Collagen-like Motifs of SasG: A Novel Fold for Protein Mechanical Strength

• Published in: Journal of molecular biology Vol. 435; no. 6; p. 167980
• Main Authors: Bruce, Alexander J.E., Paci, Emanuele, Brockwell, David J.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier Ltd 15.03.2023
• Subjects: alanine; Amino Acid Motifs; Amino Acid Substitution; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; biofilm; bone formation; brittleness; Collagen - chemistry; Collagen - genetics; collagen-related disease and osteogenesis imperfecta (OI); Humans; mechanobiology; Membrane Proteins - chemistry; Membrane Proteins - genetics; molecular biology; Osteogenesis Imperfecta - genetics; Osteogenesis Imperfecta - metabolism; Phenotype; Protein Conformation, beta-Strand; Protein Domains; Protein Folding; Protein Stability; protein unfolding; SasG; Single-molecule force spectroscopy (SMFS); Staphylococcus aureus; strength (mechanics); surface proteins; thermodynamics; mechanobiology; PBS; SMFS; SasG; CLM; PPII; AFM; CWA; FU; WLC; collagen-related disease and osteogenesis imperfecta (OI); Aap; LC-MS; protein unfolding; S. aureus; FWHM; MD; OI; WT; E. coli; Single-molecule force spectroscopy (SMFS); TS
• ISSN: 0022-2836; 1089-8638; 1089-8638
• DOI: 10.1016/j.jmb.2023.167980

[Display omitted] •The structural determinants of the high unfolding force for SasG are unclear.•Structural, thermodynamic and mechanical effects of 18 variants investigated.•Variants' ϕ-values show importance of mechanical clamp and domain interface motifs.•Study identifies and characterises collagen-like regions as a novel mechanical motif. The Staphylococcus aureus surface protein G (SasG) is associated with host colonisation and biofilm formation. As colonisation occurs at the liquid-substrate interface bacteria are subject to a myriad of external forces and, presumably as a consequence, SasG displays extreme mechanical strength. This mechanical phenotype arises from the B-domain; a repetitive region composed of alternating E and G5 subdomains. These subdomains have an unusual structure comprising collagen-like regions capped by triple-stranded β-sheets. To identify the determinants of SasG mechanical strength, we characterised the mechanical phenotype and thermodynamic stability of 18 single substitution variants of a pseudo-wildtype protein. Visualising the mechanically-induced transition state at a residue-level by ϕ-value analysis reveals that the main force-bearing regions are the N- and C-terminal ‘Mechanical Clamps’ and their side-chain interactions. This is tailored by contacts at the pseudo-hydrophobic core interface. We also describe a novel mechanical motif – the collagen-like region and show that glycine to alanine substitutions, analogous to those found in Osteogenesis Imperfecta (brittle bone disease), result in a significantly reduced mechanical strength.