The domain organization of the bacterial intermediate filament‐like protein crescentin is important for assembly and function

Abstract

Crescentin is a bacterial filament-forming protein that exhibits domain organization features found in metazoan intermediate filament (IF) proteins. Structure-function studies of eukaryotic IFs have been hindered by a lack of simple genetic systems and easily quantifiable phenotypes. Here we exploit the characteristic localization of the crescentin structure along the inner curvature of Caulobacter crescentus cells and the loss of cell curvature associated with impaired crescentin function to analyze the importance of the domain organization of crescentin. By combining biochemistry and ultrastructural analysis in vitro with cellular localization and functional studies, we show that crescentin requires its distinctive domain organization, and furthermore that different structural elements have distinct structural and functional contributions. The head domain can be functionally subdivided into two subdomains; the first (amino-terminal) is required for function but not assembly, while the second is necessary for structure assembly. The rod domain is similarly required for structure assembly, and the linker L1 appears important to prevent runaway assembly into nonfunctional aggregates. The data also suggest that the stutter and the tail domain have critical functional roles in stabilizing crescentin structures against disassembly by monovalent cations in the cytoplasm. This study suggests that the IF-like behavior of crescentin is a consequence of its domain organization, implying that the IF protein layout is an adaptable cytoskeletal motif, much like the actin and tubulin folds, that is broadly exploited for various functions throughout life from bacteria to humans. © 2011 Wiley-Liss, Inc.

Cytoskeletal Proteins in Caulobacter crescentus: Spatial Orchestrators of Cell Cycle Progression, Development, and Cell Shape

“…Also like other IF proteins, crescentin bears a conserved stutter, an interruption in the heptad repeat, in the C-terminal coiled-coil region (Ausmees et al 2003). Solubilized crescentin spontaneously assembles into filaments in vitro (Ausmees et al 2003; Esue et al 2010; Cabeen et al 2011). While self-assembly is largely unaffected by salt concentrations, crescentin filaments form most readily at a low pH of 6.5 (Fig.…”

“…At higher pH of up to 8.5, divalent cations (e.g. 5 mM Mg 2+ ) are required to stabilize polymers (Cabeen et al 2011). At pH 6.5, crescentin structures are typically 8 – 10 nm (single filaments) or 17 – 20 nm (paired bundles) in width, but can form thicker bundles, particularly in the presence of divalent cations (Fig.…”

“…At pH 6.5, crescentin structures are typically 8 – 10 nm (single filaments) or 17 – 20 nm (paired bundles) in width, but can form thicker bundles, particularly in the presence of divalent cations (Fig. 5E) (Ausmees et al 2003; Cabeen et al 2011). A study of the mechanical properties of crescentin in vitro determined that crescentin filamentous structures are elastic and solid-like, and can recover their network elasticity after shear stress (Esue et al 2010).…”

“…First, crescentin-induced cell curvature depends on its polymerization and membrane attachment (Cabeen et al 2009; Charbon et al 2009; Cabeen et al 2010; Cabeen et al 2011). Crescentin variants that fail to polymerize under physiological pH and salt conditions in vitro (e.g.…”

“…Crescentin variants that fail to polymerize under physiological pH and salt conditions in vitro (e.g. pH 7.5, 200 mM KCl) also fail to assemble into filaments in vivo , and cells producing these non-polymerizing variants are straight (Cabeen et al 2011). Cells expressing membrane-dissociated CreS ΔN27 are also straight, even though the polymerization properties of this mutant are similar to wild-type (Cabeen et al 2009; Cabeen et al 2011).…”