Team:UNSW Australia/Lab/Assembly

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Assembly

Overview

The formation of our enzyme-scaffold complex requires two stages of assembly – the formation of the alpha prefoldin and beta prefoldin hexamer and the covalent attachment of enzymes to the scaffold through SpyTag/SpyCatcher or SnoopTag/SnoopCatcher reactions. These assembly stages were characterised separately, by Size Exclusion Chromatography (SEC) and SDS-PAGE respectively. We have demonstrated the formation of alpha and beta prefoldin hexameric and other oligomeric structures, successfully covalently attached the IaaH-SpyTag protein to alpha prefoldin and gamma prefoldin scaffolds and visualised wild type gamma prefoldin, gamma prefoldin fused to SpyCatcher and gamma prefoldin fused to SpyCatcher and IaaH-SpyTag filaments.

Introduction

Self-assembly is the spontaneous organisation of individual subunits without central direction. A self-assembling protein scaffold offers an elegant method of spatially organising enzymes for co-localisation. Other approaches for enzyme scaffolding include the use of microcompartments, DNA and RNA scaffolds and fusions of aptamer domains¹. We designed experiments to investigate two distinct assembly processes for our enzyme-scaffold complex: the oligomerisation of alpha prefoldin and beta prefoldin, and the formation of isopeptide bonds between SpyTags and SnoopTags and SpyCatchers and SnoopCatchers respectively.

Alpha prefoldin (aPFD) (15.7 kDa) and beta prefoldin (bPFD) (13.8 kDa) derived from Methanobacterium thermoautotrophicum oligomerise to form heterohexameric structures (87 kDa), consisting of 2 alpha subunits and 4 beta subunits². The prefoldin hexamer is assembled through interactions between beta hairpins in each subunit. These beta hairpins form two 8 stranded up and down beta barrels. Appropriate methods for analysing the assembly of prefoldin hexamers include techniques such as Size Exclusion Chromatography (SEC), Native-PAGE and Multiangle Light Scattering (MALS), as these retain the native structure of assemblies and can distinguish between molecules of varying size. SEC was used to demonstrate the formation of aPFD and bPFD hexamers and other oligomers, and also could enable the investigation of the monodispersity of the sample. We also aimed to investigate if aPFD and bPFD could hexamerise when fused with SpyCatcher or SnoopCatcher.

Figure 1: aPFD and bPFD form hexamers. Image created using PDB ID: 1FXK²

The SpyCatcher and SpyTag system were derived from Streptococcus pyogenes fibronectin-binding protein FbaB³. A spontaneous isopeptide bond forms between a lysine residue on the SpyCatcher protein and an aspartic acid on the SpyTag peptide, covalently fusing the two components. Fusion of the SpyCatcher and SpyTag to different proteins enables their spontaneous covalent conjugation within minutes. A similar attachment mechanism was developed from the adhesin RrgA from Streptococcus pneumoniae, yielding SnoopCatcher and SnoopTag which also covalently bind to each other⁴. In addition, the attachment systems are orthogonal, as SnoopTag/SnoopCatcher does not interact with SpyTag/SpyCatcher.

Figure 2: A spontaneous isopeptide bond forms between SpyTag and SpyCatcher. Image created using PDB ID: 4MLS⁵

The use of both systems in tandem enable specific and irreversible attachment of tagged enzymes with aPFD fused to SpyCatcher or bPFD fused to SnoopCatcher. The prefoldins can then self-assemble to form an enzyme-scaffold complex. SDS-PAGE was employed to investigate the Tag/Catcher reaction, as the sodium dodecyl sulphate and heat treatment before analysis denatures proteins, while retaining covalent interactions. Any covalent interaction between an enzyme and a prefoldin-catcher fusion could be determined, indicating the binding of an enzyme to our scaffold.

Gamma prefoldin (gPFD) filaments was proposed as an alternative scaffold to alpha and beta prefoldin hexamers. These filaments are composed of a single protein, related to aPFD and bPFD, that polymerises linearly⁶. Although it is difficult to control the length of filaments^7,8 and the distribution of a mixture of fusions with SpyCatcher or SnoopCatcher, the filaments enable increased density of enzyme clustering. To investigate if gPFD filaments are able to form when fused to SpyCatcher and/or SpyCatcher and a SpyTagged enzyme, Transmission Electron Microscopy (TEM) was used to image gPFD, gPFD fused to SpyCatcher (gPFD-SpyC) and gPFD-SpyC mixed with a SpyTagged enzyme (IaaH-SpyT).

SEC, SDS-PAGE and TEM provide valuable information about the required conditions for assembly of our enzyme-scaffold complex. A multiplexed approach to the characterisation of the assembly process enable us to piece together a more complete understanding of the enzyme scaffold and its potential applications to industry and foundational research.