NMR insights into the pre-amyloid ensemble and secretion targeting of the curli subunit CsgA

Abstract

The biofilms of Enterobacteriaceae are fortified by assembly of curli amyloid fibres on the cell surface. Curli not only provides structural reinforcement, but also facilitates surface adhesion. To prevent toxic intracellular accumulation of amyloid precipitate, secretion of the major curli subunit, csgA, is tightly regulated. In this work, we have employed solution state NMR spectroscopy to characterise the structural ensemble of the pre-fibrillar state of CsgA within the bacterial periplasm, and upon recruitment to the curli pore, CsgG, and the secretion chaperone, CsgE. We show that the N-terminal targeting sequence (N) of CsgA binds specifically to CsgG and that its subsequent sequestration induces a marked transition in the conformational ensemble, which is coupled to a preference for CsgE binding. these observations lead us to suggest a sequential model for binding and structural rearrangement of CsgA at the periplasmic face of the secretion machinery.

Functional Bacterial Amyloids: Understanding Fibrillation, Regulating Biofilm Fibril Formation and Organizing Surface Assemblies

“…[63]. Monomeric CsgA is very unstructured according to hydrogen-deuterium exchange [69], light scattering, single fiber AFM-analysis [70], secondary structure determination [71] and NMR studies on the pre-amyloid state [72] and MD simulations of peptide segments of CsgA [73]. However, upon association, CsgA folds into a uniform amyloid state in what appears to be a simple two-state model [69,70].…”

Chaperones mainly suppress primary nucleation during formation of functional amyloid required for bacterial biofilm formation

“…CsgA is maintained in an unfolded state in the periplasm prior to export through dynamic and electrostatically driven 27 transient binding to the chaperone CsgC, 22 which can maintain CsgA in an unfolded state at CsgC : CsgA mole ratios as low as 1 : 40. 28 CsgC also inhibits the aggregation of the pathological amyloid a-synuclein (aSN) through a shared sequence motif. 22 Our study addresses the question of whether a set of human proteins, known to inhibit pathological amyloid formation in vitro and in a variety of physiological settings, will have a similar effect on functional amyloids.…”

Functional amyloids from bacterial biofilms – structural properties and interaction partners

“…[115][116][117] Solution NMR spectroscopy has been applied to study functional amyloids in their monomeric soluble form. 81,[118][119][120] Since most of the known functional amyloids are unfolded proteins, the use of this technique is limited to understanding the structural changes during amyloid bril formation. Nevertheless, it provides valuable information, e.g.…”