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# Function of the GspF pilus assembly factor in the Klebsiella type II secretion system

Ingrid Guilvout, Firdaus Samsudin, Roland G Huber, Peter J Bond, Benjamin

Bardiaux, Olivera Francetic

► To cite this version:

Ingrid Guilvout, Firdaus Samsudin, Roland G Huber, Peter J Bond, Benjamin Bardiaux, et al. Function of the GspF pilus assembly factor in the Klebsiella type II secretion system. Microbial Adhesion and Signal Transduction Gordon Research Conference, Jul 2023, Newport, United States. pasteur-05279137

HAL Id: pasteur-05279137

https://pasteur.hal.science/pasteur-05279137v1

Submitted on 23 Sep 2025

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# Function of the GspF pilus assembly factor in the Klebsiella type II secretion system

Ingrid Guilvout¹, Firdaus Samsudin², Roland Huber², Peter J. Bond²,³, Benjamin Bardiaux⁴,⁵* and Olivera Francetic¹*

¹Institut Pasteur, Université Paris Cité, CNRS UMR3528, Biochemistry of Macromolecular Interactions Unit, Department of Structural Biology and Chemistry, F-75724 Paris, France.
²Bioinformatics Institute (A-STAR), 30 Biopolis Str, #07-01 Matrix, Singapore 138671. ³Department of Biological Sciences, National University of Singapore, Singapore 117543
⁴Institut Pasteur, Université Paris Cité, CNRS UMR3528, Structural Bioinformatics Unit, Department of Structural Biology and Chemistry, F-75724 Paris, France.
⁵Present address: Institut Pasteur, Université Paris Cité, CNRS UMR3528, Bacterial Transmembrane Systems U5, Department of Structural Biology and Chemistry, F-75724 Paris, France.

Grant SYNERGY_T2SS
anr®

**ABSTRACT.** Polytopic membrane proteins of the GspF/PilC superfamily are central components of type II secretion (T2S) and type IV pilus (T4P) assembly systems. Current models propose that these assembly platform proteins exist as dimers which transmit movements of the ATPase motor to drive assembly of pilin subunits. Yet, so far, their structure and molecular function have remained elusive. Here we describe an all atom model of PulF, the platform protein of the Klebsiella T2S system, comprising the cytoplasmic domains N, Cyto1 and Cyto2 and three transmembrane segments (TMS). Cysteine scanning data defined TMS1 and TMS3 as part of the dimer interface. However, residue conservation analysis and additional crosslinking studies favor and validate the trimeric state of native PulF. In the trimer, TMS1 and TMS3 form a central channel lined with hydrophobic and polar residues that are fully conserved in the GspF/PilC superfamily. Molecular dynamics simulations of PulF trimer in Escherichia coli model membranes reveal a narrow, water-impermeable channel that can accommodate Na⁺ or H⁺ ions. Blocking the channel lumen with disulfide bonds abolished assembly of PulG endopilus and secretion of pullulanase, the specific substrate of this system. These data recall earlier studies showing the requirement of proton motive force for T2S function. Together with structural similarities of AP complex with the vacuolar ATPase, they suggest that PulF forms a sodium or proton channel that cooperates with the GspE/PilB ATPase to fuel (endo)pilus assembly and protein secretion.

Type IV filament (T4F) assembly nanomachines are widespread prokaryotic organelles that play key adaptive roles in motility, adherence, macromolecule trafficking. Prominent members of this family include Type IV pili (T4P), cell surface fibers that promote surface motility or DNA uptake and Type II secretion systems (T2SS) (Figure 1) that transport folded proteins from the periplasm to the surface of Gram-negative bacteria. T4F systems share common essential components of AP complex in the inner membrane, including the polytopic platform protein of the GspF/PilC family (Fig. 1). **GspF** is thought to transduce the mechanical energy generated by the assemby ATPase underlying the AP complex and to orchestrate incorporation of pilin subunits (GspG) into the growing pili. We used Robetta and coevulution analysis to build models of PulF cytoplasmic domains, and AF2 to model PulF monomer (Fig. 1) and dimer (Fig. 2).

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