Crystal structures of a polypeptide processing and secretion transporter

Abstract

Bacteria secrete peptides and proteins to communicate, to poison competitors, and to manipulate host cells. Among the various protein-translocation machineries, the peptidase-containing ATP-binding cassette transporters (PCATs) are appealingly simple. Each PCAT contains two peptidase domains that cleave the secretion signal from the substrate, two transmembrane domains that form a translocation pathway, and two nucleotide-binding domains that hydrolyse ATP. In Gram-positive bacteria, PCATs function both as maturation proteases and exporters for quorum-sensing or antimicrobial polypeptides. In Gram-negative bacteria, PCATs interact with two other membrane proteins to form the type 1 secretion system. Here we present crystal structures of PCAT1 from Clostridium thermocellum in two different conformations. These structures, accompanied by biochemical data, show that the translocation pathway is a large α-helical barrel sufficient to accommodate small folded proteins. ATP binding alternates access to the transmembrane pathway and also regulates the protease activity, thereby coupling substrate processing to translocation.

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Figure 1: Biochemical properties of PCAT1.
Figure 2: Ribbon diagram of the structure of PCAT1.
Figure 3: The translocation pathway.
Figure 4: A primed NBD dimer.
Figure 5: Conformational change upon ATP binding.
Figure 6: Functional properties of the PEP domain.
Figure 7: The alternating-access model for protein translocation.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Coordinates and structure factors have been deposited in the Protein Data Bank under accession numbers 4RY2 (nucleotide-free form) and 4S0F (ATPγS-bound form).

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Acknowledgements

We thank the staff at the Advance Photon Source GM/CA-CAT and NE-CAT for assistance with data collection, S. McCarry for editing the manuscript, R. MacKinnon and D. Kearns for helpful discussions, M. L. Oldham for help with figure preparation, and H. Zhang and W. Mi for efforts in the early stage of this project. This work was supported by the Howard Hughes Medical Institute.

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All authors designed the study and analysed the data. D.Y.-w.L. and S. H. performed cloning and biochemical experiments. D.Y.-w.L. determined the crystal structures and wrote the manuscript together with J.C.

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Correspondence to Jue Chen.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Sequence alignment of PCAT1 from Clostridium thermocellum, LagD from Lactococcus lactis, and HlyB from Escherichia coli.

Extended Data Figure 2 PCAT1 protease activities towards substrates of other Gram-positive bacteria.

PCAT1 was able to cleave its putative substrate, Cthe_0535, from C. thermocellum at 37 °C for 2 h but showed no proteolytic activities towards CA_P0072 from Clostridium acetobutylicum or ComC from Streptococcus pneumoniae.

Extended Data Figure 3 Anomalous difference Fourier electron density map.

Stereoview of the backbone of SeMet-substituted PCAT1 (grey ribbon). Methionine residues are shown in orange sticks. The blue mesh contoured at 3σ represents the superimposed anomalous difference Fourier map calculated from data collected on four different PCAT1 constructs. A total of 28 selenium sites were identified and used as markers to assist assignment of the sequence register. Out of the 21 native methionine residues, only two were not identified (Met 1 and Met 271), probably reflecting the conformational flexibility of these residues.

Extended Data Figure 4 Stereoview of the final electron density map (2FoFc, 1σ) of the E648Q mutant in complex with ATPγS.

Extended Data Figure 5 The TM tunnel in the ATP-free form is large enough to accommodate a small protein.

The bovine pancreatic trypsin inhibitor (PDB accession 4PTI) is modelled into the TM tunnel of PCAT1, shown as a blue ribbon, to illustrate the size of the cavity.

Extended Data Table 1 Data collection and refinement statistics (Molecular Replacement)

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Lin, D., Huang, S. & Chen, J. Crystal structures of a polypeptide processing and secretion transporter. Nature 523, 425–430 (2015). https://doi.org/10.1038/nature14623

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