Interfacial self-assembly of a bacterial hydrophobin

Abstract

The majority of bacteria in the natural environment live within the confines of a biofilm. The Gram-positive bacterium Bacillus subtilis forms biofilms that exhibit a characteristic wrinkled morphology and a highly hydrophobic surface. A critical component in generating these properties is the protein BslA, which forms a coat across the surface of the sessile community. We recently reported the structure of BslA, and noted the presence of a large surfaceexposed hydrophobic patch. Such surface patches are also observed in the class of surface-active proteins known as hydrophobins, and are thought to mediate their interfacial activity. However, although functionally related to the hydrophobins, BslA shares no sequence nor structural similarity, and here we show that the mechanism of action is also distinct. Specifically, our results suggest that the amino acids making up the large, surface-exposed hydrophobic cap in the crystal structure are shielded in aqueous solution by adopting a random coil conformation, enabling the protein to be soluble and monomeric. At an interface, these cap residues refold, inserting the hydrophobic side chains into the air or oil phase and forming a three-stranded β-sheet. This form then self-assembles into a wellordered 2D rectangular lattice that stabilizes the interface. By replacing a hydrophobic leucine in the center of the cap with a positively charged lysine, we changed the energetics of adsorption and disrupted the formation of the 2D lattice. This limited structural metamorphosis represents a previously unidentified environmentally responsive mechanism for interfacial stabilization by proteins.BslA | interfacial self-assembly | bacterial hydrophobin | Bacillus subtilis | biofilm

The Bacterial Hydrophobin BslA is a Switchable Ellipsoidal Janus Nanocolloid

“…4 Electron microscopic images of the BslA film have demonstrated that the most stable assembly of BslA surface layers correlates with a high degree of order between individual proteins. 12 In our simulations, the outward facing conformation (wt-BslA-L out ) exhibits the most clearly defined upright orientation at the interface in combination with the highest adsorption free energy. The deviation from clearly defined upright orientations we show here for mutant proteins is thus likely to be an important factor in the relative loss of mutant BslA layer stability in vitro, and of the observed wettability of the mutant biofilms in vivo.…”

“…4,12 Our simulations provide reliable estimates of the adsorption free energy of wild-type (wt) BslA and enable us to rationalize a number of experimental observations.…”

“…9 Experimental evidence for a subtle conformational switching process in BslA has recently also been obtained. 12 Large hydrophobic regions are usually unstable at the surface of soluble proteins as during folding, they normally organize into the shielded protein hydrophobic core by hydrophobic collapse, exposing mainly polar residues to water. 35 Interestingly, all previously characterized similar proteins stabilize their large external hydrophobic regions by forming an extended network of rigid disulfide bonds.…”

Bifunctionality of a biofilm matrix protein controlled by redox state

“…The hydrophobicity of the biofilm has been attributed to a small secreted protein named BslA (7,8), which works alongside the fibrous protein TasA (9,10) and the extracellular polysaccharide produced by the products of the epsA-O operon (5) to allow morphogenesis of the biofilm. Partial evidence of the mechanism used by BslA to confer hydrophobicity to the B. subtilis biofilm has been revealed (8,(11)(12)(13). Although transcription of bslA occurs uniformly within the population, secreted BslA is localized to the periphery of the biofilm, where it forms a hydrophobic layer at the airbiofilm surface (8,12).…”

“…Structural analysis revealed that BslA is an amphipathic protein consisting of an immunoglobin G-like scaffold appended with a hydrophobic "cap" (12) that can present in two forms: "cap in" and "cap out." This property either shields (cap in) or reveals (cap out) the hydrophobic domain in response to the local environment (11). Thus, when BslA is in the aqueous environment of the matrix, it is likely that the hydrophobic cap is "hidden" toward the interior of the protein.…”