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Enzymatic and Structural Similarities between theEscherichia coli ATP-dependent Proteases, ClpXP and ClpAP*

Open AccessPublished:May 15, 1998DOI:https://doi.org/10.1074/jbc.273.20.12476
      Escherichia coli ClpX, a member of the Clp family of ATPases, has ATP-dependent chaperone activity and is required for specific ATP-dependent proteolytic activities expressed by ClpP. Gel filtration and electron microscopy showed that ClpX subunits (M r46,000) associate to form a six-membered ring (M r ∼ 280,000) that is stabilized by binding of ATP or nonhydrolyzable analogs of ATP. ClpP, which is composed of two seven-membered rings stacked face-to-face, interacts with the nucleotide-stabilized hexamer of ClpX to form a complex that could be isolated by gel filtration. Electron micrographs of negatively stained ClpXP preparations showed side views of 1:1 and 2:1 ClpXP complexes in which ClpP was flanked on either one or both sides by a ring of ClpX. Thus, as was seen for ClpAP, a symmetry mismatch exists in the bonding interactions between the seven-membered rings of ClpP and the six-membered rings of ClpX. Competition studies showed that ClpA may have a slightly higher affinity (∼2-fold) for binding to ClpP. Mixed complexes of ClpA, ClpX, and ClpP with the two ATPases bound simultaneously to opposite faces of a single ClpP molecule were seen by electron microscopy. In the presence of ATP or nonhydrolyzable analogs of ATP, ClpXP had nearly the same activity as ClpAP against oligopeptide substrates (>10,000 min−1/tetradecamer of ClpP). Thus, ClpX and ClpA interactions with ClpP result in structurally analogous complexes and induce similar conformational changes that affect the accessibility and the catalytic efficiency of ClpP active sites.
      The Clp family of ATP-dependent chaperone-linked proteases are high molecular weight complexes composed of a protease with limited peptidase and virtually no intrinsic proteolytic activity and an ATPase that activates proteolysis by binding and unfolding protein substrates (
      • Gottesman S.
      • Wickner S.
      • Jubete Y.
      • Singh S.K.
      • Kessel M.
      • Maurizi M.R.
      ,
      • Maurizi M.R.
      ). Clp proteases were first described inEscherichia coli, where ClpAP and ClpXP were shown to consist of a common proteolytic core, ClpP, which can be activated by either of two ATPases, ClpA or ClpX (
      • Katayama Y.
      • Gottesman S.
      • Pumphrey J.
      • Rudikoff S.
      • Clark W.P.
      • Maurizi M.R.
      ,
      • Hwang B.J.
      • Woo K.M.
      • Goldberg A.L.
      • Chung C.H.
      ,
      • Gottesman S.
      • Clark W.P.
      • de Crecy-Lagard V.
      • Maurizi M.R.
      ,
      • Wojtkowiak D.
      • Georgopoulos C.
      • Zylicz M.
      ). Recently another branch of the Clp family consisting of a unique proteolytic component, ClpQ (or HslV), and the ATPase ClpY (or HslU) was described (
      • Rohrwild M.
      • Coux O.
      • Huang H.C.
      • Moerschell R.P.
      • Yoo S.J.
      • Seol J.H.
      • Chung C.H.
      • Goldberg A.L.
      ,
      • Kessel M.
      • Wu W.-F.
      • Gottesman S.
      • Kocsis E.
      • Steven A.
      • Maurizi M.R.
      ,
      • Yoo S.J.
      • Seol J.H.
      • Shin D.H.
      • Rohrwild M.
      • Kang M.-S.
      • Tanaka K.
      • Goldberg A.L.
      • Chung C.H.
      ). Despite high degrees of amino acid sequence homology, Clp ATPases appear to fall into two groups, the ClpA/ClpX-like proteins that have intrinsic chaperone activity and also act as part of proteolytic complexes and the ClpB-like proteins that appear to function solely as molecular chaperones independent of proteolytic components (
      • Schirmer E.C.
      • Glover J.R.
      • Singer M.A.
      • Lindquist S.
      ,
      • Gottesman S.
      • Maurizi M.R.
      ). Clp ATPases are widespread in eukaryotes and prokaryotes indicating that, at the least, the protein-remodeling activity of Clp ATPases is highly conserved (
      • Gottesman S.
      • Wickner S.
      • Jubete Y.
      • Singh S.K.
      • Kessel M.
      • Maurizi M.R.
      ,
      • Schirmer E.C.
      • Glover J.R.
      • Singer M.A.
      • Lindquist S.
      ).
      The two Clp proteolytic components described in E. coli, ClpP and ClpQ, are not related to each other, differing in their amino acid sequences and in their catalytic mechanisms of peptide bond cleavage (
      • Maurizi M.R.
      • Clark W.P.
      • Katayama Y.
      • Rudikoff S.
      • Pumphrey J.
      • Bowers B.
      • Gottesman S.
      ,
      • Chuang S.-E.
      • Burland III, V.G.P.
      • Daniels D.L.
      • Blattner F.R.
      ,
      • Larsen C.N.
      • Finley D.
      ). ClpP is representative of a family of serine proteases that is unique both in sequence and in the folding domains seen in the recently solved x-ray crystal structure (
      • Wang J.
      • Hartling J.A.
      • Flanagan J.
      ). The ClpP subfamily is highly conserved in prokaryotes and is found in plant chloroplasts as well as in mammalian cell mitochondria (
      • Maurizi M.R.
      • Clark W.P.
      • Kim S.-H.
      • Gottesman S.
      ). ClpQ is a member of the proteasome family (
      • Chuang S.-E.
      • Burland III, V.G.P.
      • Daniels D.L.
      • Blattner F.R.
      ,
      • Bochtler M.
      • Ditzel L.
      • Groll M.
      • Huber R.
      ). Proteasomes are multimeric proteases that not only form the proteolytic core of the major ATP-dependent protease in the eukaryotic cytosol but that are also found in eubacteria and in Archaea (
      • Lupas A.
      • Flanagan J.M.
      • Tamura T.
      • Baumeister W.
      ). ClpQ has an amino-terminal catalytically active threonine residue and a tertiary structure similar to that of the proteasomal β-subunits (
      • Bochtler M.
      • Ditzel L.
      • Groll M.
      • Huber R.
      ). Surprisingly, ClpQ subunits assemble into rings with only six subunits (
      • Kessel M.
      • Wu W.-F.
      • Gottesman S.
      • Kocsis E.
      • Steven A.
      • Maurizi M.R.
      ,
      • Rohrwild M.
      • Pfeifer G.
      • Santarius U.
      • Muller S.A.
      • Huang H.C.
      • Engel A.
      • Baumeister W.
      • Goldberg A.L.
      ), unlike the proteasome, which has seven subunits per ring. For both ClpP and ClpQ, the active sites are buried within an aqueous cavity formed by the joining of the two rings, and access to the active sites is limited to a narrow axial channel through the center of each ring. It has been proposed that binding of the chaperone component and cycles of ATP hydrolysis may alter the size and properties of the channel and increase substrate access (
      • Thompson M.W.
      • Singh S.K.
      • Maurizi M.R.
      ,
      • Thompson M.W.
      • Maurizi M.R.
      ).
      The Clp ATPases not only carry out the energy-dependent steps in protein remodeling and degradation, but they also determine the selection of protein substrates for both activities. Proteins that bind to and are remodeled by ClpX are also degraded by the corresponding holoenzyme complexes with ClpP (
      • Gottesman S.
      • Clark W.P.
      • de Crecy-Lagard V.
      • Maurizi M.R.
      ,
      • Wojtkowiak D.
      • Georgopoulos C.
      • Zylicz M.
      ). The same can be said for substrate selection in all three activities carried out by ClpA or ClpAP (
      • Wickner S.
      • Gottesman S.
      • Skowyra D.
      • Hoskins J.
      • McKenney K.
      • Maurizi M.R.
      ,
      • Wawrzynow A.
      • Wojtkowiak D.
      • Marszalek J.
      • Banecki B.
      • Jonsen M.
      • Graves B.
      • Georgopoulos C.
      • Zylicz M.
      ). Thus, it is likely that protein binding and unfolding by Clp ATPases is an integral part of their ability to promote specific protein degradation by ClpP. The structure of the ClpAP complex as revealed by electron microscopy is consistent with protein binding and enzymatic properties of the enzyme. ClpA binds on the planar surface of each ring of ClpP, controlling access to the openings of the axial channels (
      • Kessel M.
      • Maurizi M.R.
      • Kim B.
      • Trus B.L.
      • Kocsis E.
      • Singh S.K.
      • Steven A.C.
      ). Presumably, substrates must interact with ClpA and pass through or around the rings formed by the two domains of ClpA to gain access to the proteolytic active sites. We have proposed a model in which ATP-dependent protein unfolding is coupled to translocation of segments of the substrate to the interior of ClpP (
      • Gottesman S.
      • Maurizi M.R.
      ,
      • Gottesman S.
      • Wickner S.
      • Maurizi M.R.
      ). Because of the unequal number of subunits in the respective rings, the subunits in ClpA will not all be in the same register with those in ClpP. Progressive movement of different pairs of subunits into alignment during successive rounds of ATP hydrolysis may aid in translocation of protein substrates through the narrow channels.

      DISCUSSION

      ATP-dependent proteases from a variety of sources and from different evolutionary families have complex, multimeric structures. Modular assemblies (as in ClpAP and ClpXP or the complex of the proteasome with either the ATP-dependent 19 S or ATP-independent 11 S activators) and alternative subunit compositions (as in the isoforms of eukaryotic proteasomes) are now among the well established features of these proteases (
      • Larsen C.N.
      • Finley D.
      ,
      • Lupas A.
      • Flanagan J.M.
      • Tamura T.
      • Baumeister W.
      ,
      • Gottesman S.
      • Maurizi M.R.
      • Wickner S.
      ). The variety of ways in which the different components can be combined allows the specificity, the activity, and the response to regulatory signals to be fine-tuned. One goal of our studies is to distinguish the structural features that underlie the activities common to all the proteases and those that reflect unique functions of particular proteases.
      We had shown previously that ClpA formed a complex with ClpP in which a hexameric ring of ClpA was bound to each of the two heptameric rings of ClpP (
      • Kessel M.
      • Maurizi M.R.
      • Kim B.
      • Trus B.L.
      • Kocsis E.
      • Singh S.K.
      • Steven A.C.
      ). One surprising aspect of the ClpAP complex was the symmetry mismatch between the six-membered and seven-membered rings of the two components. Although intriguing speculations regarding a function of the symmetry mismatch in allowing ratcheting or rotation of the ClpA and ClpP rings about each other during catalysis were attractive, it remained possible that this structural feature was peculiar to ClpAP. Such reservations were of heightened concern because much more was known from genetic and biochemical studies about the physiological activities of a putative complex between ClpX and ClpP, two proteins that are encoded in an operon and subject to co-regulation in vivo (
      • Gottesman S.
      • Clark W.P.
      • de Crecy-Lagard V.
      • Maurizi M.R.
      ). It was thus imperative to determine the oligomeric structure of ClpX and to establish the nature of the ClpX and ClpP interaction in forming the active proteolytic complex.
      Our results demonstrate that ClpX, like ClpA, is composed of six subunits arranged in a symmetrical ring. Like ClpA, ClpX binds tightly to ClpP only in the oligomeric state stabilized by nucleotide binding. ClpX can form 1:1 and 2:1 complexes with ClpP, and the ClpXP complexes, like ClpAP, have a symmetry mismatch between the ATPase and proteolytic components. The ability to generalize this structural feature of the ATP-dependent Clp proteases suggests that, although the details are still not understood, the symmetry mismatch must be a fundamental determinant of the mechanism of chaperone-mediated proteolysis by ClpAP and ClpXP.
      The competition assays (Fig. 4) indicate that the affinities of ClpA and ClpX for ClpP are within a factor of three of each other, and thus, ClpA and ClpX should compete for ClpP in vivo. Estimates of the intracellular concentrations of ClpA, ClpX, and ClpP indicate that, in exponentially growing cells on rich media, ClpA is in slight excess of ClpX, and ClpP is limiting compared with ClpA and ClpX combined.1 Thus, the distribution of ClpP between ClpA and ClpX could affect the selection of substrates under different physiological conditions. ClpA is degraded in cells with a half-life of 1 h, but the degradation appears to be autocatalytic (
      • Katayama Y.
      • Gottesman S.
      • Pumphrey J.
      • Rudikoff S.
      • Clark W.P.
      • Maurizi M.R.
      ) and is not dependent on ClpX.
      M. R. Maurizi, unpublished observations.
      Further studies are required to understand the relative amounts of ClpAP and ClpXP and their significance in vivo.
      A unique finding of this study is the formation of a mixed complex between ClpA, ClpX, and ClpP. Because so few side views were available, we were prevented from obtaining accurate quantitation of the relative numbers of the three complexes, ClpAP, ClpXP, and ClpAPX. Experiments are under way to determine the frequency with which the mixed complexes occur. It would appear, however, that there is no bias against such complexes, i.e. no negative cooperativity between ClpA and ClpX, and thus we think it highly probable that mixed complexes existin vivo as well. Activity measurements with ClpAP had suggested that 1:1 and 2:1 complexes of ClpAP had nearly the same specific activity for casein degradation, indicating that ClpA might not translocate substrates from both sides of ClpP simultaneously (
      • Maurizi M.R.
      • Singh S.K.
      • Thompson M.W.
      • Kessel M.
      • Ginsburg A.
      ). Whether ClpA and ClpX can activate degradation of different substrates simultaneously cannot be determined from our data. It is interesting, however, that the inhibition of ClpAP activity by ClpX appeared to be cooperative, suggesting that displacing a single ClpA failed to inhibit casein degrading activity and implying that ClpAPX has casein degrading activity comparable to that of the 1:1 and 2:1 ClpAP complexes.
      The 6-fold symmetry of ClpX has been observed in all micrographs of native ClpX that we have studied. In contrast, the homologous ATPase, ClpY (HslU), formed rings with 6- or 7-fold symmetry (
      • Kessel M.
      • Wu W.-F.
      • Gottesman S.
      • Kocsis E.
      • Steven A.
      • Maurizi M.R.
      ,
      • Rohrwild M.
      • Pfeifer G.
      • Santarius U.
      • Muller S.A.
      • Huang H.C.
      • Engel A.
      • Baumeister W.
      • Goldberg A.L.
      ). Sequence homology apparently does not dictate that proteins will form rings with the same numbers of subunits. Another example of such structural deviation is the difference in symmetry between E. coli ClpQ (HslV) (6-fold) and the homologous β-subunits of proteasomes from Archaea and eukaryotic cells (7-fold) (
      • Larsen C.N.
      • Finley D.
      ,
      • Lupas A.
      • Flanagan J.M.
      • Tamura T.
      • Baumeister W.
      ). The circular alignment of active sites or binding sites produced by the ring-like structure appears to be the critical structural element rather than the exact number of such sites. In the case of ClpYQ, it is interesting to note that, if the predominant form of ClpY has seven subunits per ring (
      • Kessel M.
      • Wu W.-F.
      • Gottesman S.
      • Kocsis E.
      • Steven A.
      • Maurizi M.R.
      ,
      • Rohrwild M.
      • Pfeifer G.
      • Santarius U.
      • Muller S.A.
      • Huang H.C.
      • Engel A.
      • Baumeister W.
      • Goldberg A.L.
      ), there would be an inside out (with respect to ClpAP or ClpXP) symmetry mismatch between the ATPase and the proteolytic component (ClpQ).
      It is not yet possible to generalize about the occurrence of symmetry mismatches in other ATP-dependent proteases. Symmetry mismatch cannot be essential, because homooligomeric proteases such as Lon and FtsH, which have the ATPase and proteolytic sites in the same polypeptide, are perforce symmetrical. The structure of the ATPases in the 26 S proteasome has not been defined, but in yeast there appears to be only six such ATPases (
      • Hochstrasser M.
      ), which suggests at least a nonstoichiometric interaction between ATPase and proteasome subunits. A symmetry mismatch between the 11 S activator (PA28) and the 20 S proteasome may exist, although there is controversy regarding the number of subunits in the rings of PA28 (six or seven) (
      • Gray C.W.
      • Slaughter C.A.
      • DeMartino G.N.
      ,
      • Knowlton J.R.
      • Johnston S.C.
      • Whitby F.G.
      • Realini C.
      • Zhang Z.
      • Rechsteiner M.
      • Hill C.P.
      ). With the uncertainty regarding the numbers of subunits in ClpY and in the 11 S, it is reasonable to consider the possibility that some of these proteins may exist in both forms and that the degradative activities of the resulting complexes may vary. We would like to note that preliminary studies of ClpX structure using a His-tagged ClpX protein (kindly supplied by T. Baker, MIT) had shown rings with 7-fold symmetry.1 Structural or chemical perturbations may have significant effects on the assembly of Clp ATPases.

      References

        • Gottesman S.
        • Wickner S.
        • Jubete Y.
        • Singh S.K.
        • Kessel M.
        • Maurizi M.R.
        Cold Spring Harbor Symp. Quant. Biol. 1995; 60: 533-548
        • Maurizi M.R.
        Experientia (Basel). 1992; 48: 178-201
        • Katayama Y.
        • Gottesman S.
        • Pumphrey J.
        • Rudikoff S.
        • Clark W.P.
        • Maurizi M.R.
        J. Biol. Chem. 1988; 263: 15226-15236
        • Hwang B.J.
        • Woo K.M.
        • Goldberg A.L.
        • Chung C.H.
        J. Biol. Chem. 1988; 263: 8727-8734
        • Gottesman S.
        • Clark W.P.
        • de Crecy-Lagard V.
        • Maurizi M.R.
        J. Biol. Chem. 1993; 268: 22618-22626
        • Wojtkowiak D.
        • Georgopoulos C.
        • Zylicz M.
        J. Biol. Chem. 1993; 268: 22609-22617
        • Rohrwild M.
        • Coux O.
        • Huang H.C.
        • Moerschell R.P.
        • Yoo S.J.
        • Seol J.H.
        • Chung C.H.
        • Goldberg A.L.
        Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5808-5813
        • Kessel M.
        • Wu W.-F.
        • Gottesman S.
        • Kocsis E.
        • Steven A.
        • Maurizi M.R.
        FEBS Lett. 1996; 398: 274-278
        • Yoo S.J.
        • Seol J.H.
        • Shin D.H.
        • Rohrwild M.
        • Kang M.-S.
        • Tanaka K.
        • Goldberg A.L.
        • Chung C.H.
        J. Biol. Chem. 1996; 271: 14035-14040
        • Schirmer E.C.
        • Glover J.R.
        • Singer M.A.
        • Lindquist S.
        Trends Biochem. Sci. 1996; 21: 289-296
        • Gottesman S.
        • Maurizi M.R.
        Microbiol. Rev. 1992; 56: 592-621
        • Maurizi M.R.
        • Clark W.P.
        • Katayama Y.
        • Rudikoff S.
        • Pumphrey J.
        • Bowers B.
        • Gottesman S.
        J. Biol. Chem. 1990; 265: 12536-12545
        • Chuang S.-E.
        • Burland III, V.G.P.
        • Daniels D.L.
        • Blattner F.R.
        Gene. 1993; 134: 1-6
        • Larsen C.N.
        • Finley D.
        Cell. 1997; 91: 431-434
        • Wang J.
        • Hartling J.A.
        • Flanagan J.
        Cell. 1997; 91: 447-456
        • Maurizi M.R.
        • Clark W.P.
        • Kim S.-H.
        • Gottesman S.
        J. Biol. Chem. 1990; 265: 12546-12552
        • Bochtler M.
        • Ditzel L.
        • Groll M.
        • Huber R.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6070-6074
        • Lupas A.
        • Flanagan J.M.
        • Tamura T.
        • Baumeister W.
        Trends Biochem. Sci. 1997; 22: 399-404
        • Rohrwild M.
        • Pfeifer G.
        • Santarius U.
        • Muller S.A.
        • Huang H.C.
        • Engel A.
        • Baumeister W.
        • Goldberg A.L.
        Nat. Struct. Biol. 1997; 4: 133-139
        • Thompson M.W.
        • Singh S.K.
        • Maurizi M.R.
        J. Biol. Chem. 1994; 269: 18209-18215
        • Thompson M.W.
        • Maurizi M.R.
        J. Biol. Chem. 1994; 269: 18201-18208
        • Wickner S.
        • Gottesman S.
        • Skowyra D.
        • Hoskins J.
        • McKenney K.
        • Maurizi M.R.
        Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12218-12222
        • Wawrzynow A.
        • Wojtkowiak D.
        • Marszalek J.
        • Banecki B.
        • Jonsen M.
        • Graves B.
        • Georgopoulos C.
        • Zylicz M.
        EMBO J. 1995; 14: 1867-1877
        • Kessel M.
        • Maurizi M.R.
        • Kim B.
        • Trus B.L.
        • Kocsis E.
        • Singh S.K.
        • Steven A.C.
        J. Mol. Biol. 1995; 250: 587-594
        • Gottesman S.
        • Wickner S.
        • Maurizi M.R.
        Genes Dev. 1997; 11: 815-823
        • Lanzetta P.A.
        • Alvarez L.J.
        • Reinach P.S.
        • Candia O.A.
        Anal. Biochem. 1979; 100: 95-97
        • Maurizi M.R.
        • Thompson M.W.
        • Singh S.K.
        • Kim S.H.
        Methods Enzymol. 1994; 244: 314-331
        • Moody M.F.
        • Makowski L.
        J. Mol. Biol. 1981; 150: 217-244
        • Trus B.
        • Kocsis E.
        • Conway J.F.
        • Steven A.
        J. Struct. Biol. 1996; 116: 61-67
        • Kocsis E.
        • Trus B.L.
        • Cerritelli M.
        • Cheng N.
        • Steven A.C.
        Ultramicroscopy. 1995; 60: 219-228
        • Gottesman S.
        • Maurizi M.R.
        • Wickner S.
        Cell. 1997; 91: 435-438
        • Maurizi M.R.
        • Singh S.K.
        • Thompson M.W.
        • Kessel M.
        • Ginsburg A.
        Biochemistry. 1998; (in press)
        • Hochstrasser M.
        Annu. Rev. Genet. 1996; 30: 405-439
        • Gray C.W.
        • Slaughter C.A.
        • DeMartino G.N.
        J. Mol. Biol. 1994; 236: 7-15
        • Knowlton J.R.
        • Johnston S.C.
        • Whitby F.G.
        • Realini C.
        • Zhang Z.
        • Rechsteiner M.
        • Hill C.P.
        Nature. 1997; 390: 639-643
        • Trus B.L.
        • Unser M.
        • Pun T.
        • Steven A.C.
        Scanning Microsc. Suppl. 1992; 6: 441-451
        • Unser M.
        • Trus B.L.
        • Steven A.C.
        Ultramicroscopy. 1986; 19: 337-347