Hi guys, Thank you so much for the suggestions! I used DSXONLINE to rank the two binding sites according to binding free energy and get the prediction there, and will go on to use some other programs to see whether I get the same prediction. At the same time, I am thinking of disrupting the binding sites by mutagenesis and do ITC again as suggested by you guys. Now I have a question: which and how many residues I should mutate to disrupt the binding of the ligand. One of the two binding sites, which I am thinking of mutating first, has the following interactions with the ligand: 5 residues are involved in forming 7 pairs of the hydrogen bonds with the ligands, 1 residue is involved in electrostatic interaction with the ligand, 14 residues (including 3 that forms the hydrogen bonds with the ligands) are involved in hydrophobic contacts with the ligand. I am not sure how many residues I should mutate in order to completely abolish the ligand binding to this pocket assayed by ITC. I am looking at the other proteins from other species which could potentially bind the same ligand in the pocket, and found out that: 3 out of 5 that forms hydrogen bonds with the ligands are conserved, 1 residue that forms electrostatic interaction is conserved, 1 residue that is involved in the hydrophobic contacts is conserved.
Any suggestions or ideas about which and at least how many residues I should mutate to abolish the binding? Any rules or criteria to predict which residues might be more important and should be mutated first? Thank you so much! Best, Wei On Wed, Nov 20, 2013 at 10:13 AM, Ed Pozharski <epozh...@umaryland.edu>wrote: > Baerbel, > > Certainly, constraints of the crystal lattice affect everything. The > magnitude of the influence is what is in question here. Let's do the > numbers (this is admittedly simplistic but reflect general trends well). > > Let's say you observe difference in occupancies for two identical sites > that is 50%/75%. This means that difference in binding affinities is > due to change in free energy of binding at DDG=RTlog(K1/K2). If ligand > is in excess with respect to the protein, K~(1-occ)*L/occ. Thus the > estimate of DDG is ~0.6kcal/mol. Point is that it does not take much > free energy to result in difference in occupancy. Yet anyone who ever > done ITC would tell you that such small difference would be hard to > detect with the technique. So the prior is that Wei sees significant > DDG. > > Determining affinities from crystal soaks is, of course, overkill. > There are other easier methods that do not pose the problem you mention. > However, the original post was not about determining affinities, but > rather which of the two sites has the higher affinity. Is it possible > that lattice constraints invert the affinity ratio? Sure. But given > that the prior of the "lattice DDG"=0, it is more likely that the sign > of the affinity ratio in crystal is the same as in solution. The > likelihood of this, of course, depends on the value of "solution DDG" > with respect to "lattice DDG". > > Again, mutagenesis works too, but if crystals and beamtime are > available, such experiment makes sense, at least in my opinion. > > Cheers, > > Ed. > > On Wed, 2013-11-20 at 08:20 +0100, Bärbel Blaum wrote: > > Hi Ed, > > > > you are right about the original question, but what I mean is this: if > > the occupancies (and B-factors) differ so much in crystals with > > IDENTICAL binding sites, i.e. identical affinities, does this not show > > that occupancies (and B-factors) do not reflect affinities alone, but > > equally local packing? There might be individual cases in which such > > effects can be neglected, but generally I think trying to determine > > affinities from crystal soaks is, hmm, not very good pratice, simply > > because there are other dedicated methods to do it that suffer less > > from side effects. Including the docking approach. > > > > Kind regards, Baerbel > > > > > > Quoting Ed Pozharski <epozh...@umaryland.edu>: > > > > > If I understand the original post correctly, the binding sites in > > > question are not chemically identical. While it's possible that > lattice > > > may invert the order in which sites are occupied, it is not very likely > > > given that affinity gap is sufficient to be observable by ITC. > > > > > > Mutagenesis is a good option too. > > > > > > On Tue, 2013-11-19 at 17:12 +0100, Bärbel Blaum wrote: > > >> Hello, > > >> > > >> we work with proteins that have typically several chemically identical > > >> binding sites (viral capsid proteins fully assembled or as multimeric > > >> assembly-intermediates). Depending on how long at which concentrations > > >> they are soaked the chemically identical ligand pockets within one > > >> asymmetric unit are typically occupied to different levels purely > > >> because of individual crystal contacts and accessibility. I therefore > > >> think that neither soaking with different concentrations nor B-factor > > >> analysis are solid methods to determine some sort of relative > > >> affinities. I'd suggest to design mutants for either binding site and > > >> ITC measurements with the mutant proteins. This might also tell you if > > >> some sort of co-op exists between both sites. > > >> > > >> Baerbel > > >> > > >> Quoting Ed Pozharski <epozh...@umaryland.edu>: > > >> > > >> > IMHO, while explaining binding affinity from a structure is fun, it > does > > >> > not prove anything. Assuming that I understand your situation > > >> > correctly, you can (relatively) easily find out from experiment > which > > >> > pocket has higher affinity. Just do soaks with different ligand > > >> > concentrations - the expectation is that the weaker binding site > will > > >> > become partially occupied first. > > >> > > > >> > On Tue, 2013-11-19 at 04:58 +0000, Xiaodi Yu wrote: > > >> >> Hi Wei: > > >> >> > > >> >> Based on the structure, you can calculate the binding surface > between > > >> >> the protein and the ligand. Maybe the two binding pockets will give > > >> >> you two different numbers. And the larger one usually can have the > > >> >> higher binding affinity. You also can analyse how the ligand > > >> >> interacts with the protein though hydrophobic or electrostatic > > >> >> interaction , etc? the last, you may also compare the b factors of > > >> >> the ligand or the protein binding pocket regions after you refining > > >> >> the structure. These things may give you some hints about which > > >> >> binding site is more strong. > > >> >> > > >> >> Dee > > >> >> > > >> >> > > >> >> > ______________________________________________________________________ > > >> >> Date: Mon, 18 Nov 2013 22:45:58 -0500 > > >> >> From: wei.shi...@gmail.com > > >> >> Subject: Re: [ccp4bb] distinguish ligand binding sites within a > > >> >> protein > > >> >> To: CCP4BB@JISCMAIL.AC.UK > > >> >> > > >> >> Thank you so much for the suggestions, Tomas! Yes, my ligand is a > > >> >> small molecule. I have the crystal structure of the ligands bound > to > > >> >> the protein, do I still need to computationally dock the ligand to > the > > >> >> two pockets, can I calculate the parameters of binding directly > using > > >> >> the crystal structure? > > >> >> > > >> >> Best, > > >> >> Wei > > >> >> > > >> >> > > >> >> > > >> >> On Mon, Nov 18, 2013 at 9:03 PM, Tomas Malinauskas > > >> >> <tomas.malinaus...@gmail.com> wrote: > > >> >> Dear Wei Shi, > > >> >> is your ligand a small molecule? If it is a small > molecule, I > > >> >> would > > >> >> try to computationally dock the small molecule to two > pockets > > >> >> separately using AutoDock, and look at the estimated free > > >> >> energies of > > >> >> binding. > > >> >> Best wishes, > > >> >> Tomas > > >> >> > > >> >> On Mon, Nov 18, 2013 at 8:55 PM, Wei Shi > > >> >> <wei.shi...@gmail.com> wrote: > > >> >> > Hi all, > > >> >> > I got the crystal structure of a transcription factor, > and > > >> >> every monomer > > >> >> > binds two molecules of the same ligand in different > binding > > >> >> pockets. And I > > >> >> > also did the ITC experiment, titrating the ligand into > the > > >> >> protein, and got > > >> >> > a U-shaped curve. The binding affinity for the first > binding > > >> >> site is higher > > >> >> > than the second binding site. > > >> >> > I am wondering whether I could computationally determine > > >> >> from the > > >> >> > protein-ligand complex structure that which binding site > has > > >> >> higher affinity > > >> >> > for the ligand and correlate the binding sites with the > > >> >> parameters I got > > >> >> > from ITC experiment. > > >> >> > Thank you so much! > > >> >> > > > >> >> > Best, > > >> >> > Wei > > >> >> > > >> >> > > >> >> > > >> > > > >> > -- > > >> > Edwin Pozharski, PhD, Assistant Professor > > >> > University of Maryland, Baltimore > > >> > ---------------------------------------------- > > >> > When the Way is forgotten duty and justice appear; > > >> > Then knowledge and wisdom are born along with hypocrisy. > > >> > When harmonious relationships dissolve then respect and devotion > arise; > > >> > When a nation falls to chaos then loyalty and patriotism are born. > > >> > ------------------------------ / Lao Tse / > > >> > > > >> > > >> > > >> > > > > > > -- > > > Edwin Pozharski, PhD, Assistant Professor > > > University of Maryland, Baltimore > > > ---------------------------------------------- > > > When the Way is forgotten duty and justice appear; > > > Then knowledge and wisdom are born along with hypocrisy. > > > When harmonious relationships dissolve then respect and devotion arise; > > > When a nation falls to chaos then loyalty and patriotism are born. > > > ------------------------------ / Lao Tse / > > > > > > > > > > > > > > > > > -- > Bullseye! Excellent shot, Maurice. > Julian, King of Lemurs. >
