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Content archived on 2024-05-27

Folding and assembly of the outer membrane component PulD of the type II secretion pathway of Klebsiella oxytoca

Final Report Summary - FAPUL (Folding and assembly of the outer membrane component PulD of the type II secretion pathway of Klebsiella oxytoca)

Membrane protein folding is a complex process of which our understanding is progressing slowly compared to soluble proteins. The folding problem holds the key to the fundamental question of how sequence relates to structure and function. Many membrane proteins undergo conformational transitions during their function and in some cases assembly and folding is an intrinsic part of their function. Membrane proteins perform vital functions in health and disease. Understanding how membrane proteins attain their functional state, is therefore also of interest for drug discovery and biotechnology.

In this project we aim to elucidate the in vitro folding and assembly mechanism of the multimeric outer membrane protein PulD. PulD is a multidomain constitutive outer membrane protein that forms the exit portal for the secretion of virulence factors, enzymes and the assembly of surface pili. We aim to isolate folding intermediates along the pathway to the native state and characterise each state structurally using a range of biophysical and biochemical techniques. We also invesitgated the role of lipid membranes in membrane protein folds.

Our results demonstrate that PulD assembles via a sequential multistep process that is initiated by the association of monomers with the membrane. Stored membrane energy is utilised to kinetically trap the protein in the membrane. Variation of the membrane composition enabled trapping of an intermediate state that lacks any of the known determinants of the native state. Because of its multimeric nature and its tight association with the membrane we propose this intermediate to represent a « prepore » structure. Secondary structure analysis suggests that the prepore structure attains the native PulD state via a conformational switch. A mutant library was screened to select for destabilised PulD variants that are trapped in assembly intermediates. Two variants, one with a substitution in the transmembrane spanning C-domain and one with a substitution in the adjacent periplasmic domain, had prepore characteristics. The results led us to propose that interdomain communication underpins the transition from the prepore into the native pore.

Together with high-resolution structural data (or modeling), the identification of PulD regions that are crucial for portal assembly provide a first step towards targeting specific sites by novel antimicrobials to disarm pathogenic bacteria.