X-ray crystallography is a tool used for the structural determination of proteins and biological molecules. My specific project focuses on the zinc finger ubiquitin binding domain (Zf-UBD) of USP5; however, USP5 is just one of many USPs that contain a Zf-UBD. Table 1 shows a list of the USP Zf-UBDs and their available structures in the protein data bank (PDB). In addition, the protein BRAP also has a Zf-UBD. My recently identified USP5 inhibitors are far from being optimized, but my goal is to find compounds that are potent and selective for USP5 and that can be used by cell-biologists. I will need to test them against other Zf-UBD’s to determine their specificity. Crystal structures of other Zf-UBD’s will be useful to rational design selective compounds.
Table 1. USP Zf-UBDs & Available PDB Structures
|Target Zf-UBD||PDB ID|
I designed constructs of the different USP Zf-UBDs through analysis of the available structures and by using secondary structure prediction tools. Peter Loppnau, SGC’s cloning expert ordered primers and cloned my constructs into expression vector pET28-MHL to allow expression and purification from bacterial production. Linda Lin, another talented colleague, then did a test expression experiment using small-scale cultures to see if there was expression of the constructs. I designed 69 constructs of USP Zf-UBDs, and chose 17 that expressed well in the TestX and 12 available constructs already in the SGC database that have high expression. Next, I did large scale growth in bacteria and am now working my way through purifying them. Once proteins are pure, I set up crystal trays using our in-house screens. So far, I have identified some crystals for USP20 and USP33 Zf-UBD but they either don’t diffract or need more optimizing (Table 2). You can see full experimental details on Zenodo.
Table 2. Summary of USP Zf-UBD Crystallization Pipeline To Date
|Clone||Target||AA||Vector||Media||Amount Grown (L)||Purification Yield (mg)||Crystal Plate Setup (Y/N)||Crystals? (Y/N)||Diffraction (Y/N)|
|TOC013D03||USP20||1-110||pET28-MHL||M9||6||13.2 mg||Y||Y: optimizing|
|Constructs previously available in SGC database that have high expression:|
I still have some more constructs to grow and purify and hopefully I’ll be able to crystallize and solve some novel USP Zf-UBDs. More on this later!