocessing of these precursors is an essential step in the life cycle of the virus (2Skalka A.M. Cell. 1989; 56: 911-913Abstract Full Text PDF PubMed Scopus (90) Google Scholar, 3Kay J. Dunn B.M. Biochem. BioPhys. Acta. 1990; 1048: 1-18Crossref PubMed Scopus (125) Google Scholar). In HIV, HIV-1 protease (PR) plays a crucial role in virus maturation by processing these precursors into functional proteins (4Kohl N.E. Emini E.A. Schleif W.A. Davis L.J. Heimbach J.C. Dixon R.A.F. Scolnick E.M. Sigal I.S. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4686-4690Crossref PubMed Scopus (1279) Google Scholar, 5Kramer R.A. Schaber M.D. Skalka A.M. Ganguly K. Wong-Staal F. Reddy E.P. Science. 1986; 231: 1580-1584Crossref PubMed Scopus (232) Google Scholar). The HIV-1 PR is a 22-kDa homodimeric aspartyl protease, with each monomer having 99 amino acids and contributing the conserved catalytic sequence Asp-(Ser/Thr)-Gly (6Seelmeier S. Schmidt H. Turk V. Von der Helm K. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6612-6616Crossref PubMed Scopus (313) Google Scholar, 7Le Grice S.F.J. Mills J. Mous J. EMBO J. 1988; 7: 2547-2553Crossref PubMed Scopus (98) Google Scholar, 8Darke P.L. Leu C.T. Davis L.J. Heimbach J.C. Diehl R.E. Hill W.S. Dixon R.A. Sigal I.S. J. Biol. Chem. 1989; 264: 2307-2312Abstract Full Text PDF PubMed Google Scholar, 9Wlodawer 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 (1052) Google Scholar). As the HIV-1 protease, which is flanked by the highly variable p6pol at its N terminus and by the reverse transcriptase (RT) at its C terminus (10Oroszlan S. Luftig R.B. Curr. Top. Microbiol. Immunol. 1990; 157: 153-185PubMed Google Scholar, 11Candotti D. Chappey C. Rosenheim M. M'Pele P. Huraux J.M. Agut H. C. R. Acad. Sci. III. 1994; 317: 183-189PubMed Google Scholar), is responsible for all cleavages in the Gag-Pol precursor, its dimerization and autocatalytic release from the Gag-Pol are critical steps in the viral life cycle (4Kohl N.E. Emini E.A. Schleif W.A. Davis L.J. Heimbach J.C. Dixon R.A.F. Scolnick E.M. Sigal I.S. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4686-4690Crossref PubMed Scopus (1279) Google Scholar, 13Burstein H. Bizub D. Kotler M. Schatz G. Vogt V.M. Skalka A.M. J. Virol. 1992; 66: 1781-1785Crossref PubMed Google Scholar, 14Louis J.M. McDonald R.A. Nashed N.T. Wondrak E.M. Jerina D.M. Oroszlan S. Mora P.T. Eur. J. Biochem. 1991; 199: 361-369Crossref PubMed Scopus (61) Google Scholar). Earlier studies have shown that premature activation or partial inhibition of the protease leads to retarded viral maturation (15Kaplan 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, 16Rose J.R. Babe L.M. Craik C.S. J. Virol. 1995; 69: 2751-2758Crossref PubMed Google Scholar, 17Karacostas V. Wolffe E.J. Nagashima K. Gonda M.A. Moss B. Virology. 1993; 193: 661-671Crossref PubMed Scopus (184) Google Scholar, 18Krausslich H.G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3213-3217Crossref PubMed Scopus (176) Google Scholar). Hence understanding the exact sequence of protease maturation from the Gag-Pol precursor has gained importance in recent years because of its intrinsic importance in viral maturation and as a target for drugs against AIDS (19Wondrak 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). Pettit et al. (20Pettit S.C. Everitt L.E. Choudhury S. Dunn B.M. Kaplan A.H. J. Virol. 2004; 78: 8477-8485Crossref PubMed Scopus (110) Google Scholar) have recently shown, by co-expressing equivalent amounts of substituted Gag-Pol constructs, that the initial cleavage of the HIV-1 Gag-Pol precursor is intramolecular. Moreover, they showed that competitive active site inhibition by the drug retonavir was 10,000-fold less for the protease embedded in the precursor than for the mature free protease (20Pettit S.C. Everitt L.E. Choudhury S. Dunn B.M. Kaplan A.H. J. Virol. 2004; 78: 8477-8485Crossref PubMed Scopus (110) Google Scholar). Earlier, kinetic studies on the model precursor system MBP-ΔTF-Protease-ΔRT showed that the protease maturation takes place in two steps. (ΔTF and ΔRT are short native sequences from the transframe protein and the reverse transcriptase, respectively. MBP stands for maltose-binding protein of Escherichia coli containing two native cleavage sites, p6pol/PR at the N terminus and PR/RT at the C terminus.) The first step involves an intramolecular cleavage of the N terminus that is followed by intermolecular cleavage of the C terminus (19Wondrak 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, 21Louis 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). A relatively low Km for peptide substrates representing the p6*-PR (where p6* is TFP+p6pol) cleavage site, compared with that for oligopeptides corresponding to other Gag or Pol cleavage sites (23Dunn B.M. Gustchina A. Wlodawer A. Kay J. Methods Enzymol. 1994; 241: 254-278Crossref PubMed Scopus (68) Google Scholar) supports the view that the N-terminal cleavage is an early event in the proteolytic cascade. The activity of the protease-ΔRT was found to be nearly equal to that of the mature PR, though its conformational stability was much less than that of PR (19Wondrak 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). However a 600-fold decrease in catalytic activity was seen in MBP-ΔTF-protease-ΔRT compared with mature PR (19Wondrak 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, 21Louis 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). Thus the flanking N terminus of the protease seems to have important consequences with maturation. The N-terminal transframe region (TFR) consisting of a conserved N-terminal transframe octapeptide (TFP) and a 48–60 amino acid long variable p6pol, with a protease cleavage site at the intersection, does not have any stable secondary or tertiary structure in free solution (24Beissinger M. Paulus C. Bayer P. Wolf H. Rosch P. Wagner R. Eur. J. Biochem. 1996; 237: 383-392Crossref PubMed Scopus (28) Google Scholar), though some tendency for helix formation has been seen. However, when present with the PR, TFR does seem to act as a regulator for the autoprocessing of the protease (11Candotti D. Chappey C. Rosenheim M. M'Pele P. Huraux J.M. Agut H. C. R. Acad. Sci. III. 1994; 317: 183-189PubMed Google Scholar, 23Dunn B.M. Gustchina A. Wlodawer A. Kay J. Methods Enzymol. 1994; 241: 254-278Crossref PubMed Scopus (68) Google Scholar, 25Louis J.M. Dyda F. Nashed N.T. Kimmel A.R. Davies D.R. Biochemistry. 1998; 37: 2105-2110Crossref PubMed Scopus (84) Google Scholar, 26Paulus C. Hellebrand S. Tessmer U. Wolf H. Krauslich H.G. Wagner R. J. Biol. Chem. 1999; 274: 21539-21543Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 27Dautin N. Karimova G. Ladant D. J. Virol. 2003; 77: 8216-8226Crossref PubMed Scopus (15) Google Scholar, 28Louis 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, 29Louis M. Clore G.M. Gronenborn A.M. Nat. Stuct. Biol. 1999; 6: 868-875Crossref PubMed Scopus (162) Google Scholar). Interaction of recombinant p6* protein with HIV-1 PR was found to specifically inhibit its activity, and the inhibition was dependent on the C-terminal cleavage site residues SFNF in the p6*. In separate experiments with the precursor, these residues blocked the substrate binding cleft in HIV1-PR after N-terminal autoprocessing of the precursor. At the same time it was also observed that the p6* stabilized the dimer, as the relative amount of dimer increased by 12% in its presence (25Louis J.M. Dyda F. Nashed N.T. Kimmel A.R. Davies D.R. Biochemistry. 1998; 37: 2105-2110Crossref PubMed Scopus (84) Google Scholar). Functional characterization of the model precursor ΔTFP-p6pol-PR (ΔTFP is a 5-residue variant of TFP) by examination of the mechanism and the pH rate profile of the autocatalytic reaction to produce mature PR shows that full-length TFR with its native cleavage sites is critical for the regulated autoprocessing of Gag-Pol and for optimal catalytic activity (28Louis 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 extensive study by Dautin et al. (27Dautin N. Karimova G. Ladant D. J. Virol. 2003; 77: 8216-8226Crossref PubMed Scopus (15) Google Scholar) on functional modulations due to N- and C-terminal extensions to PR, using an E. coli genetic assay for proteolytic activity and a bacterial two-hybrid system, shows that the TFR can restore enzymatic activity to a dimerization-deficient HIV protease variant. Experiments with various deletion and addition mutants of PR and its precursors, Gag-Pol, TFR-PR, also give insights into folding and dimerization of PR (29Louis M. Clore G.M. Gronenborn A.M. Nat. Stuct. Biol. 1999; 6: 868-875Crossref PubMed Scopus (162) Google Scholar). For example, deletion of the first four residues in PR led to >90% unfolded ΔPR. Similar destabilization was observed for PR with additional residues in the N terminus (29Louis M. Clore G.M. Gronenborn A.M. Nat. Stuct. Biol. 1999; 6: 868-875Crossref PubMed Scopus (162) Google Scholar). Earlier, it has also been shown that removal of the p6pol domain from the Gag-Pol polyprotein leads to a significantly higher rate of processing of the Gag-ΔPol precursor (31Partin K. Zybarth G. Ehrlich L. DeCrombrugghe M. Wimmer E. Carter C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4776-4780Crossref PubMed Scopus (64) Google Scholar). The studies discussed so far are mainly based on enzymatic activity assays for the HIV-1 PR and its precursors using the chromogenic peptide substrate Lys-Ala-Arg-Val-Nle-Phe(p-NO2)-Glu-Ala-Nle-NH2 (19Wondrak 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, 21Louis 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, 32Richards 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), or immunoblotting assays of the autolytic products. These give very good quantitative as well as qualitative information with regard to the working of the various precursors of HIV-1 protease. However, there are very few reports about the residue level structural characteristics of these precursors, which is crucial to understanding the molecular mechanism of protease maturation (29Louis M. Clore G.M. Gronenborn A.M. Nat. Stuct. Biol. 1999; 6: 868-875Crossref PubMed Scopus (162) Google Scholar, 33Ishima R. Torchia D.A. Shannon M.L. Angela M.G. Louis J.M. J. Biol. Chem. 2003; 278: 43311-43319Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Louis et al. (29Louis M. Clore G.M. Gronenborn A.M. Nat. Stuct. Biol. 1999; 6: 868-875Crossref PubMed Scopus (162) Google Scholar) have earlier shown, through NMR, how the N-terminal TFR extension to the HIV-1 PR does not allow it to fold even in the presence of DMP323, which is one of the tightest binding inhibitors. Detailed NMR structural characterization of wildtype TFP-p6pol-PR was not possible because of its autolytic property. Hence, in a later study, an active site D25N mutation was introduced, and the HSQC spectra were seen to have many peaks at the same chemical shifts as in the spectra of the folded PRD25N, though, they also had many intense peaks in a narrow region of amide proton chemical shifts (8.0–8.5 ppm), presumably belonging to the TFR residues. This indicated that the PR region folded properly, although the TFR region could not be characterized because of insufficient dispersion of the peaks (33Ishima R. Torchia D.A. Shannon M.L. Angela M.G. Louis J.M. J. Biol. Chem. 2003; 278: 43311-43319Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). It was suggested that the TFR region was largely unstructured. Thus, all the above studies demonstrate the importance of TFR on the folding and maturation of the protease. However, the mechanistic details at the residue level are still not understood. In this context we present here investigations on a precursor TFP-p6pol-PR-Cnn, where Cnn is a non-native pentapeptide extension at the C terminus of PR. Bacterial expression and MALDI analysis of the precursor show that TFR does not hamper the autoprocessing of the precursor so as to release the PR. Deletion of Cnn enhanced autoprocessing, indicating that the non-native C-terminal extension interferes in the cleavage mechanism. We carried out extensive NMR investigations on the precursor containing an active site mutation D25N, which was stable for several weeks for NMR experiments. We monitored the intrinsic folding propensities of the precursor by studying the graded changes in the dynamic as well as structural characteristics of the equilibrium intermediates, created by use of different concentrations of the chemical denaturant, urea. These results have significant implications for the regulation mechanism of the autoprocessing reaction of HIV-1 protease precursors. Protein Preparation—Starting with the clone for the TFR-PR-tethered dimer (TFR-PR-Cnn-PR), kindly supplied by Dr. M. V. Hosur of Bhabha Atomic Research Centre, Mumbai, we introduced an active site mutation, D25N, in PR, using a standard PCR-based site-directed mutagenesis strategy; this mutation does not affect PR folding but prevents its autocleavage. From this the TFR-PR-Cnn region was selected and introduced into the NdeI/BamH1 multiple cloning site of a pET11a plasmid. The inclusion of the Cnn, besides providing a non-native flanking C terminus, has a practical advantage. It has the sequence GGSSG, and the glycines have a special significance in the NMR assignment strategy. At the same time, Cnn is known not to affect the folding characteristics of PR in the tethered dimer, which folds similarly to the native homodimer (34Pillai B. Kannan K.K. Hosur M.V. PROTEINS, Struct. Funct. Genet. 2001; 43: 57-64Crossref PubMed Scopus (60) Google Scholar). Similarly, the TFR-PR construct was also prepared. The desired wild-type constructs were prepared by PCR amplification of TFR-PR and TFR-PR-Cnn regions from the full clone (TFR-PR-Cnn-PR) and introducing them into a pET11a plasmid as described above. The constructs were sequenced to verify that there were no inadvertent PCR-induced errors. The plasmid was transformed into E. coli strain BL21(DE3) for protein overexpression. Transformed bacteria were grown at 37 °C in M9 medium to OD600 of ∼0.8, and then induced for production of the desired proteins using 1 mm isopropyl-1-thio-β-d-galactopyranoside. Uniformly 15N- and 15N/13C-labeled protein samples were prepared by growing bacteria in M9 minimal media supplemented with 1 g liter-1 15NH Cl and 4 g liter-1 [13C]glucose. Protein was purified as described previously (35Panchal S.C. Pillai B. Hosur M.V. Hosur R.V. Curr. Sci. 2000; 79: 1684-1695Google Scholar). MALDI analysis of the protein showed peaks at the expected molecular mass (17.3 kDa). The NMR samples contained 1 mm protein in 50 mm acetate buffer (pH 5.2) containing 5 mm EDTA, 20 mm dithiothreitol, and different concentrations of urea in 90% H2O, 10% D2O. Gel Electrophoresis—The recombinant protein was induced in BL21(DE3) E. coli bacterial cells as described in the section on protein preparation. Aliquots were taken at two different induction times, 3 and 5 h, and analyzed on 12% SDS-PAGE. Capillary Electrophoresis—The purified protein was concentrated to ∼1 mm and analyzed by neutral capillary electrophoresis on a Beckmann-Coulter capillary electrophoresis system in the presence and absence of the denaturants, urea and guanidine hydrochloride. Mass Spectroscopy—MALDI-TOF mass spectrometry analyses were carried out with Micromass (UK) MALDI-TOF Spec 2E spectrometer equipped with a UV nitrogen laser (337 nm) and a dual microchannel microplate detector. The samples were prepared by mixing 1 μl of protein solution (∼20 μm) with 1 μl of freshly prepared matrix solution (10 mg/ml of 2,5-dihydroxybenzoic acid in 3:2 0.1% trifluoroacetic acid/acetonitrile). A total of 1 μl of this mixture was placed on the stainless steel probe plate and allowed to dry at room temperature. The spectra were recorded in the positive reflector linear mode at an accelerated voltage of 20 kV in the range from 4000 to 30,000 Da. For each measurement, the spectra were externally calibrated using myoglobin and trypsinogen. NMR Spectroscopy—All NMR experiments were performed at 32 °C on a Varian Unity-plus 600 MHz NMR spectrometer equipped with pulse-shaping and pulse-field gradient capabilities. For the HNN spectrum the delays TN, and TC, were both set to 28 ms. 40 complex points were used along the t1 and t2 dimensions. The HN(C)N spectrum was recorded with the same TN and TC parameters, the same number of t1 and t2 points, and the TCC delay was set to 9 ms. TOCSY-HSQC was recorded with a mixing time of 60 ms, 32 complex points along the 15N (t1) dimension and 64 complex points along the 1H (t2) dimension. CBCANH and CBCA(CO)NH were recorded with 40 complex t1 points (15N) and 64 complex t2 points (13C). HNCO was recorded with 40 complex points along t1 and t2. An HSQC was recorded with 256 t1 increments. For the high resolution HSQC data, required for coupling constant measurements, 8192 and 512 complex points were acquired along the t2 and t1 dimensions, respectively. For the relaxation measurements 2048 and 256 complex points were collected along the two dimensions. For R2 measurements, the following Carr-Purcell-Meiboom-Gill (CPMG) delays were used: 10, 30, 50, 90, 130, 150, 190, 230 ms and spectra duplicated at 50 and 150 ms. The R2 values were extracted by fitting the peak intensities to the equation I(t) = B exp(-R2t). The experiments were carried out using the pulse sequences described by Farrow et al. (36Farrow N.A. Muhandiram R. Singer A.U. Pascal S.M. Kay C.M. Gish G. Shoelson S.E. Pawson T. Forman-Kay J.D. Kay L.E. Biochemistry. 1994; 33: 5984-6003Crossref PubMed Scopus (2018) Google Scholar). C-terminal Extension at PR Retards the Autoprocessing Activity of the Precursor—We checked the autoproteolytic activity of TFR-PR and TFR-PR-Cnn precursors in vivo (Fig. 1, lanes II and VII). For TFR-PR, we see no trace of the precursor in the SDS-PAGE after 6 h of induction. This is clear evidence that the TFR does not prevent the autoprocessing activity of the precursor (lane VII). However, in the case of TFR-PR-Cnn we see the presence of the intact precursor in the gel (lane II). Thus the C-terminal extension in our TFR-PR precursor slowed down autoprocessing. Our MALDI result with the purified protein also points to the same fact (Fig. 2). For the TFR-PR we see only an ∼11-kDa peak for the PR and a ∼7-kDa peak for the TFR part; however for the TFR-PR-Cnn we see a peak at ∼18 kDa corresponding to the precursor. This seems to suggest that the C-terminal extension possibly interacts with the PR region; either it interferes with dimer formation or it blocks the active site as has been observed for the SFNF stretch at the C terminus of the TFR in an earlier study (26Paulus C. Hellebrand S. Tessmer U. Wolf H. Krauslich H.G. Wagner R. J. Biol. Chem. 1999; 274: 21539-21543Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar).Fig. 2MALDI-TOF analysis of precursor activity. Top panel, MALDI spectrum of TFR-PR-Cnn containing the D25N active site mutation. Peaks attributed to singly [M1]+, doubly [M1]2+, and triply [M1]3+ charged species of the precursor are seen. Middle panel, MALDI spectrum of the precursor without the D25N mutation. The fragment peaks [M2]+ and [M3]+ corresponding to the monomer PR and TFR are seen. A small peak corresponding to [M1]+ is also visible. Bottom panel, MALDI spectrum of the precursor TFR-PR. The intact precursor peak [M1]+ is not seen. Only the fragment peaks [M2]+ and [M3]+ are seen. In both the middle and the bottom panels doubly charged species of PR are also seen.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Intrinsic Folding Characteristics of the Precursor—Since for the autocleavage reaction, the precursor has to become active by forming a dimer with the correct fold and generate an active site, we attempted to determine the structure of the precursor by NMR in solution. For this purpose we first prepared a D25N mutant of the precursor, which is inactive as the mutation is at the active site of PR. At the same time it is also known that the D25N mutation does not affect the folding of the protease (37Louis 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). This mutant precursor is thus stable for weeks together and is ideally suited for structural characterization by NMR. However, it turned out that the protein had a high tendency to aggregate, as seen by dynamic light scattering, capillary electrophoresis (data not shown), and also by NMR (see below), over a wide range of experimental conditions of pH and temperature. Deleting the C-terminal extension also did not make any difference with regard to this behavior. This is at variance with the earlier report by Ishima et al. (33Ishima R. Torchia D.A. Shannon M.L. Angela M.G. Louis J.M. J. Biol. Chem. 2003; 278: 43311-43319Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) on a precursor, which had the mutations Q7K, D25N, L33I, L63I, C67A in the PR region, and three residues at the N terminus of TFR that were different from those in our precursor. Ishima et al. (33Ishima R. Torchia D.A. Shannon M.L. Angela M.G. Louis J.M. J. Biol. Chem. 2003; 278: 43311-43319Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) found the protein to be a monomer and stable even at the high NMR concentration, and the spectra displayed features of native-like fold for the PR region. Therefore to investigate the intrinsic folding characteristics of our present precursor, we undertook to elucidate the structural characteristics in 8 m urea and the transitions therefrom by NMR, and the various equilibrium intermediates were created by systematically varying the urea concentration. In the following, we first describe the sequence-specific resonance assignments for the various urea denatured states and then present the structural and dynamic characterizations of the precursor at pH 5.2 and 32 °C. Resonance Assignments—The TFR-PR-Cnn precursor is 161 residues long, of which the first 57 residues belong to the TFR portion. The next 99 residues, that is, 58–156 actually constitute the PR portion. Hence residues 59–62, 152–156 form the dimerization domain, 82–84 form the active site, 138–140 form the substrate binding cleft, 100–106 form the hinge region, and 109–112 constitute the mobile flaps in the PR (35Panchal S.C. Pillai B. Hosur M.V. Hosur R.V. Curr. Sci. 2000; 79: 1684-1695Google Scholar). The final five residues (157–161) having the sequence GGSSG, constitute an extension to the PR at the C terminus. Henceforth we will use these numbers for structural discussion. Conventionally, backbone assignment in proteins has been achieved by a combination of several three-dimensional triple resonance experiments, typically, HNCA, HN(CO)CA, CBCANH, and CBCA(CO)NH (reviewed recently in Ref. 38Ferentz A.E. Wagner G. Quart. Rev. Biophys. 2000; 33: 29-65Crossref PubMed Scopus (210) Google Scholar). These experiments display correlations between HN, 15N, and (Cα, Cb) nuclei along the protein backbone. The success of this approach depends critically on the dispersion of the Cα, Cβ chemical shifts, and therefore for unfolded proteins, where this dispersion is very poor, the method has serious limitations. Our methodology of assignment is based on the recently described triple resonance experiments HNN and HN(C)N (39Panchal S.C. Bhavesh N.S. Hosur R.V. J. Biomol. NMR. 2001; 20: 135-147Crossref PubMed Scopus (175) Google Scholar). The most significant feature of these experiments is the observation of different patterns of positive and negative peaks in the (F1, F3) planes depending on the residue types at i-1, i, and i+1 positions. These have been discussed in detail earlier (39Panchal S.C. Bhavesh N.S. Hosur R.V. J. Biomol. NMR. 2001; 20: 135-147Crossref PubMed Scopus (175) Google Scholar, 40Bhavesh N.S. Panchal S.C. Hosur R.V. Biochemistry. 2001; 40: 14727-14735Crossref PubMed Scopus (64) Google Scholar); suffice it to say here that glycines and prolines play important roles in this regard, the former because of the absence of the Cβ, and the latter because of the absence of the amide proton. Triplets containing these residues produce very characteristic patterns in the (F1, F3) planes, which can be termed as fixed points. These provide many starts and check points for the sequential walk, and hence it is less crucial to obtain side chain assignment to validate the backbone assignments. Nevertheless, simultaneous analysis" @default.
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