Folding Regulates Autoprocessing of HIV-1 Protease Precursor

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. 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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, for maltose-binding protein of containing two native cleavage at the N terminus and at the C The first step an cleavage of the N terminus that is by 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, J.M. Nashed N.T. Jerina D.M. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar). for the is cleavage with that for to Gag or cleavage B.M. A. A. J. 1994; PubMed Scopus Google Scholar) the that the N-terminal cleavage is an in the The activity of the was to be to that of the mature PR, its was 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). a in catalytic activity was in 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, J.M. Nashed N.T. Jerina D.M. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar). the N terminus of the protease to have important with The N-terminal transframe region of a conserved N-terminal transframe and a amino variable with a protease cleavage site at the have or in free solution M. C. P. H. P. R. Eur. J. Biochem. 1996; PubMed Scopus Google Scholar), some tendency for has However, with the PR, TFR to as a for the autoprocessing of the protease D. Chappey C. Rosenheim M. M'Pele P. Huraux J.M. Agut H. C. R. Acad. Sci. III. 1994; 317: 183-189PubMed Google Scholar, B.M. A. A. J. 1994; PubMed Scopus Google Scholar, J.M. F. Nashed N.T. PubMed Scopus Google Scholar, C. S. U. H. H.G. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, G. D. J. Virol. PubMed Scopus Google Scholar, J.M. Wondrak E.M. P.T. Nashed N.T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. A.M. Biol. PubMed Scopus Google Scholar). of protein with HIV-1 PR was to its and the inhibition was on the C-terminal cleavage site in the In with the precursor, these the in N-terminal autoprocessing of the the same was that the the as the of by in its J.M. F. Nashed N.T. PubMed Scopus Google Scholar). characterization of the model precursor is a 5-residue of by of the mechanism and the pH of the autocatalytic reaction to mature PR shows that TFR with its native cleavage is critical for the autoprocessing of Gag-Pol and for catalytic activity J.M. Wondrak E.M. P.T. Nashed N.T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). 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Chem. 1990; Full Text PDF PubMed Google Scholar), or of the These as as information with to the of the precursors of HIV-1 protease. However, are the residue level structural characteristics of these which is crucial to understanding the molecular mechanism of protease maturation M. A.M. Biol. PubMed Scopus Google Scholar, R. D.A. Louis J.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Louis et al. M. A.M. Biol. PubMed Scopus Google Scholar) have shown, the N-terminal TFR to the HIV-1 PR to fold in the of which is of the NMR structural characterization of was because of its in a an active site D25N was and the to have many at the same as in the of the folded they many in a region of to the TFR that the PR region folded the TFR region be because of of the R. D.A. Louis J.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). was that the TFR region was all the studies the importance of TFR on the folding and maturation of the protease. However, the at the residue level are In this context we here investigations on a precursor Cnn is a non-native at the C terminus of and of the precursor that TFR the autoprocessing of the precursor as to release the of Cnn enhanced indicating that the non-native C-terminal in the cleavage NMR investigations on the precursor containing an active site which was for for NMR the intrinsic folding propensities of the precursor by the in the as as structural characteristics of the by use of different of the These results have for the regulation mechanism of the autoprocessing reaction of HIV-1 protease with the for the by M. V. of we an active site mutation, in PR, a this PR folding its this the TFR-PR-Cnn region was and into the site of a The of the a non-native C has a has the sequence and the have a in the NMR the same Cnn is known to the folding characteristics of PR in the which to the native B. PubMed Scopus Google Scholar). the was The wild-type by of and TFR-PR-Cnn from the and into a as The to that The was into E. for protein at °C in to of and for of the proteins and protein by in minimal with and was as S.C. B. Curr. Sci. Scholar). of the protein showed at the molecular The NMR protein in containing and different of in protein was in E. as in the on protein at two different and and on protein was to and by on a system in the and of the and with with a laser and a The by of protein solution with of solution of in total of of this was on the and to at The in the at an of in the from to each the and NMR NMR at 32 °C on a NMR with and the the and both to used the and The was with the same and the same of and and the was to was with a of 32 the and the and with and was with and was with the high for and acquired the and the and the two the and at and The by the to the The the sequences by et al. R. G. L.E. 1994; PubMed Scopus Google Scholar). C-terminal at PR the Autoprocessing of the the activity of and TFR-PR-Cnn precursors in and we of the precursor in the of is that the TFR the autoprocessing activity of the precursor However, in the of TFR-PR-Cnn we the of the precursor in the the C-terminal in precursor with the protein to the same the we an for the PR and a for the TFR for the TFR-PR-Cnn we a at to the to that the C-terminal with the PR region; with or the active site as has for the at the C terminus of the TFR in an C. S. U. H. H.G. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google of precursor of TFR-PR-Cnn containing the D25N active site to and of the precursor are of the precursor the D25N The and to the monomer PR and TFR are to is of the precursor The precursor is the and are In both the and the of PR are of the for the the precursor has to active by a with the fold and an active we to the of the precursor by NMR in this we first a D25N mutant of the precursor, which is as the is at the active site of the same is known that the D25N the folding of the protease J.M. R. D.A. A.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). mutant precursor is for and is for structural characterization by However, that the protein a high tendency to as by and by NMR a of conditions of pH and the C-terminal with to this is at with the report by et al. R. D.A. Louis J.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) on a precursor, which the in the PR and at the N terminus of TFR that different from in et al. R. D.A. Louis J.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) the protein to be a monomer and at the high NMR and the of fold for the PR to the intrinsic folding characteristics of precursor, we to the structural characteristics in and the by and the by the In the we first the for the and the structural and of the precursor at pH 5.2 and 32 TFR-PR-Cnn precursor is of which the first to the TFR The 99 that the PR Hence the dimerization the active the the and the in the PR S.C. B. Curr. Sci. Scholar). The having the sequence an to the PR at the C we use these for structural in proteins has by a of and recently in G. Rev. PubMed Scopus Google Scholar). These and the protein The of this on the of the and for this is the has of is on the recently and S.C. J. PubMed Scopus Google Scholar). The of these is the of different of and in the on the residue at and These have in S.C. J. PubMed Scopus Google Scholar, S.C. PubMed Scopus Google to here that and important roles in this the because of the of the and the because of the of the containing these in the which be as These many and for the and is less crucial to to the of an S. Rev. 1990; PubMed Scopus Google Scholar) in in the because of of the is in the are to their and the of the be TFR-PR-Cnn has and which are the of the are a of to shows an the and displays the of the the and are shown in the in in of the precursor TFR-PR-Cnn containing the D25N in the PR the at pH 5.2 and 32 the and as the from the known and S. A. J. 1992; Scholar, S. A. J. Chem. 1992; Scopus Google Scholar). The two the many for the from in a use of the and the in have in of the The at to the at and be by of at is that the of proteins are with or propensities for J. Curr. Sci. Scholar, D. Curr. 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J. 1995; PubMed Scopus Google Scholar) at pH for the in by the from the for all the in These are shown in The to indicate the of of the of many short with of The are for which preferences in the are many short of than by the in the and these be to be having propensities for are with which indicate to the in the The of the with and propensities are shown by and in the The TFR of the protein to two short and two short on structural characteristics of M. C. P. H. P. R. Eur. J. Biochem. 1996; PubMed Scopus Google Scholar, J.M. Wondrak E.M. P.T. Nashed N.T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) that the TFR be in to that be at a of some in an The PR of the protein many and The of the is the same as in the native PR the C this to the in the of the to the in the dimeric of PR B. PubMed Scopus Google Scholar). The in the results from to a extent. In both the with and propensities are at the same as is the with The short of the or a be to the that the are and the of the and to preferences be the are in with the of is used for The at is and this to the of the native protease B. PubMed Scopus Google Scholar). the and all of which indicate that the protein is of in The of the to the structural preferences are with the The of at certain we that in at pH 5.2 and 32 the is with short of structural propensities in a the by different to the folding the folding of a protein. shows the of the precursor as a of the at and in the of that the protein has a high tendency to be unfolded. the in the are in the and at a that However, are some in the at all the which the of that be folded The of these that the by the conserved in the would have in the characteristics under the different The in the of shows which is with the of the protein have the structural from the to the in precursor and The are to and on the In many these have into and in which in to the folding S. PubMed Scopus Google Scholar, R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, V. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, J. M. S.B. J. E. L.J. H. Science. 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The in the of as the is on the the sequence that to and native by of native or of non-native shows the for the TFR-PR-Cnn as the is from to The indicating different of the in the structural this we that the of for some of the is by the in because of and having than have in the are less than shows the in the as we and indicate or in and and as as the protein is and are the are in would be by The in be into and The is to the in the and be as that as we from the propensities for by and The important these are: at the N at the C both of which are cleavage of the and at the of the PR As we to the same the are for the at the active which shows indicating The indicate a tendency of as we to we a in the from the at to to indicate of characterization would and this is by the tendency of the protein to and The which has the is an NMR that be to an into the structural that the preferences K. NMR of and Scholar). in the of to to and which is an of the to is that the for residue is by its N-terminal and two of have for each the N-terminal to of the two of C. J. R.A. L.J. J. Biol. PubMed Scopus Google Scholar) and under of the from the which we as to would on the structural propensities the would indicate propensities and would indicate shows the of the in the from which the The from to the sequence in all the from to as shown in and the by is the same the in the is The are shown in As many of the are or to because of are that or than these are the at in and at and in which be to indicate some These have on the by and all of to in the PR region are non-native type. all the with to the in urea, which intrinsic initial and the folding as the is important that be from these at a is that folding is a in that a at to all the in the the folded is to that the TFR region in the precursor, which is to be has some intrinsic These the preferences in the PR domain as by The PR domain has native preferences many non-native which be for the protein to fold to its native In the PR this to and a folded dimeric protein is However, in the precursor, to the TFR removal of non-native leads to the TFR and PR regions, which in results in a highly to at the NMR have here to residue level into the intrinsic preferences in the precursor and the characteristics of the folding by the structural and in the by of in the The protein to have intrinsic preferences for many non-native in to native and the of TFR folding of the protein to the native dimeric However, these non-native to cleavage of the TFR in the of this cleavage by an active site to removal of the non-native which in leads to and of the protein. is an the TFR region and the PR which a have of an C. S. U. H. H.G. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). in precursor was that the C-terminal to some of its autoprocessing as shown by the and the an the C-terminal and the PR domain as These results in the of results by et precursor, which from the here by of and in the TFR sequence at the N that the sequence plays a major role in the folding propensities of the In in HIV-1 protease precursors because of the of the reverse transcriptase of the As the different mutant proteins would have different folding they be to have different and to and that folding plays a crucial role in the regulation of precursor and in the protease in the viral life cycle. to the first residue level of folding characteristics of an HIV-1 protease precursor, and into folding have a regulatory role in its autoprocessing and here that this is the and by NMR