6QB2

Crystal structure of the cystatin-based engineered protein scaffold SQT-1C

Summary for 6QB2

• Entry DOI: 10.2210/pdb6qb2/pdb
• Descriptor: Monomer of SQT-1C (1 entity in total)
• Functional Keywords: engineered scaffold protein, de novo protein
• Biological source: synthetic construct
• Total number of polymer chains: 1
• Total formula weight: 15321.17

Authors

Levy, C.W. (deposition date: 2018-12-20, release date: 2019-07-03, Last modification date: 2024-01-24)

Primary citation

Zalar, M.,Indrakumar, S.,Levy, C.W.,Tunnicliffe, R.B.,Peters, G.H.J.,Golovanov, A.P.
Studies of the oligomerisation mechanism of a cystatin-based engineered protein scaffold.
Sci Rep, 9:9067-9067, 2019

PubMed Abstract: Engineered protein scaffolds are an alternative to monoclonal antibodies in research and drug design due to their small size, ease of production, versatility, and specificity for chosen targets. One key consideration when engineering such proteins is retaining the original scaffold structure and stability upon insertion of target-binding loops. SQT is a stefin A derived scaffold protein that was used as a model to study possible problems associated with solution behaviour of such aptamers. We used an SQT variant with AU1 and Myc insertion peptides (SQT-1C) to study the effect of peptide insertions on protein structure and oligomerisation. The X-ray structure of monomeric SQT-1C revealed a cystatin-like fold. Furthermore, we show that SQT-1C readily forms dimers and tetramers in solution. NMR revealed that these oligomers are symmetrical, with inserted loops comprising the interaction interface. Two possible mechanisms of oligomerisation are compared using molecular dynamics simulations, with domain swap oligomerisation being thermodynamically favoured. We show that retained secondary structure upon peptide insertion is not indicative of unaltered 3D structure and solution behaviour. Therefore, additional methods should be employed to comprehensively assess the consequences of peptide insertions in all aptamers, particularly as uncharacterized oligomerisation may alter binding epitope presentation and affect functional efficiency.

PubMed: 31227800
DOI: 10.1038/s41598-019-45565-6

Experimental method

X-RAY DIFFRACTION (2.5 Å)