I don't think there is a satisfactory solution if you are strapped for resources and/or equipment. Assuming crystallyzing the protein:ligand complex and doing x-ray diffraction is out of the question, if you have other ligands binding the same protein whose interaction site is known, you can run competition assays. Or you can run the structure of your protein through one of the many binding pockets/druggability predictors online servers currently available, and do site-directed mutagenesis on the predicted sites to see if the interaction is really disturbed. If you do have access to mass spectrometry, I suppose you could dissolve the protein in deuterated water in the presence or absence of the ligand, then digest the protein and look for differences in proton exchange rates between the resulting peptides (this can be combined with pocket predictions to narrow the search).

Hope it helps!

Thanks Alejandro.

Of course we have mass spectroscopy. Do you think SAXS (small angle X-ray diffraction) will also help in determining the interaction site ?

No idea, honestly. Sorry I can't be of more help.

X-ray crystallography is the best method. Perhaps you can find a collaborator to help with this.

For large protein complexes, cryoelectron microscopy can also be used. Obviously, you would have to collaborate with an expert in the field.

Multidimensional protein NMR can be used to identify sites of interaction of ligands with a protein if the resonances are assigned to specific residues. This is a very complicated and specialized study, so you would need to collaborate with an expert.

I don't know whether this applies to your situation, but it sometimes happens that if you select for mutant cells resistant to a drug, the mutations occur in the target protein in and around the drug binding site, but not always.

If you create a modified version of the ligand that has a mildly reactive chemical moiety, or a photoaffinity analog, you may be able to identify residues in or near the ligand binding site using proteolytic digestion and mass spectrometry by comparison to the unlabeled protein.

If you have some idea of residues that might be involved in binding, then site-directed mutagenesis can be used to test hypotheses. However, these results are not definitive without structural information.

I agree with Adam that if you want to know the exact interaction sites, you must use structural biology technologies, including X-ray crystallography, NMR, and cryo-electron microscopy to solve this problem. I don’t know the specific information of your target protein, and different technologies have their own pros and cons, you need to choose the appropriate method based on your protein properties. If you don’t have the resources or equipment, you can work together with a reliable partner to solve this problem.

Recommend an article for you, maybe can help you choose experimental method to determine the structure, https://www.creative-biostructure.com/comparison-of-crystallography-nmr-and-em_6.htm

Hope it helps!