The structure of the complex between the kinase CheA and response regulator CheY has been solved. Two of the pertinent structures are 1eay (2.0 Angstrom structure by the Dahlquist group) and 1a0o (2.95 Angstrom structure by the Samana group). The 1eay contains four polypeptide chains and the inspection of the PDB file revelas that the chains A and B correspond to two molecules of CheY while the chains C and D define the P2 domain of CheA. Further reading of the PDB file reveals that 1eay has a number of missing side-chain atoms (Arg 19, Lys 26, Glu 118 in CheY-chainB, and Lys 168 and Glu 224 in CheA-chainD). Missing aroms of charged sidechains present a serious problem for APBS, as we will see later. Reading the PDB file 1a0o reveals that this structure consists of eight chains: A, C, E, and G for CheY, and B, D, F, H for CheA. Further reading of file 1a0o shows that all CheY residues are defined but some residues of CheA have missing atoms. The best scenario involves chains D and H for which only the residue Ile 203 lacks its methyl groups. It seems that 1a0o is a better structure for the electrostatic potential analysis despite its lower resolution. We will generate the potential for the bound CheY first.
Inspect the structure. Notice that the negatively charged P2 domain of CheY is indeed bound to the positively charged surface crevice of CheY. Recall from our previous analysis that residues Lys 92, Lys119, Lys122, and Lys 126, and Arg 73 were likely to be involved in binding. Let's visualize these along with their interaction partners:
Zoom out (right mouse button) and adjust the slab (mouse wheel) so that the interface is clearly visible. Inspect the structure. Notice that two glutamic acid residues (Glu 171 and Glu 217) in CheA make direct contacts with lysines in CheY. In addition, Glu 177 and Glu 178 of CheA points to a positively charged pocket near Lys 119 of CheY. Examine the binding pocket of Phe 214. This hydrophobic residue is bound to a positively charged pocket such that the pi-electron cloud of the phenyl ring is near the -NH3+ of Lys119. This could well be an example of a cation-Pi interaction.
We will now show the electrostatic potential surface for CheA. Recall that the residue Ile 203 lacks the methyl groups. This is a problem for APBS, which gets easily confused by non-standard residues (although newer plugins try to implement clever solutions). The crudest solution to "get the work done" is to delete the offending residue altogether. However, deleting a residue in PyMOL will create new amino terminus and a new carboxy terminus, which are charged and will certainly distort the electrostatic potential surface. In other words, we do not want to model isoleucine as an ion pair between residues 202 and 204. As a rule of thumb, one should not simply delete residues to get the potential energy surface. The next best approach, used in programs such as UCSF Chimera, is to truncate strange residues to alanine. Another alternative is to take advantage of PyMOL's mutagenesis wizard and recreate the Ile residue with all the atoms. We will later see that often the bestsolution is to use the PDB2PQR web server that performs the automatic reconstruction of the molecule and optimization of the H-bonding network when adding hydrogens. For now, we will mutate the "bad" Ile 203 into a "good" Ile 203 with PyMOL.
The view we have created suggests that CheA has a complementary negatively charged surface region and the binding of these two proteins is largely driven by charge-charge attractions.