Structures of the Xcp and Hxc T2SS endopili provide new insights into type IV pili subfamilies.

Type IV filaments (T4F) are widespread bacterial appendages involved in adhesion, motility, DNA uptake, and protein secretion. In the type II secretion system (T2SS), a short pilus-like structure termed the endopilus drives protein export across the outer membrane. Despite its important function, the specific molecular determinants of endopili remain poorly understood. Here, we used cryo-electron microscopy to determine the structures of two endopili assembled by the independent Hxc and Xcp T2SS systems of Pseudomonas aeruginosa. Comparative analyses reveal defining structural and chemical features of T2SS endopili, including a unique surface property of the XcpG endopilus that explains its distinctive bundling behavior. Mapping inter-subunit interactions further identifies residues governing selective assembly within coexisting secretion systems. Comparisons with related type IVa pili define a conserved architectural framework and constrained helical parameters characteristic of the T2SS endopilus family, providing insight into how subtle chemical variations encode functional specificity and efficient protein export.