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.

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SummaryThe type III secretion system (TTSS) is a modular apparatus assembled by many pathogenic Gramnegative bacteria and is designed to translocate proteins through the bacterial cell wall into the eukaryotic host cell. The conserved components of the TTSS comprise stacks of rings spanning the inner and outer bacterial membrane and a narrow, needlelike structure projecting outwards. The TTSS of enteropathogenic E. coli is unique in that one of the translocator proteins, EspA, polymerizes to form an extension to the needle complex which interacts with the host cell. In this study we present the 3D structure of EspA filaments to c. 26 Å resolution determined from electron micrographs of negatively stained preparations by image processing. The structure comprises a helical tube with a diameter of 120 Å enclosing a central channel of 25 Å diameter through which effector proteins may be transported. The subunit arrangement corresponds to a one-start helix with 28 subunits present in five turns of the helix and an axial rise of 4.6 Å per subunit. This is the first report of a 3D structure of a filamentous extension to the TTSS.

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