The folding and aggregation properties of a single KH-domain protein: Ribosome binding factor A (RbfA) from Pseudomonas aeruginosa

• Published in: Biochimica et biophysica acta. General subjects 2021-02, Vol.1865 (2), p.129780, Article 129780
• Main Authors: Santorelli, D., Rocchio, S., Fata, F., Silvestri, I., Angelucci, F., Imperi, F., Marasco, D., Diaferia, C., Gigli, L., Demitri, N., Federici, L., Di Matteo, A., Travaglini-Allocatelli, C.
• Format: Article
• Language: English
• Subjects: Aggregation; Amyloid fibrils; Crystallography, X-Ray; Folding mechanism; Humans; KH domains; Models, Molecular; Protein Aggregates; Protein Domains; Protein Folding; Pseudomonas aeruginosa - chemistry; Pseudomonas Infections - microbiology; Thermodynamics
• ISSN: 0304-4165; 1872-8006
• DOI: 10.1016/j.bbagen.2020.129780

Ribosome-binding factor A from the pathogenic bacterium Pseudomonas aeruginosa (PaRbfA) is a small ribosome assembly factor, composed by a single KH domain, involved in the maturation of the 30S subunit. These domains are characterized by the ability to bind RNA or ssDNA and are often located in proteins involved in a variety of cellular functions. However, although the ability of proteins to fold properly, to misfold or to aggregate is of paramount importance for their cellular functions, limited information is available on these dynamic properties in the case of KH domains. PaRbfA thermodynamic stability and folding mechanism: Far-UV CD and fluorescence spectroscopy, stopped-flow kinetics and chevron plot analysis, site-directed mutagenesis. Fibrils characterization: FT-IR spectroscopy, Thioflavin T fluorescence, Transmission Electron Microscopy (TEM) and X-ray fibrils diffraction. Quantitative analysis of the (un)folding kinetics of PaRbfA show that, in vitro, the protein folds via a 3-states mechanism involving a transiently populated folding intermediate. We also provide experimental evidences that PaRbfA can form ordered fibrils endowed with cross-β structure even in mild conditions. These results lead to the hypothesis that the folding intermediate of PaRbfA may expose (some of) the predicted amyloidogenic regions, which could act as aggregation nuclei in the fibrillogenesis. The methodological approach presented herein could be readily adapted to verify the ability of other KH domain proteins to form cross-β structured fibrils and to transiently populate a folding intermediate. •The biophysical characterization of PaRbfA•A KH domain protein from P. aeruginosa, shows that the protein transiently populates a folding intermediate and can form ordered fibrils•A remarkable feature that could be of general interest for other proteins containing KH-domain(s).