Gatekeeper residues in the major curlin subunit modulate bacterial amyloid fiber biogenesis

Abstract

Amyloid fibers are filamentous protein structures commonly associated with neurodegenerative diseases. Unlike disease-associated amyloids, which are the products of protein misfolding, Escherichia coli assemble membrane-anchored functional amyloid fibers called curli. Curli fibers are composed of two proteins, CsgA and CsgB. In vivo, the polymerization of the major curli subunit protein, CsgA, is dependent on CsgB-mediated nucleation. The amyloid core of CsgA features five imperfect repeats (R1-R5), and R1 and R5 govern CsgA responsiveness to CsgB nucleation and self-seeding by CsgA fibers. Here, the specificity of bacterial amyloid nucleation was probed, revealing that certain aspartic acid and glycine residues inhibit the intrinsic aggregation propensities and nucleation responsiveness of R2, R3, and R4. These residues function as "gatekeepers" to modulate CsgA polymerization efficiency and potential toxicity. A CsgA molecule lacking gatekeeper residues polymerized in vitro significantly faster than wild-type CsgA and polymerized in vivo in the absence of the nucleation machinery, resulting in mislocalized fibers. This uncontrolled polymerization was associated with cytotoxicity, suggesting that incorrectly regulated CsgA polymerization was detrimental to the cell.A myloids are ordered proteinaceous fibers commonly associated with mammalian neurodegenerative diseases and prion-based encephalopathies (1). Amyloid fibers have distinct biochemical and biophysical properties, such as remarkable resistance to chemical and thermal denaturation, and specific tinctorial properties when bound to Congo red and thioflavin T (ThT) (1). The molecular basis of neurodegenerative disease development induced by amyloid propagation remains elusive, partially because of the seemingly erratic and uncontrolled nature of amyloidogenesis. An emerging focus of amyloid biosynthesis has shown that amyloids can also be an integral part of physiology found in different organisms including bacteria, fungi, and mammals (2, 3). How nature coordinates functional amyloid propagation and reduces the associated cytotoxicity is poorly understood.Curli, a bacterially produced functional amyloid, is an important component of the extracellular matrix and is involved in bacterial community behaviors (4). Because of the amyloid properties of curli fibers (5, 6), the colonies of curli-producing Escherichia coli stain red when grown on Congo red indicator plates, which provides a convenient assay to monitor curli assembly in vivo (7). In E. coli, at least six proteins are dedicated to directing efficient curli formation. Curli fibers are composed of a major subunit CsgA and a minor subunit CsgB. CsgA remains unpolymerized until it encounters the surface-tethered nucleator CsgB, which initiates CsgA polymerization (8). CsgD is a transcriptional activator for the csgBA operon (4). CsgG, CsgE, and CsgF are nonfiber structural accessory proteins involved in secretion and stabilization of the fiber subunits and modulation of fiber assembly (6). CsgG is prop...

Specific Chaperones and Regulatory Domains in Control of Amyloid Formation

“…CsgA and CsgB each have five stacked ␤-strand-turn- ␤-strand motifs formed by imperfect repeats. In the more amyloidogenic CsgA, aggregation propensities of the repeats are fine-tuned by gatekeeper residues that reduce self-assembly (55). Recently, the CsgC protein was identified as a chaperone that prevents the intracellular aggregation of unsecreted CsgA.…”

Protein aggregation in bacteria: the thin boundary between functionality and toxicity

“…The aggregation-prone proteins are under strict transcription, translation and degradation control to make sure that the levels of these proteins are low. Furthermore, gatekeepers play a regulatory role in the assembly process of the major curli subunit CsgA and reduce the potential cytotoxicity of functional amyloids (Wang et al, 2010). It has been suggested that the differential control of aggregation-prone proteins may be a part of a general regulatory framework that not only minimizes unwanted aggregation but also keeps functional aggregation in check (Gsponer & Babu, 2012).…”

The TDP‐43 N‐terminal domain structure at high resolution

“…Similar multiple S/G-Y-S/G-like motifs are also found in the C-terminal portion of TDP-43 and flank its Q/N rich motif. Although the N-terminal domain of TDP-43 appears to be unique among hnRNP proteins, it may work similar to the domains or accessory proteins that govern amyloidogenesis in functional amyloids, including curlins [41], Sup35 [42] and CPEB/Orb2A [43]. Therefore, the high-resolution structure of the NTD of TDP-43 may represent the first example of an emerging class of protein domains that regulate functional amyloid formation.…”