Protein Folding Mediated by an Intramolecular Chaperone: Energy Landscape for Unimolecular Pro-Subtilisin E Maturation

Abstract

Efficient and precise assembly of polypeptides into native functional states is critical for normal cellular processes. Understanding how a specific structure is encoded in the polypeptide sequence and what drives the structural progression to the native state is essential to deciphering the folding problem. Several prokaryotic and eukaryotic proteins require their propeptide-domains to function as dedicated intramolecular chaperones (IMCs). In this manuscript, we investigate the elementary steps in the IMC mediated maturation of Subtilisin E, a bacterial serine protease, and a prototype for the eukaryotic proprotein convertases (PCs). Through detailed analyses, we have attempted to define the unimolecular folding energy landscape for SbtE to understand how the stabilization of folding intermediates influences the maturation process, an aspect that is difficult to study in eukaryotic PCs. Our studies demonstrate that a rapid hydrophobic collapse precedes acquisition of tertiary structure during the folding of Pro-SbtE and results in formation of a molten-globule like intermediate. Induction of structure within the IMC stabilizes both the molten globule-like folding intermediate and the native state, and appears to expedite initial stages of folding, purely through thermodynamic stabilization of the folded state. While the induced structure does not affect the activation energies in the unimolecular folding reaction, it is detrimental to the autoproteolytic cleavage of the precursor and subsequent release and degradation of the inhibitory IMC-domain since both these stages require some degree of unfolding. Completion of Pro-SbtE maturation results in the formation of a kinetically trapped and extremely stable native state. Hence, our results suggest that the SbtE IMC appears to have evolved to be intrinsically unstructured and to bind with its cognate protease with a specific affinity that is critical for biological regulation.

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Subbian, E. , Williamson, D. and Shinde, U. (2015) Protein Folding Mediated by an Intramolecular Chaperone: Energy Landscape for Unimolecular Pro-Subtilisin E Maturation. Advances in Bioscience and Biotechnology, 6, 73-88. doi: 10.4236/abb.2015.62008.

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1] Wolynes, P.G. (2014) Evolution, Energy Landscapes and the Paradoxes of Protein Folding. Biochimie.
http://dx.doi.org/10.1016/j.biochi.2014.12.007
[2] Alm, E., et al. (2002) Simple Physical Models Connect Theory and Experiment in Protein Folding Kinetics. Journal of Molecular Biology, 322, 463-476.
http://dx.doi.org/10.1016/S0022-2836(02)00706-4
[3] Dobson, C.M. (2004) Principles of Protein Folding, Misfolding and Aggregation. Seminars in Cell Developmental Biology, 15, 3-16.
http://dx.doi.org/10.1016/j.semcdb.2003.12.008
[4] Dobson, C.M. (2003) Protein Folding and Misfolding. Nature, 426, 884-890. http://dx.doi.org/10.1038/nature02261
[5] Onuchic, J.N. and Wolynes, P.G. (2004) Theory of Protein Folding. Current Opinion in Structural Biology, 14, 70-75.
http://dx.doi.org/10.1016/j.sbi.2004.01.009
[6] Religa, T.L., et al. (2005) Solution Structure of a Protein Denatured State and Folding Intermediate. Nature, 437, 1053-1056.
http://dx.doi.org/10.1038/nature04054
[7] Bartlett, A.I. and Radford, S.E. (2009) An Expanding Arsenal of Experimental Methods Yields an Explosion of Insights into Protein Folding Mechanisms. Nature Structural Molecular Biology, 16, 582-588.
http://dx.doi.org/10.1038/nsmb.1592
[8] Bartlett, A.I. and Radford, S.E. (2010) Desolvation and Development of Specific Hydrophobic Core Packing during Im7 Folding. Journal of Molecular Biology, 396, 1329-1345.
http://dx.doi.org/10.1016/j.jmb.2009.12.048
[9] Plaxco, K.W., et al. (2000) Topology, Stability, Sequence, and Length: Defining the Determinants of Two-State Protein Folding Kinetics. Biochemistry, 39, 11177-11183.
http://dx.doi.org/10.1021/bi000200n
[10] Kuwajima, K. (1989) The Molten Globule State as a Clue for Understanding the Folding and Cooperativity of Globular-Protein Structure. Proteins, 6, 87-103.
http://dx.doi.org/10.1002/prot.340060202
[11] Shastry, M.C. and Udgaonkar, J.B. (1995) The Folding Mechanism of Barstar: Evidence for Multiple Pathways and Multiple Intermediates. Journal of Molecular Biology, 247, 1013-1027.
http://dx.doi.org/10.1006/jmbi.1994.0196
[12] Nishimura, C., Prytulla, S., Dyson, H.J. and Wright, P.E. (2000) Conservation of Folding Pathways in Evolutionarily Distant Globin Sequences. Nature Structural Biology, 7, 679-686.
http://dx.doi.org/10.1038/77985
[13] Matagne, A., Jamin, M., Chung, E.W., Robinson, C.V., Radford, S.E. and Dobson, C.M. (2000) Thermal Unfolding of an Intermediate Is Associated with Non-Arrhenius Kinetics in the Folding of Hen Lysozyme. Journal of Molecular Biology, 297, 193-210.
http://dx.doi.org/10.1006/jmbi.2000.3540
[14] Raschke, T.M. and Marqusee, S. (1997) The Kinetic Folding Intermediate of Ribonuclease H Resembles the Acid Molten Globule and Partially Unfolded Molecules Detected under Native Conditions. Nature Structural Biology, 4, 298-304.
http://dx.doi.org/10.1038/nsb0497-298
[15] Shinde, U. and Inouye, M. (2000) Intramolecular Chaperones: Polypeptide Extensions That Modulate Protein Folding. Seminars in Cell & Developmental Biology, 11, 35-44.
http://dx.doi.org/10.1006/scdb.1999.0349
[16] Eder, J., Rheinnecker, M. and Fersht, A.R. (1993) Folding of Subtilisin BPN': Role of the Pro-Sequence. Journal of Molecular Biology, 233, 293-304.
http://dx.doi.org/10.1006/jmbi.1993.1507
[17] Bryan, P.N. (2002) Prodomains and Protein Folding Catalysis. Chemical Reviews, 102, 4805-4816.
http://dx.doi.org/10.1021/cr010190b
[18] Yabuta, Y., Subbian, E., Oiry, C. and Shinde, U. (2003) Folding Pathway Mediated by an Intramolecular Chaperone. A Functional Peptide Chaperone Designed Using Sequence Databases. Journal of Biological Chemistry, 278, 15246-15251.
http://dx.doi.org/10.1074/jbc.M212003200
[19] Williamson, D.M., Elferich, J., Ramakrishnan, P., Thomas, G. and Shinde, U. (2013) The Mechanism by Which a Propeptide-Encoded pH Sensor Regulates Spatiotemporal Activation of Furin. The Journal of Biological Chemistry, 288, 19154-19165.
http://dx.doi.org/10.1074/jbc.M112.442681
[20] Dillon, S.L., Williamson, D.M., Elferich, J., Radler, D., Joshi, R., Thomas, G. and Shinde, U. (2012) Propeptides Are Sufficient to Regulate Organelle-Specific pH-Dependent Activation of Furin and Proprotein Convertase 1/3. Journal of Molecular Biology, 423, 47-62.
http://dx.doi.org/10.1016/j.jmb.2012.06.023
[21] Shinde, U. and Thomas, G. (2011) Insights from Bacterial Subtilases into the Mechanisms of Intramolecular Chaperone-Mediated Activation of Furin. Methods in Molecular Biology, 768, 59-106.
http://dx.doi.org/10.1007/978-1-61779-204-5_4
[22] Feliciangeli, S.F., Thomas, L., Scott, G.K., Subbian, E., Hung, C.-H., Molloy, S.S., et al. (2006) Identification of a pH Sensor in the Furin Propeptide That Regulates Enzyme Activation. Journal of Biological Chemistry, 281, 16108-16116.
http://dx.doi.org/10.1074/jbc.M600760200
[23] Ikemura, H., Takagi, H. and Inouye, M. (1987) Requirement of Pro-Sequence for the Production of Active Subtilisin E in Escherichia coli. The Journal of Biological Chemistry, 262, 7859-7864.
[24] Eder, J., Rheinnecker, M. and Fersht, A.R. (1993) Folding of Subtilisin BPN': Characterization of a Folding Intermediate. Biochemistry, 32, 18-26.
http://dx.doi.org/10.1021/bi00052a004
[25] Li, Y., Hu, Z., Jordan, F. and Inouye, M. (1995) Functional Analysis of the Propeptide of Subtilisin E as an Intramolecular Chaperone for Protein Folding. Refolding and Inhibitory Abilities of Propeptide Mutants. The Journal of Biological Chemistry, 270, 25127-25132.
http://dx.doi.org/10.1074/jbc.270.42.25127
[26] Yabuta, Y., Takagi, H., Inouye, M. and Shinde, U. (2001) Folding Pathway Mediated by an Intramolecular Chaperone: Propeptide Release Modulates Activation Precision of Pro-Subtilisin. The Journal of Biological Chemistry, 276, 44427-44434.
http://dx.doi.org/10.1074/jbc.M107573200
[27] Subbian, E., Yabuta, Y. and Shinde, U.P. (2005) Folding Pathway Mediated by an Intramolecular Chaperone: Intrinsically Unstructured Propeptide Modulates Stochastic Activation of Subtilisin. Journal of Molecular Biology, 347, 367-383.
http://dx.doi.org/10.1016/j.jmb.2005.01.028
[28] Subbian, E., Yabuta, Y. and Shinde, U. (2004) Positive Selection Dictates the Choice between Kinetic and Thermodynamic Protein Folding and Stability in Subtilases. Biochemistry, 43, 14348-14360.
http://dx.doi.org/10.1021/bi048397x
[29] Siezen, R.J. and Leunissen, J.A. (1997) Subtilases: The Superfamily of Subtilisin-Like Serine Proteases. Protein Science, 6, 501-523.
http://dx.doi.org/10.1002/pro.5560060301
[30] Shinde, U. and Inouye, M. (1993) Intramolecular Chaperones and Protein Folding. Trends in Biochemical Sciences, 18, 442-446.
http://dx.doi.org/10.1016/0968-0004(93)90146-E
[31] Anderson, E.D., Molloy, S.S., Jean, F., Fei, H., Shimamura, S. and Thomas, G. (2002) The Ordered and Compartment-Specific Autoproteolytic Removal of the Furin Intramolecular Chaperone Is Required for Enzyme Activation. Journal of Biological Chemistry, 277, 12879-12890.
http://dx.doi.org/10.1074/jbc.M108740200
[32] Elferich, J., Williamson, D.M., Krishnamoorthy, B. and Shinde, U. (2013) Propeptides of Eukaryotic Proteases Encode Histidines to Exploit Organelle pH for Regulation. The FASEB Journal, 27, 2939-2945.
http://dx.doi.org/10.1096/fj.12-226886
[33] Seidah, N.G. and Prat, A. (2012) The Biology and Therapeutic Targeting of the Proprotein Convertases. Nature Reviews Drug Discovery, 11, 367-383.
http://dx.doi.org/10.1038/nrd3699
[34] Khatib, A.M., Siegfried, G., Prat, A., Luis, J., Chrétien, M., Metrakos, P. and Seidah, N.G. (2001) Inhibition of Proprotein Convertases Is Associated with Loss of Growth and Tumorigenicity of HT-29 Human Colon Carcinoma Cells: Importance of Insulin-Like Growth Factor-1 (IGF-1) Receptor Processing in IGF-1-Mediated Functions. Journal of Biological Chemistry, 276, 30686-30693.
http://dx.doi.org/10.1074/jbc.M101725200
[35] Seidah, N.G., Khatib, A.M. and Prat, A. (2006) The Proprotein Convertases and Their Implication in Sterol and/or Lipid Metabolism. Biological Chemistry, 387, 871-877.
http://dx.doi.org/10.1515/BC.2006.110
[36] Seidah, N.G., Mayer, G., Zaid, A., Rousselet, E., Nassoury, N., Poirier, S., et al. (2008) The Activation and Physiological Functions of the Proprotein Convertases. The International Journal of Biochemistry & Cell Biology, 40, 1111-1125.
http://dx.doi.org/10.1016/j.biocel.2008.01.030
[37] Seidah, N.G. and Prat, A. (2002) Precursor Convertases in the Secretory Pathway, Cytosol and Extracellular Milieu. Essays in Biochemistry, 38, 79-94.
[38] Jaswal, S.S., Sohl, J.L., Davis, J.H. and Agard, D.A. (2002) Energetic Landscape of Alpha-Lytic Protease Optimizes Longevity through Kinetic Stability. Nature, 415, 343-346.
http://dx.doi.org/10.1038/415343a
[39] Bryan, P., Alexander, P., Strausberg, S., Schwarz, F., Lan, W., Gilliland, G. and Gallagher, D.T. (1992) Energetics of Folding Subtilisin BPN'. Biochemistry, 31, 4937-4945.
http://dx.doi.org/10.1021/bi00136a003
[40] Shinde, U. and Inouye, M. (1995) Folding Pathway Mediated by an Intramolecular Chaperone: Characterization of the Structural Changes in Pro-Subtilisin E Coincident with Autoprocessing. Journal of Molecular Biology, 252, 25-30.
http://dx.doi.org/10.1006/jmbi.1995.0472
[41] Jain, S.C., Shinde, U., Li, Y.Y., Inouye, M. and Berman, H.M. (1998) The Crystal Structure of an Autoprocessed Ser221Cys-Subtilisin E-Propeptide Complex at 2.0 Å Resolution. Journal of Molecular Biology, 284, 137-144.
http://dx.doi.org/10.1006/jmbi.1998.2161
[42] Yabuta, Y., Subbian, E., Takagi, H., Shinde, U. and Inouye, M. (2002) Folding Pathway Mediated by an Intramolecular Chaperone: Dissecting Conformational Changes Coincident with Autoprocessing and the Role of Ca2+ in Subtilisin Maturation. Journal of Biochemistry, 131, 31-37.
http://dx.doi.org/10.1093/oxfordjournals.jbchem.a003074
[43] Chu, N.M., Chao, Y. and Bi, R.C. (1995) The 2 Å Crystal Structure of Subtilisin E with PMSF Inhibitor. Protein Engineering, Design and Selection, 8, 211-215.
http://dx.doi.org/10.1093/protein/8.3.211
[44] Comellas-Bigler, M., Maskos, K., Huber, R., Oyama, H., Oda, K. and Bode, W. (2004) 1.2 Å Crystal Structure of the Serine Carboxyl Proteinase Pro-Kumamolisin. Structure of an Intact Pro-Subtilase. Structure, 12, 1313-1323.
http://dx.doi.org/10.1016/j.str.2004.04.013
[45] Shinde, U. and Inouye, M. (1995) Folding Mediated by an Intramolecular Chaperone: Autoprocessing Pathway of the Precursor Resolved via a Substrate Assisted Catalysis Mechanism. Journal of Molecular Biology, 247, 390-395.
http://dx.doi.org/10.1006/jmbi.1994.0147
[46] Engelhard, M. and Evans, P.A. (1995) Kinetics of Interaction of Partially Folded Proteins with a Hydrophobic Dye: Evidence That Molten Globule Character Is Maximal in Early Folding Intermediates. Protein Science, 4, 1553-1562.
http://dx.doi.org/10.1002/pro.5560040813
[47] Ali, V., Prakash, K., Kulkarni, S., Ahmad, A., Madhusudan, K.P. and Bhakuni, V. (1999) 8-Anilino-1-Naphthalene Sulfonic Acid (ANS) Induces Folding of Acid Unfolded Cytochrome c to Molten Globule State as a Result of Electrostatic Interactions. Biochemistry, 38, 13635-13642.
http://dx.doi.org/10.1021/bi9907835
[48] Marie-Claire, C., Yabuta, Y., Suefuji, K., Matsuzawa, H. and Shinde, U. (2001) Folding Pathway Mediated by an Intramolecular Chaperone: The Structural and Functional Characterization of the Aqualysin I Propeptide. Journal of Molecular Biology, 305, 151-165.
http://dx.doi.org/10.1006/jmbi.2000.4233
[49] Buevich, A.V., Shinde, U.P., Inouye, M. and Baum, J. (2001) Backbone Dynamics of the Natively Unfolded Pro-Peptide of Subtilisin by Heteronuclear NMR Relaxation Studies. Journal of Biomolecular NMR, 20, 233-249.
http://dx.doi.org/10.1023/A:1011243116136
[50] Shinde, U., Li, Y., Chatterjee, S. and Inouye, M. (1993) Folding Pathway Mediated by an Intramolecular Chaperone. Proceedings of the National Academy of Sciences of the United States of America, 90, 6924-6928.
http://dx.doi.org/10.1073/pnas.90.15.6924
[51] Subbian, E., Yabuta, Y. and Shinde, U. (2005) Folding Pathway Mediated by an Intramolecular Chaparone: Intrinsically Unstructured Propeptide Modulates Stochastic Activation of Subtilisin. Journal of Molecular Biology, 347, 367-383.
http://dx.doi.org/10.1016/j.jmb.2005.01.028
[52] Ruvinov, S., Wang, L., Ruan, B., Almog, O., Gilliland, G.L., Eisenstein, E. and Bryan, P.N. (1997) Engineering the Independent Folding of the Subtilisin BPN' Prodomain: Analysis of Two-State Folding versus Protein Stability. Biochemistry, 36, 10414-10421.
http://dx.doi.org/10.1021/bi9703958
[53] Wang, L., Ruan, B., Ruvinov, S. and Bryan, P.N. (1998) Engineering the Independent Folding of the Subtilisin BPN' Pro-Domain: Correlation of Pro-Domain Stability with the Rate of Subtilisin Folding. Biochemistry, 37, 3165-3171.
http://dx.doi.org/10.1021/bi972741r
[54] Li, Y. and Inouye, M. (1996) The Mechanism of Autoprocessing of the Propeptide of Prosubtilisin E: Intramolecular or Intermolecular Event? Journal of Molecular Biology, 262, 591-594.
http://dx.doi.org/10.1006/jmbi.1996.0537
[55] Zhu, X.L., Ohta, Y., Jordan, F. and Inouye, M. (1989) Pro-Sequence of Subtilisin Can Guide the Refolding of Denatured Subtilisin in an Intermolecular Process. Nature, 339, 483-484.
http://dx.doi.org/10.1038/339483a0
[56] Shinde, U.P., Liu, J.J. and Inouye, M. (1997) Protein Memory through Altered Folding Mediated by Intramolecular Chaperones. Nature, 389, 520-522.
http://dx.doi.org/10.1038/39097
[57] Fu, X., Inouye, M. and Shinde, U. (2000) Folding Pathway Mediated by an Intramolecular Chaperone. The Inhibitory and Chaperone Functions of the Subtilisin Propeptide Are Not Obligatorily Linked. The Journal of Biological Chemistry, 275, 16871-16878.
http://dx.doi.org/10.1074/jbc.275.22.16871
[58] Fujiwara, K., Arai, M., Shimizu, A., Ikeguchi, M., Kuwajima, K. and Sugai, S. (1999) Folding-Unfolding Equilibrium and Kinetics of Equine Beta-Lactoglobulin: Equivalence between the Equilibrium Molten Globule State and a Burst-Phase Folding Intermediate. Biochemistry, 38, 4455-4463.
http://dx.doi.org/10.1021/bi982683p
[59] Takei, J., Chu, R.A. and Bai, Y. (2000) Absence of Stable Intermediates on the Folding Pathway of Barnase. Proceedings of the National Academy of Sciences of the United States of America, 97, 10796-10801.
http://dx.doi.org/10.1073/pnas.190265797
[60] Mizuguchi, M., Arai, M., Ke, Y., Nitta, K. and Kuwajima, K. (1998) Equilibrium and Kinetics of the Folding of Equine Lysozyme Studied by Circular Dichroism Spectroscopy. Journal of Molecular Biology, 283, 265-277.
http://dx.doi.org/10.1006/jmbi.1998.2100
[61] Inouye, M., Fu, X. and Shinde, U. (2001) Substrate-Induced Activation of a Trapped IMC-Mediated Protein Folding Intermediate. Nature Structural Biology, 8, 321-325.
http://dx.doi.org/10.1038/86194

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