New compounds developed by Meds4Kids (M4K) and other collaborators against ALK2 would benefit from being crystallised so we can look at the structure of the complex and explain exactly how these compounds bind. This helps us work out how to improve them for both potency and selectivity and explain why some bind better than others. This requires protein crystals and this in turn requires pure protein.
For details methods for this work, please click here.
Last week I purified two proteins – ALK2 and FKBP12. FKBP12 is a protein associated with ALK2 and is thought to help inhibit it under normal circumstances. I used a construct of ALK2 which has both the kinase domain and the GS loop, and which will therefore bind to FKBP12. FKBP12 sits on top of the ALK2, we think this interaction may be interrupted by the R206H FOP mutation and other mutations in that area.
Figure 1: J Biol Chem. 2012 Oct 26; 287(44): 36990–36998. Showing FKBP12 bound to the top of ALK2 and with dorsomorphin bound to the active site of ALK2.
ALK2 is expressed in insect cells while FKBP12 is expressed in E.coli cells. I had cell pellets of each from which I extracted pure protein before mixing the two and then isolating the complex of the two bound together. After several steps I had a relatively pure sample of complex as shown in the gel beneath.
Fig. 2: SDS PAGE gel showing the purified complex from the output of a superdex 200 size exclusion column, separated from uncleaved FKBP12-GST/ALK2 complex and free FKBP12.
I concentrated these fractions down and I ended up with enough pure protein to set up 24 plates – four for each compound I wanted to look at, (PK012002a, PK008982b, PK012055a, PK012057a, PK010701a and PK006099b). I then put two plates to incubate at 20°C and two plates to incubate at 4°C for each of the compounds.
I checked the drops after the weekend and I found some crystals! There are some that look good enough to mount for four of the compounds so far, although it’s possible more might appear over time.
Here are some pictures of the best crystals so far:
Fig. 3: Examples of Crystals of ALK2 with FKBP12, co-crystallised with a variety of compounds.
The next step will be to see if they grow any bigger, then mount them, shoot them in the x-ray beam and see if they diffract well enough to get any structural data from them!