CLP-AP:Substrate interactions studied by cryo-electron microscopy

F. Beuron*, A. C. Steven, M. Kessel, F. B. Buoy, S. Wickner, M. R. Maufoi

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

Energy-dependent proteolysis is a central regulatory function in all cells. How protein substrates interact with the regulatory components of ATP-dependent proteases and are delivered to the proteolytic active sites is of great interest. Several ATP-dependent proteases, including the 26S proteasome, resemble E. coli CIpAP in structure. ClpAP forms a stack of coaxial cylinders, with double heptameric rings of ClpP in the center and bilobed hexameric rings of ClpA bound to each face of ClpP. The proteolytic active sites lie within an internal aqueous cavity in ClpP and are accessible only through narrow axial channels. Access to those sites is facilitated by ClpA, which has ATP-dependent chaperone activity. We have used cryo-electron microscopy to study ClpA and ClpAP assembled in the presence of ATPyS and the complexes formed between ClpA and ClpAP and the phage PI protein, RepA. RepA is a model substrate for the ClpA chaperone activity as well as the ClpAP proteolytic activity. Our 3-D density map of ClpA reveals a cavity centrally placed between two lobes. En face views of ClpA with RepA bound show additional mass in the center of the ClpA ring, but, in the absence of side views, do not specify the axial position of the RepA dimer. Averaged side views of ClpAP complexes with bound RepA show additional density along the axis of the cylinder. Definitive identification of the mass attributable to RepA is complicated by differences in the conformations of the ClpA and the ClpP when RepA is bound to the complex. Preliminary data also suggest major conformational changes when ATP is added to the CIpAP/RepA complex formed in the presence of ATPyS. These data are consistent with models in which protein substrates pass through axial channels in ClpA and ClpP en route to the active sites in the ClpP interior.

Original languageEnglish
Pages (from-to)A1419
JournalFASEB Journal
Volume12
Issue number8
StatePublished - 1998
Externally publishedYes

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