Energy-dependent protein degradation is carried out by large multimeric protein complexes such as the proteasomes of eukaryotic and archaeal cells and the ATP-dependent proteases of eubacterial cells. Clp protease, a major multicomponent protease of Escherichia coli, consists of a proteolytic component, ClpP, in association with an ATP-hydrolyzing, chaperonin-like component, ClpA. To provide a structural basis for understanding the regulation and mechanism of action of Clp protease, we have used negative staining electron microscopy and image analysis to examine ClpA and ClpP separately, as well as active ClpAP complexes. Digitized images of ClpP and ClpA were analyzed using a novel algorithm designed to detect rotational symmetries. ClpP is composed of two rings of seven subunits superimposed in bipolar fashion along the axis of rotational symmetry. This structure is similar to that formed by the beta subunits of the eukaryotic and archaeal proteasomes. In the presence of MgATP, ClpA forms an oligomer with 6-fold symmetry when viewed en face. Side views of ClpA indicate that the subunits are bilobed with the respective domains forming two stacked rings. ClpAP complexes contain a tetradecamer of ClpP flanked at one or both ends with a hexamer of ClpA, resulting in a symmetry mismatch between the axially aligned molecules. Our findings demonstrate that, despite the lack of sequence similarity between ClpAP and proteasomes, these multimeric proteases nevertheless have a profound similarity in their underlying architecture that may reflect a common mechanism of action.

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 r 46,000) associate to form a six-membered ring (M r ϳ 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 (ϳ2-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

SummaryBordetella pertussis establishes infection by attaching to epithelial cells of the respiratory tract. One of its adhesins is filamentous haemagglutinin (FHA), a 500-Å -long secreted protein that is rich in b-structure and contains two regions, R1 and R2, of tandem 19-residue repeats. Two models have been proposed in which the central shaft is (i) a hairpin made up of a pairing of two long antiparallel b-sheets; or (ii) a b-helix in which the polypeptide chain is coiled to form three long parallel b-sheets. We have analysed a truncated variant of FHA by electron microscopy (negative staining, shadowing and scanning transmission electron microscopy of unstained specimens): these observations support the latter model. Further support comes from detailed sequence analysis and molecular modelling studies. We applied a profile search method to the sequences adjacent to and between R1 and R2 and found additional 'covert' copies of the same motifs that may be recognized in overt form in the R1 and R2 sequence repeats. Their total number is sufficient to support the tenet of the b-helix model that the shaft domain -a 350 Å rod -should consist of a continuous run of these motifs, apart from loop inserts. The N-terminus, which does not contain such repeats, was found to be weakly homologous to cyclodextrin transferase, a protein of known immunoglobulin-like structure. Drawing on crystal structures of known b-helical proteins, we developed structural models of the coil motifs putatively formed by the R1 and R2 repeats. Finally, we applied the same profile search method to the sequence database and found several other proteins -all large secreted proteins of bacterial provenance -that have similar repeats and probably also similar structures.

Chaperonins GroEL and GroES form two types of hetero-oligomers in vitro that can mediate the folding of proteins. Chemical cross-linking and electron microscopy showed that in the presence of adenosine triphosphate (ATP), two GroES7 rings can successively bind a single GroEL14 core oligomer. The symmetric GroEL14(GroES7)2 chaperonin, whose central cavity appears obstructed by two GroES7 rings, can nonetheless stably bind and assist the ATP-dependent refolding of RuBisCO enzyme. Thus, unfolded proteins first bind and possibly fold on the external envelope of the chaperonin hetero-oligomer.

The ClpAP protease from Escherichia coli consists of the ATP-binding regulatory component, ClpA (subunit Mr 84 165), and the proteolytic component, ClpP (subunit Mr 21 563). Our hydrodynamic studies demonstrate that the predominant forms of these proteins in solution correspond to those observed by electron microscopy. ClpP and proClpP(SA), which in electron micrographs appear to have subunits arranged in rings of seven subunits, were found by ultracentrifugation to have s20,w values of 12.2 and 13.2 S and molecular weights of 300 000 and 324 000 +/- 3000, respectively, indicating that the native form of each consists of two such rings. The two intact rings of ClpP were separated in the presence of >/= 0.1 M sulfate at low temperatures, suggesting that ring-ring contacts are polar in nature and more easily disrupted than subunit contacts within individual rings. Sedimentation equilibrium analysis indicated that ClpA purified without nucleotide exists as an equilibrium mixture of monomers and dimers with Ka = (1.0 +/- 0.2) x 10(5) M-1 and that, upon addition of MgATP or adenosine 5'-O-(3-thiotriphosphate), ClpA subunits associated to a form with Mr 505 000 +/- 5000, consistent with the hexameric structure seen by electron microscopy. Sedimentation velocity and gel-filtration analysis showed that the nucleotide-promoted hexamer of ClpA (s20,w = 17.2 S) binds tightly to ClpP producing species with s20,w values of 21 and 27 S (f/f0 = 1.5 and 1.8, respectively), consistent with electron micrographs of ClpAP that show a single tetradecamer of ClpP associated with either one or two ClpA hexamers [Kessel et al. (1995) J. Mol. Biol. 250, 587-594]. Under assay conditions in the presence of ATP and Mg2+, the apparent dissociation constant of hexameric ClpA and tetradecameric ClpP was approximately 4 +/- 2 nM. By the method of continuous variation, the optimal ratio of ClpA to ClpP in the active complex was 2:1. The specific activities of limiting ClpA and ClpP determined in the presence of an excess of the other component indicated that the second molecule of ClpA provides very little additional activation of ClpP.

SummaryRapid proteolysis plays an important role in regulation of gene expression. Proteolysis of the phage CII transcriptional activator plays a key role in the lysis-lysogeny decision by phage . Here we demonstrate that the E. coli ATP-dependent protease FtsH, the product of the host ftsH/hflB gene, is responsible for the rapid proteolysis of the CII protein. FtsH was found previously to degrade the heat-shock transcription factor 32 . Proteolysis of 32 requires, in vivo, the presence of the DnaK-DnaJ-GrpE chaperone machine. Neither DnaK-DnaJ-GrpE nor GroEL-GroES chaperone machines are required for proteolysis of CII in vivo. Purified FtsH carries out specific ATP-dependent proteolysis of CII in vitro. The degradation of CII is at least 10-fold faster than that of 32 . Electron microscopy revealed that purified FtsH forms ringshaped structures with a diameter of 6-7 nm.

Intracellular protein degradation, which must be tightly controlled to protect normal proteins, is carried out by ATP-dependent proteases. These multicomponent enzymes have chaperone-like ATPases that recognize and unfold protein substrates and deliver them to the proteinase components for digestion. In ClpAP, hexameric rings of the ClpA ATPase stack axially on either face of the ClpP proteinase, which consists of two apposed heptameric rings. We have used cryoelectron microscopy to characterize interactions of ClpAP with the model substrate, bacteriophage P1 protein, RepA. In complexes stabilized by ATP␥S, which bind but do not process substrate, RepA dimers are seen at near-axial sites on the distal surface of ClpA. On ATP addition, RepA is translocated through Ϸ150 Å into the digestion chamber inside ClpP. Little change is observed in ClpAP, implying that translocation proceeds without major reorganization of the ClpA hexamer. When translocation is observed in complexes containing a ClpP mutant whose digestion chamber is already occupied by unprocessed propeptides, a small increase in density is observed within ClpP, and RepA-associated density is also seen at other axial sites. These sites appear to represent intermediate points on the translocation pathway, at which segments of unfolded RepA subunits transiently accumulate en route to the digestion chamber.ATP-dependent protease ͉ chaperone ͉ protein unfoldase ͉ processivity ͉ cryoelectron microscopy

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