Journal:CHEMBIOINT:1

The four-helix bundle in cholinesterase dimers: structural and energetic determinants of stability

Dana A. Novichkova, Sofya V. Lushchekina, Orly Dym, Patrick Masson, Israel Silman and Joel L. Sussman ^[1]

Molecular Tour
The crystal structures of truncated forms of cholinesterases provide good models for assessing the role of non-covalent interactions in dimer assembly in the absence of cross-linking disulfide bonds. These structures identify the four-helix bundle that serves as the interface for formation of acetylcholinesterase and butyrylcholinesterase dimers. Here we performed a theoretical comparison of the structural and energetic factors governing dimerization. This included identification of inter-subunit and intra-subunit hydrogen bonds and hydrophobic interactions, evaluation of solvent-accessible surfaces, and estimation of electrostatic contributions to dimerization. To reveal the contribution to dimerization of individual amino acids within the contact area, free energy perturbation alanine screening was performed. Markov state modelling shows that the loop between the α13 and α14 helices in BChE is unstable, and occupies 4 macro-states. The order of magnitude of mean first passage times between these macrostates is ~10-8 seconds. Replica exchange molecular dynamics umbrella sampling calculations revealed that the free energy of human BChE dimerization is -15.5 kcal/mol, while that for human AChE is -26.4 kcal/mol. Thus, the C-terminally truncated human butyrylcholinesterase dimer is substantially less stable than that of human acetylcholinesterase.

: pink, hAChE (PDB ID 4ey4); gray, mAChE (PDB ID 1j06); blue, TcAChE (PDB ID 1w76); green, DmAChE (PDB ID 1qo9). .

All crystal structures of AChE deposited in the PDB (https://www.rcsb.org) display dimers in which the monomers associate through an antiparallel four-helix bundle. With respect to BChE, crystal structures have been obtained only for monomeric forms of hBChE. In the first hBChE crystal structure obtained (PDB ID 1p0i), of partially glycosylated enzyme expressed in CHO cells, dimers assembling via a . Subsequently, however, a (PDB ID 4aqd) was obtained for fully glycosylated hBChE expressed in Drosophila S2 cells. This dissimilarity of the two crystal structures reveals the high sensitivity of hBChE dimerization to the expression system and crystallization conditions.

In silico alanine screening (FEP method) for hAChE. . The amino acids, which substitution by alanine led |ΔΔG|>1 kcal/mol are shown and colored in green or red. . These scenes show that in the case of hAChE, stabilization occurs with both hydrophobic residues of helices, and the salt bridge, and electrostatic interactions on helices. Mutations of His381 and Phe531 to alanine are advantageous. , and their interactions appear to be stabilizing.

In silico alanine screening (FEP method) for BChE. . The amino acids, which substitution by alanine led |ΔΔG|>1 kcal/mol are shown and colored in green or red. . BChE has less stabilizing amino acids in loops compared to hAChE. Amino acids unfavorable for BChE dimerization are 1) polar ones (i.e., serine) instead of hydrophobic in helices 2) not exposed to the contact interface (Leu370) 3) Asp375 and Asp378, which repel each other and destabilize the loop. His372 in BChE (analog of His381 in hAChE) forms hydrogen bond with Gln518, and is more buried from the dimerization interface, thus it does not have the destabilizing effect observed in hAChE. Generally, in silico alanine screening shows that hAChE has less amino acid residues with high contribution to both stabilization and destabilization of the dimer, rather than BChE. In spite of a few residues on helices of BChE, extrahelical loops contribute to destabilization of the dimer.

. Red loop — first macrostate, blue loop — second macrostate, green loop — third macrostate, yellow loop - forth macrostate. .

Pathways between macrostates of BChE 374-382 loop with the percentage of paths. In gray boxes mean first passage times. The more populated the macrostate, the larger the size of the circle.

BChE 374-382 loop:

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This study was supported by Russian Foundation for Basic Research (project №19-03-00043).

References

1. ↑ Novichkova DA, Lushchekina SV, Dym O, Masson P, Silman I, Sussman JL. The four-helix bundle in cholinesterase dimers: Structural and energetic determinants of stability. Chem Biol Interact. 2019 Jun 13. pii: S0009-2797(19)30450-8. doi:, 10.1016/j.cbi.2019.06.012. PMID:31202688 doi:http://dx.doi.org/10.1016/j.cbi.2019.06.012