USP Zf-UBD Crystallography Pipeline

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!

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