5TTD

Minor pilin FctB from S. pyogenes with engineered intramolecular isopeptide bond

Summary for 5TTD

• Entry DOI: 10.2210/pdb5ttd/pdb
• Related: 3KLQ
• Related PRD ID: PRD_900009
• Descriptor: Maltose-binding periplasmic protein,Pilin isopeptide linkage domain protein, alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose, FORMIC ACID, ... (4 entities in total)
• Functional Keywords: cell adhesion, isopeptide bond, protein engineering, autocatalytic reaction
• Biological source: Escherichia coli; More
• Total number of polymer chains: 2
• Total formula weight: 111414.97

Authors

Young, P.G.,Kwon, H.,Squire, C.J.,Baker, E.N. (deposition date: 2016-11-02, release date: 2017-03-01, Last modification date: 2024-10-23)

Primary citation

Kwon, H.,Young, P.G.,Squire, C.J.,Baker, E.N.
Engineering a Lys-Asn isopeptide bond into an immunoglobulin-like protein domain enhances its stability.
Sci Rep, 7:42753-42753, 2017

PubMed Abstract: The overall stability of globular protein structures is marginal, a balance between large numbers of stabilizing non-covalent interactions and a destabilizing entropic term. Higher stability can be engineered by introduction of disulfide bonds, provided the redox environment is controlled. The discovery of stabilizing isopeptide bond crosslinks, formed spontaneously between lysine and asparagine (or aspartic acid) side chains in certain bacterial cell-surface proteins suggests that such bonds could be introduced by protein engineering as an alternative protein stabilization strategy. We report the first example of an isopeptide bond engineered de novo into an immunoglobulin-like protein, the minor pilin FctB from Streptococcus pyogenes. Four mutations were sufficient; lysine, asparagine and glutamic acid residues were introduced for the bond-forming reaction, with a fourth Val/Phe mutation to help steer the lysine side chain into position. The spontaneously-formed isopeptide bond was confirmed by mass spectrometry and X-ray crystallography, and was shown to increase the thermal stability by 10 °C compared with the wild type protein. This novel method for increasing the stability of IgG-like proteins has potential to be adopted by the field of antibody engineering, which share similar β-clasp Ig-type domains.

PubMed: 28202898
DOI: 10.1038/srep42753

Experimental method

X-RAY DIFFRACTION (2 Å)