The accumulation-associated protein (Aap) from Staphylococcus epidermidis is a biofilm-related protein that was found to be a critical factor for infection using a rat catheter model. The B-repeat superdomain of Aap, composed of 5-17 B-repeats, each containing a Zn21-binding G5 and a spacer subdomain, is responsible for Zn21-dependent assembly leading to accumulation of bacteria during biofilm formation. We previously demonstrated that a minimal B-repeat construct (Brpt1.5) forms an antiparallel dimer in the presence of 2-3 Zn21 ions. More recently, we have reported the presence of functional amyloid-like fibrils composed of Aap within S. epidermidis biofilms and demonstrated that a biologically relevant construct containing five and a half B-repeats (Brpt5.5) forms amyloid-like fibrils similar to those observed in the biofilm. In this study, we analyze the initial assembly events of the Brpt5.5 construct. Analytical ultracentrifugation was utilized to determine hydrodynamic parameters of reversibly associating species and to perform linked equilibrium studies. Linkage studies indicated a mechanism of Zn21-induced dimerization similar to smaller constructs; however, Brpt5.5 dimers could then undergo further Zn21-induced assembly into a previously uncharacterized tetramer. This led us to search for potential Zn21-binding sites outside of the dimer interface. We developed a Brpt5.5 mutant that was unable to form the tetramer and was concordantly incapable of amyloidogenesis. CD and dynamic light scattering indicate that a conformational transition in the tetramer species is a critical step preceding amyloidogenesis. This mechanistic model for B-repeat assembly and amyloidogenesis provides new avenues for potential therapeutic targeting of staphylococcal biofilms.
CITATION STYLE
Yarawsky, A. E., & Herr, A. B. (2020, September 11). The staphylococcal biofilm protein Aap forms a tetrameric species as a necessary intermediate before amyloidogenesis. Journal of Biological Chemistry. American Society for Biochemistry and Molecular Biology Inc. https://doi.org/10.1074/jbc.RA120.013936
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