Hello

Do you have diffraction data from your copmlexe?

If yes

1. First resolve the protein structure from these data.

2. If the inhibitor is present then you will have an extra electron density around its position somewhere on your protein.

3. You construct the inhibitor within this extra density and thats all. 

Hi Nicolas,

Thanks for your reply. I do not have diffraction data yet. I am worried that it will not crystallize because it has never before been crystallized without DNA bound. Previous studies have performed screening assays but with no success. I am wondering how to crystallize it. 

Γειά σου, Amanda,

Do you have a PDB code for the complex with DNA structure?

Can you share the information?

In many cases proteins bind to a DNA using a DNA-binding domain, which is HTH (Helix-Turn-Helix structure). Nothing special about this domain, but you will never guess that it may bind a DNA, and it is about 60-80 amino acids long. The distinct feature of this domain is, that it has Lys and Arg residues sticking out. Those positively charged residues interact with oxygen atoms of phosphate groups of DNA. The way how most inhibitors will work for this case, would be competing for binding to those positively charged residues or modifying them (for example, phosphorylation, methylation or other modification). Actually, that is the way how those proteins are regulated in living organisms.

So, if your protein binds DNA using HTH domain, then the first thing you can do, will be to try to identify which atoms of your inhibitor could interact with the protein. Maybe a protein-DNA complex structure can help you to orient or even dock your compound into protein structure.

Back to the question, why is so hard to get nice screening assays... Because, the location of Lys and Arg is arranged the way, that when one of Lys (Arg) will interact with phosphate group of DNA, the next Lys (Arg) will be close to the next DNA phosphate group, they work cooperatively. Imagine the buttons on your shirt. When one is on the place, the next one (by design) will match the position of the next hole.  For small compounds to be in right place, even if one is already in proper position will not work, they bind independently. Chances are very low, and that is why is hard to compete with DNA. And that is why the Nature has solution for this. When there is no need in binding DNA,  the residues responsible for interaction will be modified (methylated or acetylated).

That is why is so hard to get complexes of HTH domain with compounds, even harder to crystallize them. I would suggest to try phosphate or sulfate buffers at low ionic strength for crystallization experiments, they are natural replacements of phosphate groups of RNA and DNA. Incubate your protein with inhibitor for 30 min (1 hr) and then setup crystallization plates.

It may work, but there is no guarantee, sorry.

Good luck!

George.

Hi George, 

I can not share the identity of the structure. The protein binds to DNA at the HTH domain. Thank you for this information. I will try your suggestion. We will see how this works!

Best wishes,

Amanda