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 |
| USP3 | N/A |
| USP5 | 2G43 (X-ray)
2G45 (X-ray) 3IHP (X-ray) 6DXH (X-ray) 6DXT (X-ray) |
| USP13 | 2L80 (NMR) |
| USP16 | 2I50 (NMR) |
| USP20 | 5Z4I (NMR) |
| USP22 | N/A |
| USP33 | 2UZG (NMR) |
| USP39 | N/A |
| USP44 | N/A |
| USP45 | N/A |
| USP49 | N/A |
| USP51 | N/A |
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) |
| TOC013E09 | USP44 | 27-110 | pET28-MHL | M9 | 12 | No/low expression | |||
| TOC013F02 | USP49 | 15-105 | pET28-MHL | M9 | 12 | 0.25 mg | Y | N | |
| TOC013D10 | USP33 | 37-124 | pET28-MHL | M9 | 6 | 15.7 mg | Y | Y | N |
| TOC013D11 | USP33 | 38-124 | pET28-MHL | M9 | 6 | 0.35 mg | Y | N | |
| TOC013E02 | USP39 | 97-197 | pET28-MHL | M9 | 6 | 1.6 mg | Y | ||
| TOC013E05 | USP39 | 97-205 | pET28-MHL | M9 | 6 | ||||
| TOC013C02 | USP13 | 188-292 | pET28-MHL | M9 | 6 | Insoluble | |||
| TOC013C03 | USP13 | 208-292 | pET28-MHL | M9 | 6 | Insoluble | |||
| TOC013C04 | USP13 | 208-308 | pET28-MHL | TB | 6 | ||||
| TOC013B11 | USP03 | 10-152 | pET28-MHL | TB | 6 | ||||
| TOC013C08 | USP16 | 26-146 | pET28-MHL | M9 | 6 | 0.7 mg | Y | N | |
| TOC013C09 | USP16 | 32-146 | pET28-MHL | TB | 6 | ||||
| TOC013C11 | USP20 | 1-100 | pET28-MHL | M9 | 6 | 4.8 mg | Y | N | |
| TOC013D03 | USP20 | 1-110 | pET28-MHL | M9 | 6 | 13.2 mg | Y | Y: optimizing | |
| TOC013D04 | USP20 | 8-110 | pET28-MHL | M9 | 6 | ||||
| TOC013-D07 | USP22 | 43-142 | pET28-MHL | M9 | 6 | No/low expression | |||
| TOC013D08 | USP22 | 49-142 | pET28-MHL | M9 | 6 | ||||
| Constructs previously available in SGC database that have high expression: | |||||||||
| SDC093D04 | USP13 | 183-307 | pET28-MHL | TB | 6 | ||||
| SDC093D05 | USP13 | 187-301 | pET28-MHL | TB | 6 | ||||
| SDC024F07 | USP16 | 34-185 | pET28-LIC | TB | 6 | ||||
| SDC024F09 | USP16 | 16-185 | pET28-LIC | TB | 6 | ||||
| SDC024F10 | USP16 | 8-185 | pET28-LIC | TB | |||||
| SDC036B06 | USP20 | 1-141 | pET28-LIC | TB | 6 | ||||
| SDC042G05 | USP33 | 7-139 | pET28-LIC | TB | 6 | ||||
| SDC042G06 | USP33 | 7-167 | pET28-LIC | TB | 6 | ||||
| SDC042G07 | USP33 | 7-183 | pET28-LIC | TB | |||||
| SDC168E10 | USP44 | 1-167 | pET28-MHL | TB | |||||
| SDC231F11 | USP51 | 160-350 | pET28-LIC | TB | |||||
| SDC231-G03 | USP51 | 176-350 | pET28-LIC | TB | 6 | ||||
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!