Structure and dynamic association of an assembly platform subcomplex of the bacterial type II secretion system

doi:10.1016/j.str.2022.12.003

Structure

Volume 31, Issue 2, 2 February 2023, Pages 152-165.e7

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Highlights

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Structures of two proteins from a bacterial type II secretion system assembly platform
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Each protein alone forms a homodimer, but together they assemble as a heterodimer
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The X-ray and NMR heterodimer structures are compatible with membrane insertion
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Heterodimer assembly validated by in vivo approaches is key for secretion

Summary

Type II secretion systems (T2SSs) allow diderm bacteria to secrete hydrolytic enzymes, adhesins, or toxins important for growth and virulence. To promote secretion of folded proteins, T2SSs assemble periplasmic filaments called pseudopili or endopili at an inner membrane subcomplex, the assembly platform (AP). Here, we combined biophysical approaches, nuclear magnetic resonance (NMR) and X-ray crystallography, to study the Klebsiella AP components PulL and PulM. We determined the structure and associations of their periplasmic domains and describe the structure of the heterodimer formed by their ferredoxin-like domains. We show how structural complementarity and plasticity favor their association during the secretion process. Cysteine scanning and crosslinking data provided additional constraints to build a structural model of the PulL-PulM assembly in the cellular context. Our structural and functional insights, together with the relative cellular abundance of its components, support the role of AP as a dynamic hub that orchestrates pilus polymerization.

Graphical abstract

Keywords

type II secretion system

assembly platform

type IV pili

protein-protein interaction

ferredoxin-like fold

Data and code availability

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Atomic coordinates and structure factors have been deposited in the RCSB Protein Data Bank under the accession codes PDB: 8A9W (PulL_CTD), PDB: 7ZE0 (PulM_CTD, NMR), PDB: 8A9X (PulM_CTD, X-ray) and PDB: 8AB1 (PulL_CTD-PulM_CTD). PulM ¹³C/¹⁵N/¹H resonance assignments were deposited to the BMRB under accession number BMRB: 34719. Pullulanase secretion assay dataset shown in Figure 5 have been deposited in Zenodo and is publicly available as of the date of publication (https://doi.org/10.5281/zenodo.7351675).
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This paper does not report original code.
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Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

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⁶: Lead contact