Journal:Protein Science:3

Torpedo californica acethylcholinesterase is stabilized by binding of a divalent metal ion to a novel and versatile 4D motif

Israel Silman, Valery L. Shnyrov, Yacov Ashani, Esther Roth, Anne Nicolas, Joel L Sussman, and Lev Weiner ^[1]

Molecular Tour
Metal ions are often involved in catalytic functions in proteins, but may also serve to stabilize them. Thus, Torpedo californica acetylcholinesterase (TcAChE) is strongly stabilized against thermal denaturation by the divalent cations, Mg⁺², Mn⁺², and Ca⁺². Solution of the crystal structures of the complexes of TcAChE with Mg⁺² and Ca⁺². revealed that stabilization is achieved by binding of the divalent metal ions to a cluster of carboxylate groups of four aspartates that has been called a 4D motif. The complex contains, in addition, several water molecules, and while the metal ions bind directly to two of the Asp carboxylates, they bind to the other two indirectly, via waters.

The 4D motif is a novel motif, which has not been described before. The ASSAM server ^[2], which identifies structural motifs in proteins, revealed that many other proteins contain the 4D motif, and in a substantial percentage of them solution of their crystal structures reveals one of the three metal ions referred to above, or also Zn⁺². Whereas in TcAChE the 4D motif contains a single divalent ion, together with the waters, in some such complexes two or three metal ions are seen. The 4D motif is thus a versatile motif with respect to the number of ions and waters that it contains.

4D motif in TcAChE:

• (PDB code 1ea5)^[3].
• (PDB code 7b38)^[1].
• (PDB code 7b8e)^[1].
• (PDB code 7b2w)^[1].

The 4 Asp residues, D326, D389, D392 and D393, are shown as sticks, with carbons in green, oxygens in red, and nitrogens in blue. Solvent waters are shown as blue spheres, and the metal ions as magenta spheres, with their sizes proportionate to their Van der Waals radii; the oxygens of the uranyl moiety are shown as red spheres. Non-covalent hydrogen bonds and ionic bonds are shown as dashed white lines.

. Ribbon diagram of the Mg⁺²/TcAChE complex. The representation shows the entire structure, with the , residues 4-305 in cyan, and the , residues 306-535, in red. It is oriented looking into the active-site gorge, with , in the peripheral anionic site (PAS), at the top of the gorge, and , in the catalytic anionic site (CAS) towards the back, adjacent to . All these residues are depicted as sticks. The , against which the D4 pocket is glued, is in grey, and the two helices that contribute to the 4-helix bundle of the dimer, D365-Y375 and V518-T535, are in yellow. , showing the interactions of the active site, the D4 pocket, and the conserved water, shown as an orange sphere. The in the 4D pocket is in magenta, and is surrounded by in blue. A H-bonds with , of the 4D motif, with , in the catalytic triad, and with the main-chain nitrogen of , which, in turn, contributes to the CAS. This water which is homologous to water 623 in Koellner et al^[4], is shown as an orange sphere.

Fig. 7. Sequence alignments of residues 320-400 in several AChEs and in hBChE. The numbering used is that of TcAChE. Fully conserved residues are in white on a red background. The columns for the four residues corresponding to the 4D motif in TcAChE and zebrafish acetylcholinesterase are framed in green, and it can be seen that the motif is conserved only in these three AChEs. TcAChE, Torpedo californica AChE; TmAChE, Torpedo marmorata AChE; EeAChE, Electrophorus electricus AChE; DrAChE, Danio rerio AChE; BfAChE, Bungarus fasciatus AChE; HuAChE, human AChE; BoAChE, bovine AChE; MoAChE, mouse AChE; HdAChE, designed HuAChE, D4 variant^[5]; HuBChE, human BChE; AgAChE, Anopheles gambiae AChE; DmAChE, Drosophila melanogaster AChE.

Pocket in BfAChE that is homologous to the 4D pocket in TcAChE:

• Crystal structure (PDB code 4qww)^[6], .
• with the distal oxygens displayed as red and green balls, respectively.

4D motifs in four proteins retrieved from the ASSAM server:

• (PDB code 2yeq)^[7].
• (PDB code 4a01)^[8].
• (PDB code 4lfg)^[9].
• (PDB code 5xsp)^[10].

. All metal ions and waters are removed, and only the Asp residues are displayed in stick format. Apo TcAChE in green; Ca⁺²/TcAChE in red; Mg⁺²/TcAChE in blue; UO₂⁺²/TcAChE in yellow.

Overlays on Apo TcAChE of the 4D motifs of the four proteins retrieved from the ASSAM server:

• (PDB code 2yeq).
• (PDB code 4a01).
• (PDB code 4lfg).
• (PDB code 5xsp).

All metal ions and waters are removed, and only the Asp residues are displayed in stick format. Apo TcAChE is displayed as green sticks, and the retrieved proteins as red sticks, with the distal oxygens shown as green and yellow balls, respectively.

References

1. ↑ ^{1.0 1.1 1.2 1.3} Silman I, Shnyrov VL, Ashani Y, Roth E, Nicolas A, Sussman JL, Weiner L. Torpedo californica acetylcholinesterase is stabilized by binding of a divalent metal ion to a novel and versatile 4D motif. Protein Sci. 2021 Mar 8. doi: 10.1002/pro.4061. PMID:33686648 doi:http://dx.doi.org/10.1002/pro.4061
2. ↑ ASSAM Amino acid pattern Search for Substructures And Motifs
3. ↑ Dvir H, Jiang HL, Wong DM, Harel M, Chetrit M, He XC, Jin GY, Yu GL, Tang XC, Silman I, Bai DL, Sussman JL. X-ray structures of Torpedo californica acetylcholinesterase complexed with (+)-huperzine A and (-)-huperzine B: structural evidence for an active site rearrangement. Biochemistry. 2002 Sep 3;41(35):10810-8. PMID:12196020
4. ↑ Koellner G, Kryger G, Millard CB, Silman I, Sussman JL, Steiner T. Active-site gorge and buried water molecules in crystal structures of acetylcholinesterase from Torpedo californica. J Mol Biol. 2000 Feb 18;296(2):713-35. doi: 10.1006/jmbi.1999.3468. PMID:10669619 doi:http://dx.doi.org/10.1006/jmbi.1999.3468
5. ↑ Goldenzweig A, Goldsmith M, Hill SE, Gertman O, Laurino P, Ashani Y, Dym O, Unger T, Albeck S, Prilusky J, Lieberman RL, Aharoni A, Silman I, Sussman JL, Tawfik DS, Fleishman SJ. Automated Structure- and Sequence-Based Design of Proteins for High Bacterial Expression and Stability. Mol Cell. 2016 Jul 21;63(2):337-346. doi: 10.1016/j.molcel.2016.06.012. Epub 2016, Jul 14. PMID:27425410 doi:http://dx.doi.org/10.1016/j.molcel.2016.06.012
6. ↑ Bourne Y, Renault L, Marchot P. Crystal Structure of Snake Venom Acetylcholinesterase in Complex with Inhibitory Antibody Fragment Fab410 bound at the Peripheral Site: Evidence for Open and Closed States of a Backdoor Channel. J Biol Chem. 2014 Nov 19. pii: jbc.M114.603902. PMID:25411244 doi:http://dx.doi.org/10.1074/jbc.M114.603902
7. ↑ Rodriguez F, Lillington J, Johnson S, Timmel CR, Lea SM, Berks BC. Crystal Structure of the Bacillus subtilis Phosphodiesterase PhoD Reveals an Iron and Calcium-Containing Active Site. J Biol Chem. 2014 Sep 12. pii: jbc.M114.604892. PMID:25217636 doi:http://dx.doi.org/10.1074/jbc.M114.604892
8. ↑ Lin SM, Tsai JY, Hsiao CD, Huang YT, Chiu CL, Liu MH, Tung JY, Liu TH, Pan RL, Sun YJ. Crystal structure of a membrane-embedded H+-translocating pyrophosphatase. Nature. 2012 Mar 28;484(7394):399-403. doi: 10.1038/nature10963. PMID:22456709 doi:10.1038/nature10963
9. ↑ Patskovsky, Y., Toro, R., Bhosle, R., Hillerich, B., Seidel, R.D., Washington, E., Scott Glenn, A., Chowdhury, S., Evans, B., Hammonds, J., Imker, H.J., Al Obaidi, N., Stead, M., Love, J., Poulter, C.D., Gerlt, J.A., Almo, S.C. Crystal Structure of Geranylgeranyl Diphosphate Synthase from Streptococcus Uberis 0140J To be published.
10. ↑ Wang F, He Q, Su K, Wei T, Xu S, Gu L. Structural and biochemical characterization of the catalytic domains of GdpP reveals a unified hydrolysis mechanism for the DHH/DHHA1 phosphodiesterase. Biochem J. 2018 Jan 5;475(1):191-205. doi: 10.1042/BCJ20170739. PMID:29203646 doi:http://dx.doi.org/10.1042/BCJ20170739