The genes for AChE and BChE encode the proteins responsible for enzyme activity. Additional gene products, PRiMA and PRaD, anchor AChE and BChE proteins into membranes. Soluble AChE and BChE tetramers are composed of 4 identical subunits plus one polyproline-rich peptide. Dilution does not release the polyproline-rich peptide from tetramers. However, protein denaturation, for example heating in a boiling water bath, dissociates the polyproline-rich peptide. Using mass spectrometry to sequence peptides released from soluble AChE and BChE tetramers, we find sequences that correspond to proline-rich regions from a variety of proteins. A typical peptide sequence contains 20 consecutive prolines in a 23-residue peptide LPPPPPPPPPPPPPPPPPPPPLP. There is no single, common consensus sequence i.e., no specific gene appears to be responsible for the polyproline-rich peptides found in soluble AChE and BChE tetramers. We propose that during metabolic turnover, protein fragments containing polyproline-rich sequences are scavenged by AChE and BChE dimers, to make stable AChE and BChE tetramers. The 40-residue, alpha-helical C-terminus of AChE or BChE is the tetramerization domain that binds the polyproline-rich peptide. Four parallel alpha helices wrap around a single antiparallel polyproline peptide to lock the tetramer in place. This organization was established by classical X-ray crystallography for isolated C-termini in complex with a proline-rich peptide. The organization was confirmed for intact, tetrameric BChE using cryoelectron microscopy. When 40 amino acids are deleted from the carboxy terminus, monomeric enzymes are created that retain full enzymatic activity.
It appears that homodimers of dimers in BuChE tetramers are held by tight reversible interactions of the C-terminus dominated by hydrophobic interactions of polyproline peptides that tolerate a degree of promiscuity. Monomers of dimers, however, appear to be held by covalent disulfide bonds (very C-terminal) and by hydrophobic interactions of the four helix bundle (located slightly less C-terminal), in addition to polyproline interactions.
1) Would you expect that disruption in the four helix bundles of dimers could lead to sufficient structural destabilization and eventual dissociation of tetramers ?
In our work with human AChE we are finding the four helix bundle interaction to be sensitive to distortion of the acyl pocket triggered by covalent binding of some OPs. This leads to dissociation of hAChE dimer truncated at the C-terminus where only hydrophobic interactions of the four helix bundle and no covalent bonds and no polyproline peptides are involved in stabilization. Assuming that hAChE forms tetramers similar to hBuChE we are wondering whether dissociation that we observed could have direct physiological significance in the nervous tissue?
2) Monomers of hBuChE in some of crystallographic homodimers of hBChE (for example PDB ID : 1P0I) appear to associate with different four helix bundle interaction interface compared to CryoEM dimers in the tetramer. The four helix bundle looks different and less optimally associated compared to CryoEM. Would you agree that difference comes from different extent of glycosylation in two structures, partial in X-ray structure and complete in CryoEM?
Dear Dr. Zoran Radic,
You found that truncated recombinant AChE is a dimer linked through a 4 helix bundle, and that organophosphates disrupt interactions in the 4 helix bundle. This caused the truncated AChE dimer to dissociate to monomers. You posed the possibility that the 4 helix bundle in BChE tetramers could also be disrupted by covalent binding of OP. If that were to happen, the tetramer would dissociate into dimers linked by the C-terminal disulfide bond, assuming that the polyproline interactions with the C-terminal alpha helices are weaker than the interactions in the 4 helix bundle. I discussed this possibility with my collaborator, Dr. Patrick Masson. We agree that BChE tetramers covalently bound to soman or DFP do not dissociate. I interpret this to mean that the polyproline interactions are stronger than the 4 helix bundle interactions. The polyproline interactions are the driving force for stabilizing the tetramer.
Your question raised additional questions for me. I wondered why boiled BChE tetramers do not behave like dimers on a native gel. We know that boiling releases polyproline from BChE tetramers. Without polyproline the BChE should be a disulfide linked dimer. However, boiled BChE behaves like a distorted tetramer on a native gel. One possibility to test is that the polyproline reunites with the boiled BChE.
Your second question was about the orientation of the 4 helix bundle, which is slightly different in truncated recombinant BChE X-ray structures compared to the Cryo-EM structure of full-length native BChE tetramers. I agree with your suggestion that the difference could be attributed to incomplete glycosylation of recombinant BChE.
Thank you for your comments.
Oksana Lockridge
