In Work Package 1 significant efforts were invested into solving numerous problems with protein production and purification for CHIKV proteins. This included creation and implementation of new approaches for enhancing protein solubility by promoting correct folding, identifying mutants and fusion constructs that would result in sufficient amounts of monodisperse proteins needed for crystallization trials. In addition, another set of vectors for co-expression of bacterial chaperones and folding modulators (disulphide isomerases, peptidyl-prolyl isomerases etc) was created and employed throughout the project. The total number of expression constructs for different CHIKV proteins created and tested in this project exceeds 400 and such systematic screening approach allowed us to identify the best expression constructs, the best expression conditions and the best purification schemes for CHIKV proteins which is an absolute necessity for any further studies aiming screening and validation of virus inhibitors. During this project almost 130 96-well crystallization trays using different purified alphavirus proteins were set up and analysed, which constitutes a significant effort in understanding what approaches might be working when CHIKV proteins are concerned. It was found particularly promising to employ fusion partner driven crystallization, which in combination with surface entropy reduction and buffer conditions optimisation by TSA appears to be first choice strategy for difficult targets: to address this, corresponding set of vectors facilitating production of protein of interest fused to crystallization helpers such as MBP, GFP, Trx, Sumo or T4 lysozyme has been created. The obtained deliverables include: purified nsP2 and its domains Pro and N174, several structures of CHIKV Pro alone and as part of fusion construct as well as novel structure of N-terminal domain of nsP2.
Work Package 2 was focused of expression and purification of large polyproteins, which benefited from using codon-optimised genes, introducing different mutations to prevent proteolytic processing and prevent aggregation. In parallel individual CHIKV proteins nsP1 and nsP3 were expressed and purified. Attempts to generate complexes of nsP2 protease and N174 domains with nsP1 and nsP3 were performed using mixed purified components and analytical size exclusion chromatography. Purified nsP1, nsP3 and fusion of nsP1 with N174 or Pro with nsP3 were submitted to crystallization trials and SAXS analysis. The obtained deliverables include: purified polyproteins CHIKV P12, P123, purified proteins nsP1, nsP1-N174, nsP3 and Pro-334 and their SAXS analysis. Importantly, thanks to the development of cryo-EM technique the obtained protein preparations of P12 and P123 can now be analysed using in-house cryo-EM microscope commissioned in April 2018.
In Work Package 3 after optimization of conditions thermal shift assay was performed using CHIKV Pro and in-house library of fragments encompassing 960 chemical compounds. This led to identification of several molecules that act at increasing protein stability, suggesting their binding to the target protein, and large number of compounds potentially destabilising protease domain, possibly binding at the interface between two subdomains of CHIKV Pro and promoting protein unfolding therein. These compounds will be used in the future rounds of validation by complimentary biophysical techniques such as ligand-based NMR.
Finally, Work package 4 dealt with the assay development, whereas protease activity assay based on measurements of changes in fluorescence polarization upon substrate cleavage was created.
The obtained preliminary results have already been used to apply for additional round of funding and it is also expected that novel structural information for N174 domain of CHIKV nsP2 will provide with new ideas regarding its possible role in of CHIKV replication, possibilities for its inhibition and will constitute the basis for future research proposal. The resources and methods developed during this project are expected to push the CHIKV research field further and should also be useful in other research projects dealing with difficult proteins. The data obtained within this project will be made publicly available by different means. In particular, sets of expression vectors developed in this study are being deposited to Addgene non-profit plasmid repository and will be described in the corresponding section of the research group website (
http://hyvonen.bioc.cam.ac.uk/(se abrirá en una nueva ventana)). After structure validation the corresponding data will be deposited to PDB. Results regarding novel structure of CHIKV N-terminal domain will be published as scientific publication and presented at future scientific meetings.
