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2008 | 55 | 2 | 281-296
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Limited proteolysis of E. coli ATP-dependent protease Lon - a unified view of the subunit architecture and characterization of isolated enzyme fragments

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We carried out chymotryptic digestion of multimeric ATP-dependent Lon protease from Escherichia coli. Four regions sensitive to proteolytic digestion were located in the enzyme and several fragments corresponding to the individual structural domains of the enzyme or their combinations were isolated. It was shown that (i) unlike the known AAA+ proteins, the ATPase fragment (A) of Lon has no ATPase activity in spite of its ability to bind nucleotides, and it is monomeric in solution regardless of the presence of any effectors; (ii) the monomeric proteolytic domain (P) does not display proteolytic activity; (iii) in contrast to the inactive counterparts, the AP fragment is an oligomer and exhibits both the ATPase and proteolytic activities. However, unlike the full-length Lon, its AP fragment oligomerizes into a dimer or a tetramer only, exhibits the properties of a non-processive protease, and undergoes self-degradation upon ATP hydrolysis. These results reveal the crucial role played by the non-catalytic N fragment of Lon (including its coiled-coil region), as well as the contribution of individual domains to creation of the quaternary structure of the full-length enzyme, empowering its function as a processive protease.
Physical description
  • Shemyakin Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia
  • Shemyakin Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia
  • Macromolecular Crystallography Laboratory, National Cancer Institute at Frederick, Frederick, MD, USA
  • Shemyakin Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia
  • Shemyakin Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia
  • Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD, USA
  • Cardiology Research Center, Institute of Experimental Cardiology, Moscow, Russia
  • Cardiology Research Center, Institute of Experimental Cardiology, Moscow, Russia
  • Shemyakin Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia
  • Amerik AYu, Antonov VK, Ostroumova NI, Rotanova TV, Chistiakova LG (1990) Cloning, structure and expression of the full-size lon gene in Escherichia coli coding for ATP-dependent La-protease. Bioorg Khim 16: 869-880.
  • Amerik AYu, Antonov VK, Gorbalenya AE, Kotova SA, Rotanova TV, Shimbarevich EV (1991) Site-directed mutagenesis of La protease. A catalytically active serine residue. FEBS Lett 287: 211-214.
  • Bencini DA, Wild JR, O'Donovan GA (1983) Linear one-step assay for the determination of orthophosphate. Anal Biochem 132: 254-258.
  • Botos I, Melnikov EE, Cherry S, Khalatova AG, Rasulova FS, Tropea JE, Maurizi MR, Rotanova TV, Gustchina A, Wlodawer A (2004a) Crystal structure of the AAA^+ α domain of E. coli Lon protease at 1.9Å resolution. J Struct Biol 146: 113-122.
  • Botos I, Melnikov EE, Cherry S, Tropea JE, Khalatova AG, Rasulova F, Dauter Z, Maurizi MR, Rotanova TV, Wlodawer A, Gustchina A (2004b) The catalytic domain of Escherichia coli Lon protease has a unique fold and a Ser-Lys dyad in the active site. J Biol Chem 279: 8140-8148.
  • Botos I, Melnikov EE, Cherry S, Kozlov S, Makhovskaya OV, Tropea JE, Gustchina A, Rotanova TV, Wlodawer A (2005) Atomic-resolution crystal structure of the proteolytic domain of Archaeoglobus fulgidus Lon reveals the conformational variability in the active sites of Lon proteases. J Mol Biol 351: 144-157.
  • Burton RE, Baker TA, Sauer RT (2005) Nucleotide-dependent substrate recognition by the AAA^+ HslUV protease. Nat Struct Mol Biol 12: 245-251.
  • Charette MF, Henderson GW, Markovitz A (1981) ATP hydrolysis-dependent protease activity of the lon (capR) protein of Escherichia coli K-12. Proc Natl Acad Sci USA 78: 4728-4732.
  • Chung CH, Goldberg AL (1981) The product of the lon (capR) gene in Escherichia coli is the ATP-dependent protease, protease La. Proc Natl Acad Sci USA 78: 4931-4935.
  • Dauter Z, Botos I, LaRonde-LeBlanc N, Wlodawer A (2005) Pathological crystallography: case studies of several unusual macromolecular crystals. Acta Crystallogr D Biol Crystallogr 61: 967-975.
  • Ebel W, Skinner MM, Dierksen KP, Scott JM, Trempy JE (1999) A conserved domain in Escherichia coli Lon protease is involved in substrate discriminator activity. J Bacteriol 181: 2236-2243.
  • Edmunds T, Goldberg AL (1986) Role of ATP hydrolysis in the degradation of proteins by protease La from Escherichia coli. J Cell Biochem 32: 187-191.
  • Enemark EJ, Joshua-Tor L (2006) Mechanism of DNA translocation in a replicative hexameric helicase. Nature 442: 270-275.
  • Erlanger BF, Kokowsky N, Cohen W (1961) The preparation and properties of two new chromogenic substrates of trypsin. Arch Biochem Biophys 95: 271-278.
  • Fischer H, Glockshuber R (1993) ATP hydrolysis is not stoichiometrically linked with proteolysis in the ATP-dependent protease La from Escherichia coli. J Biol Chem 268: 22502-22507.
  • Frickey T, Lupas AN (2004) Phylogenetic analysis of AAA proteins. J Struct Biol 146: 2-10.
  • Goldberg AL (1992) The mechanism and functions of ATP-dependent proteases in bacterial and animal cells. Eur J Biochem 203: 9-23.
  • Goldberg AL, Moerschell RP, Chung CH, Maurizi MR (1994) ATP-dependent protease La (lon) from Escherichia coli. Methods Enzymol 244: 350-375.
  • Gottesman S (2003) Proteolysis in bacterial regulatory circuits. Annu Rev Cell Dev Biol 19: 565-587.
  • Gottesman S, Wickner S, Jubete Y, Singh SK, Kessel M, Maurizi M (1995) Selective, energy-dependent proteolysis in Escherichia coli. Cold Spring Harb Symp Quant Biol 60: 533-548.
  • Halsall HB (1967) Atassi-Gandhi sedimentation coefficient and molecular weight relationships. Nature 215: 880-881.
  • Im YJ, Na Y, Kang GB, Rho SH, Kim MK, Lee JH, Chung CH, Eom SH (2004) The active site of a Lon protease from Methanococcus jannaschii distinctly differs from the canonical catalytic dyad of Lon proteases. J Biol Chem 279: 53451-53457.
  • Iyer LM, Leipe DD, Koonin EV, Aravind L (2004) Evolutionary history and higher order classification of AAA^+ATPases. J Struct Biol 146: 11-31.
  • Joshi SA, Hersch GL, Baker TA, Sauer RT (2004) Communication between ClpX and ClpP during substrate processing and degradation. Nat Struct Mol Biol 11: 404-411.
  • Kenniston JA, Baker TA, Sauer RT (2005) Partitioning between unfolding and release of native domains during ClpXP degradation determines substrate selectivity and partial processing. Proc Natl Acad Sci USA 102: 1390-1395.
  • Lee JY, Yang W (2006) UvrD helicase unwinds DNA one base pair at a time by a two-part power stroke. Cell 127: 1349-1360.
  • Lee AY, Hsu CH, Wu SH (2004a) Functional domains of Brevibacillus thermoruber Lon protease for oligomerization and DNA binding: role of N-terminal and sensor and substrate discrimination domains. J Biol Chem 279: 34903-34912.
  • Lee AY, Tsay SS, Chen MY, Wu SH (2004b) Identification of a gene encoding Lon protease from Brevibacillus thermoruber WR-249 and biochemical characterization of its thermostable recombinant enzyme. Eur J Biochem 271: 834-844.
  • Li M, Rasulova F, Melnikov EE, Rotanova TV, Gustchina A, Maurizi MR, Wlodawer A (2005) Crystal structure of the N-terminal domain of E. coli Lon protease. Protein Sci 14: 2895-2900.
  • Lupas AN, Martin J (2002) AAA proteins. Curr Opin Struct Biol 12: 746-753.
  • Lupas A, Van Dyke M, Stock J (1991) Predicting coiled coils from protein sequences. Science 252: 1162-1164.
  • Martin A, Baker TA, Sauer RT (2005) Rebuilt AAA^+ motors reveal operating principles for ATP-fuelled machines. Nature 437: 1115-1120.
  • Maupin-Furlow JA, Gil MA, Humbard MA, Kirkland PA, Li W, Reuter CJ, Wright AJ (2005) Archaeal proteasomes and other regulatory proteases. Curr Opin Microbiol 8: 720-728.
  • Maurizi MR, Li CC (2001) AAA proteins: in search of a common molecular basis. International Meeting on Cellular Functions of AAA Proteins. EMBO Rep 2: 980-985.
  • McGuffin LJ, Bryson K, Jones DT (2000) The PSIPRED protein structure prediction server. Bioinformatics 16: 404-405.
  • Melnikov EE, Tsirulnikov KB, Rotanova TV (2000) Coupling of proteolysis with ATP hydrolysis by Escherichia coli Lon protease. I. Kinetic aspects of ATP hydrolysis. Bioorg Khim 26: 530-538.
  • Melnikov EE, Tsirulnikov KB, Rotanova TV (2001) Coupling of proteolysis and hydrolysis of ATP upon functioning of Lon protease of Escherichia coli. II. Hydrolysis of ATP and activity of peptide hydrolase sites of the enzyme. Bioorg Khim 27: 120-129.
  • Menon AS, Goldberg AL (1987) Protein substrates activate the ATP-dependent protease La by promoting nucleotide binding and release of bound ADP. J Biol Chem 262: 14929-14934.
  • Neuwald AF, Aravind L, Spouge JL, Koonin EV (1999) AAA^+: A class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes. Genome Res 9: 27-43.
  • Ogura T, Whiteheart SW, Wilkinson AJ (2004) Conserved arginine residues implicated in ATP hydrolysis, nucleotide-sensing, and inter-subunit interactions in AAA and AAA^+ ATPases. J Struct Biol 146: 106-112.
  • Ogura T, Wilkinson AJ (2001) AAA^+ superfamily ATPases: common structure - diverse function. Genes Cells 6: 575-597.
  • O'Sullivan WJ, Smither GW (1979) Stability constants for biologically important metal-ligand complexes. Methods Enzymol 63: 294-336.
  • Park SC, Jia B, Yang JK, Van D, Shao YG, Han SW, Jeon YJ, Chang CH, Cheong GW (2005) Oligomeric structure of the ATP-dependent protease La (Lon) of Escherichia coli. Mol Cells 21: 129-134.
  • Patterson J, Vineyard D, Thomas-Wohlever J, Behshad R, Burke M, Lee I (2004) Correlation of an adenine-specific conformational change with the ATP-dependent peptidase activity of Escherichia coli Lon. Biochemistry 43: 7432-7442.
  • Ramachandran R, Hartmann C, Song HK, Huber R, Bochtler M (2002) Functional interactions of HslV (ClpQ) with the ATPase HslU (ClpY). Proc Natl Acad Sci USA 99: 7396-7401.
  • Rasulova FS, Dergousova NI, Starkova NN, Melnikov EE, Rumsh LD, Ginodman LM, Rotanova TV (1998) The isolated proteolytic domain of Escherichia coli ATP-dependent protease Lon exhibits the peptidase activity. FEBS Lett 432: 179-181.
  • Reid BG, Fenton WA, Horwich AL, Weber-Ban EU (2001) ClpA mediates directional translocation of substrate proteins into the ClpP protease. Proc Natl Acad Sci USA 98: 3768-3772.
  • Rivett AJ (1989) High molecular mass intracellular proteases. Biochem J 263: 625-633.
  • Rosenfeld SS, Houdusse A, Sweeney HL (2005) Magnesium regulates ADP dissociation from myosin V. J Biol Chem 280: 6072-6079.
  • Rotanova TV (1999) Structural and functional characteristics of ATP-dependent Lon-proteinase from Escherichia coli. Bioorg Khim 25: 883-891.
  • Rotanova TV (2002) Peptide hydrolases with catalytic dyad Ser-Lys. Similarity and distinctions of the active centers of ATP-dependent Lon proteases, LexA repressors, signal peptidases and C-terminal processing proteases. Vopr Med Khim 48: 541-552.
  • Rotanova TV, Melnikov EE, Tsirulnikov KB (2003) Catalytic dyad Ser-Lys at the active site of Escherichia coli ATP-dependent Lon-proteinase. Bioorg Khim 29: 97-99.
  • Rotanova TV, Melnikov EE, Khalatova AG, Makhovskaya OV, Botos I, Wlodawer A, Gustchina A (2004) Classification of ATP-dependent proteases Lon and comparison of the active sites of their proteolytic domains. Eur J Biochem 271: 4865-4871.
  • Rouiller I, DeLaBarre B, May AP, Weis WI, Brunger AT, Milligan RA, Wilson-Kubalek EM (2002) Conformational changes of the multifunction p97 AAA ATPase during its ATPase cycle. Nat Struct Biol 9: 950-957.
  • Rudyak SG, Brenowitz M, Shrader TE (2001) Mg^2+-linked oligomerization modulates the catalytic activity of the Lon (La) protease from Mycobacterium smegmatis. Biochemistry 40: 9317-9323.
  • Sauer RT, Bolon DN, Burton BM, Burton RE, Flynn JM, Grant RA, Hersch GL, Joshi SA, Kenniston JA, Levchenko I, Neher SB, Oakes ES, Siddiqui SM, Wah DA, Baker TA (2004) Sculpting the proteome with AAA^+ proteases and disassembly machines. Cell 119: 9-18.
  • Schechter I, Berger A (1967) On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun 27: 157-162.
  • Schmidt M, Lupas AN, Finley D (1999) Structure and mechanism of ATP-dependent proteases. Curr Opin Chem Biol 3: 584-591.
  • Stahlberg H, Kutejova E, Suda K, Wolpensinger B, Lustig A, Schatz G, Engel A, Suzuki CK (1999) Mitochondrial Lon of Saccharomyces cerevisiae is a ring-shaped protease with seven flexible subunits. Proc Natl Acad Sci USA 96: 6787-6790.
  • Swamy KH, Goldberg AL (1981) E. coli contains eight soluble proteolytic activities, one being ATP dependent. Nature 292: 652-654.
  • Takeda S, Yamashita A, Maeda K, Maeda Y (2003) Structure of the core domain of human cardiac troponin in the Ca^2+-saturated form. Nature 424: 35-41.
  • Tsilibaris V, Maenhaut-Michel G, Van Melderen L (2006) Res Microbiol 157: 701-713.
  • Vale RD (2000) AAA proteins. Lords of the ring. J Cell Biol 150: F13-19.
  • Van Holde KE, Weischet WO (1978) Boundary analysis of sedimentation-velocity experiments with monodisperse and paucidisperse solutes. Biopolymers 17: 1387-1403.
  • Vasilyeva OV, Kolygo KB, Leonova YF, Potapenko NA, Ovchinnikova TV (2002) Domain structure and ATP-induced conformational changes in Escherichia coli protease Lon revealed by limited proteolysis and autolysis. FEBS Lett 526: 66-70.
  • Vasilyeva OV, Martynova NIu, Potapenko NA, Ovchinnikova TV (2004) Isolation and characterization of fragments of ATP-dependent protease Lon from Escherichia coli obtained by limited proteolysis. Bioorg Khim 30: 341-349.
  • Vineyard D, Zhang X, Lee I (2006) Transient kinetic experiments demonstrate the existence of a unique catalytic enzyme form in the peptide-stimulated ATPase mechanism of Escherichia coli Lon protease. Biochemistry 45: 11432-11443.
  • Weber-Ban EU, Reid BG, Miranker AD, Horwich AL (1999) Global unfolding of a substrate protein by the Hsp100 chaperone ClpA. Nature 401: 90-93.
  • Wickner S, Maurizi MR, Gottesman S (1999) Posttranslational quality control: folding, refolding, and degrading proteins. Science 286: 1888-1893.
  • Zehnbauer BA, Foley EC, Henderson GW, Markovitz A (1981) Identification and purification of the Lon^+ (capR^+) gene product, a DNA-binding protein. Proc Natl Acad Sci USA 78: 2043-2047.
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