type of docking

So you do not like your output and you are moddelling a pre-activation complex of an ACP and fab enzyme, yes?

I would say ligand-protein first as using ACPs from different species seems to not have a deleterious effect on catalysis IIRC, so the protein-protein interaction is probably very weak.

The transition state would have the phosphopantetheine cysteine and the enzyme’s cysteine forming a tetraheydral hemi-thioacetyal thinggy —totally modellable as amino acids can have a third connect, albeit that it need to be declared in the PDB block with the LINK record. So you’d want your pre-activation complex to be close to that as possible.

For the pre-activation complex I would say do a docking of the transferred acyl (or physiological acyl substrate if you are trying to engineer specificity towards something else) to see where the atoms roughly ought to go. I’ve docked an acyl-CoA and an enzyme in a few steps into one of the anabolic enzymes —dont remember which, fabB?— and it was very clear cut because the keto groups of the acyls seemed to have a clear binding mode for substrates.

And then do your protein protein docking with constraints for the omega carbon and the sulfur —sparks fly below 3 Å, but if you push them too much up that activation hill, you get odd stuff happening unfortunately, so I’d say a harmomic constraint with a very low sigma (say 0.1) and a mean that is 3.5 Å works best. One thing to be aware of it the deprotonation of the catalytic cysteine (residue CYZ), which in turn requires a specific tautomer of histidine (if the base is a histidine).

0./3.

You are totally right, I made a silly mistake and my answer is completely invalid, hence the utter confusion.

Only the synthase (the malonyl transfer one that does the decarboxylative Claisen condensation) has a cysteine to which one of the substrates gets transferred over to, the rest do not form a covalent transition state —i.e. the acyl does not get passed onto the enzyme and then returned back, wherein the passing over goes through a tetrahedral two sulfurs on a carbon transition state. For some reason I thought this was the default.

Not having a covalent transition state means that my worries about strain in the pre-reaction complex are not applicable. The transition state for the dehydrogenases and dehydratase is just ketone to hydroxylate ion, so on worth modelling unless you are worried that your active site may be affected by a mutation. To clarify my incorrect train of thought, Were it covalent, two atoms would need to get close for “sparks to fly” (bond forming), however this proximity would result in a highly unfavourable screaming Lenard-Jones (attraction-repulsion) term of the force-field, which would have causes issues with the calculations. But this does not apply.

Consequently, the only constraints you need is between the ketone and the acid / conjugate acid and the proton and the base / conjugate base. This however, may have protonation issues —GLU is glutamate, while GLU:protonated (patch) is glutamic acid and getting the wrong one will cause repulsions. I do not know how to declare patches not in pyrosetta and I have been ultra lazy in the past and used a nnca for protonated glutamic acid.

So you could simply do protein-protein docking with constraints without a preceeding step. Do note however, that constraints apply to full-atoms and centroid differently and the centroid residue params (in the database) differ —most amino acids have a weird beta-carbon only as a sidechain. I mention that, because protein-protein docking has a centroid step.

However, if you do protein ligand docking first, you can constrain the position of more atoms and better place the ACP and reduce substantially the freedom of movement of the protein-protein docking (more human time, less CPU time).

4.

“Eats, leaves and shoots” — sorry for my bad punctionation. I meant a constraint for the omega carbon and a separate one for the sulfur of the enzyme’s cysteine —not applicable as mentioned.

The omega carbon constraint may be good because from your question I am guessing the acyl was not going in the active site. So a rough constraint (WELL or BOUNDED_HARMONIC) would be good.

1.

I meant using ACP and enzyme from different species may not have any effect on activity despite requiring a protein-protein interaction.

I do not remember the paper, but there is one where wet lab folk did experiments with E. coli ACP and probably something like B. subtilis ACP and there was not a difference in activity of a target E. coli enzyme. I have the feeling it was in vivo only —damn wet lab folk! This could mean that either ACPs from different bacterial phyla are ultra-conserved on their surface or that the ACP–enzyme interface is weak. I do not remember any papers that did DLS or similar to get a K_D for it. Caveat: I was reading about this five years ago as I was worried that an enzyme from a different bacteria phyla may not work in E. coli —I changed jobs subsequently so I don’t know if it did! I did not do a sequence alignment of ACPs to see conservation either. But I was baffled by the fact that the scarce info about the location of the Fab pathway suggests it is not membrane bound —odd, right?—, making me really confused about this. But the takehome message is that there is a chance the ACP-enzyme interface is very weak.

2.

Docking is defined with a nifty A_B schema. You can have AC_Z say. But I would keep it simple and have everything of the ACP in a single chain ID. After all this is what PDB or CCP4 recomends for cofactors.

5.

Histidine tautomerisation is weird as its basically treated like a different rotamer, so whereas you can force delta protonation by mutating to HIS_D, this does not guarantee it won’t flip back. I actually asked a question myself about it a few months ago.

It’s an interesting conversation, so no thankses needed.

Ah, I follow. So, like the cysteine in the synthase at the beginning of this thread, pushing the attacking atom close to model right when “sparks fly” can have some weird effects due to the strain of the restraint. So I’d say AtomPair OD2 281A C6 36B HARMONIC 3.5 0.2 at first and only during refinement of the docked complex you can push it.

Keeping the active site residues restrained is not at all a bad idea —after all, where they to move, one’d repeat it—, however, histidine and asparagine may be the wrong way round in crystal structures, so it’d require some manual inspection to be certain that they are flipped correctly.

Protein protein docking makes sense, but I’ll think aloud once more though.

So if it is a ping-pong reaction there would be four steps:

1. pre-reaction complex, with proper protonations, between your acyl-ACP and your enzyme
2. tetrahedral chiral hemi-(thio)actal intermediate `acyl-C(-O) (S-ACP)(C()-ASP)`
3. acyl-aspartate with no ACP 
4. tetrahedral achiral hemi-actal intermediate (following an attack by hydroxylate ion)
5. product

Step 5 is the easiest to model, followed by 3. If you are after making an exogenous ACP more native, hence the protein-protein then 1 is the best model… However…If you are designing specificity for a different ligand then #3 would be the better starting choice —without worrying about ACP (which without acyl group may even bind weakly). However, it assumes that the mechanism is correct —playing devil’s advocate, say, why isn’t your mechanism simply protonation of thioester’s oxygen (via a protonated aspartic acid residue) and deprotonation of water via a histidine whose other ring nitrogen is protonated and hydrogen bonding with another aspartate/glumatate or maybe asparagine/glutamine. In which case the protein-protein docking is still better.

EDIT: the word flexible baffled me and I just realised a possible misunderstanding. I don’t think you meant to refer to it —I mean it did not even cross my mind—, but just in case flexible peptide docking is a for a short peptide in a groove that is calculated from scratch and has nothing to do with this.