5hq3: Difference between revisions

Latest revision as of 10:50, 16 August 2023

Stable, high-expression variant of human acetylcholinesterase

Structural highlights

5hq3 is a 2 chain structure with sequence from Homo sapiens. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance.
Method:	X-ray diffraction, Resolution 2.6Å
Ligands:	MES, VX
Resources:	FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT

Function

ACES_HUMAN Terminates signal transduction at the neuromuscular junction by rapid hydrolysis of the acetylcholine released into the synaptic cleft. Role in neuronal apoptosis.^[1] ^[2] ^[3] ^[4]

Publication Abstract from PubMed

Upon heterologous overexpression, many proteins misfold or aggregate, thus resulting in low functional yields. Human acetylcholinesterase (hAChE), an enzyme mediating synaptic transmission, is a typical case of a human protein that necessitates mammalian systems to obtain functional expression. We developed a computational strategy and designed an AChE variant bearing 51 mutations that improved core packing, surface polarity, and backbone rigidity. This variant expressed at approximately 2,000-fold higher levels in E. coli compared to wild-type hAChE and exhibited 20 degrees C higher thermostability with no change in enzymatic properties or in the active-site configuration as determined by crystallography. To demonstrate broad utility, we similarly designed four other human and bacterial proteins. Testing at most three designs per protein, we obtained enhanced stability and/or higher yields of soluble and active protein in E. coli. Our algorithm requires only a 3D structure and several dozen sequences of naturally occurring homologs, and is available at https://pross.weizmann.ac.il.

Automated Structure- and Sequence-Based Design of Proteins for High Bacterial Expression and Stability.,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 Mol Cell. 2016 Jul 21;63(2):337-346. doi: 10.1016/j.molcel.2016.06.012. Epub 2016, Jul 14. PMID:27425410^[5]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

References

↑ Chhajlani V, Derr D, Earles B, Schmell E, August T. Purification and partial amino acid sequence analysis of human erythrocyte acetylcholinesterase. FEBS Lett. 1989 Apr 24;247(2):279-82. PMID:2714437
↑ Velan B, Grosfeld H, Kronman C, Leitner M, Gozes Y, Lazar A, Flashner Y, Marcus D, Cohen S, Shafferman A. The effect of elimination of intersubunit disulfide bonds on the activity, assembly, and secretion of recombinant human acetylcholinesterase. Expression of acetylcholinesterase Cys-580----Ala mutant. J Biol Chem. 1991 Dec 15;266(35):23977-84. PMID:1748670
↑ Shafferman A, Kronman C, Flashner Y, Leitner M, Grosfeld H, Ordentlich A, Gozes Y, Cohen S, Ariel N, Barak D, et al.. Mutagenesis of human acetylcholinesterase. Identification of residues involved in catalytic activity and in polypeptide folding. J Biol Chem. 1992 Sep 5;267(25):17640-8. PMID:1517212
↑ Yang L, He HY, Zhang XJ. Increased expression of intranuclear AChE involved in apoptosis of SK-N-SH cells. Neurosci Res. 2002 Apr;42(4):261-8. PMID:11985878
↑ 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:https://dx.doi.org/10.1016/j.molcel.2016.06.012

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OCA

[1] Chhajlani V, Derr D, Earles B, Schmell E, August T. Purification and partial amino acid sequence analysis of human erythrocyte acetylcholinesterase. FEBS Lett. 1989 Apr 24;247(2):279-82. PMID:2714437

[2] Velan B, Grosfeld H, Kronman C, Leitner M, Gozes Y, Lazar A, Flashner Y, Marcus D, Cohen S, Shafferman A. The effect of elimination of intersubunit disulfide bonds on the activity, assembly, and secretion of recombinant human acetylcholinesterase. Expression of acetylcholinesterase Cys-580----Ala mutant. J Biol Chem. 1991 Dec 15;266(35):23977-84. PMID:1748670

[3] Shafferman A, Kronman C, Flashner Y, Leitner M, Grosfeld H, Ordentlich A, Gozes Y, Cohen S, Ariel N, Barak D, et al.. Mutagenesis of human acetylcholinesterase. Identification of residues involved in catalytic activity and in polypeptide folding. J Biol Chem. 1992 Sep 5;267(25):17640-8. PMID:1517212

[4] Yang L, He HY, Zhang XJ. Increased expression of intranuclear AChE involved in apoptosis of SK-N-SH cells. Neurosci Res. 2002 Apr;42(4):261-8. PMID:11985878

[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:https://dx.doi.org/10.1016/j.molcel.2016.06.012

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[2]

[3]

[4]

[5]

@@ Line 1: / Line 1: @@
 ==Stable, high-expression variant of human acetylcholinesterase==
-<StructureSection load='5hq3' size='340' side='right' caption='[[5hq3]], [[Resolution|resolution]] 2.60&Aring;' scene=''>
+<StructureSection load='5hq3' size='340' side='right'caption='[[5hq3]], [[Resolution|resolution]] 2.60&Aring;' scene=''>
 == Structural highlights ==
-<table><tr><td colspan='2'>[[5hq3]] is a 2 chain structure with sequence from [http://en.wikipedia.org/wiki/Human Human]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=5HQ3 OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=5HQ3 FirstGlance]. <br>
+<table><tr><td colspan='2'>[[5hq3]] is a 2 chain structure with sequence from [https://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=5HQ3 OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=5HQ3 FirstGlance]. <br>
-</td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><scene name='pdbligand=MES:2-(N-MORPHOLINO)-ETHANESULFONIC+ACID'>MES</scene>, <scene name='pdbligand=VX:O-ETHYLMETHYLPHOSPHONIC+ACID+ESTER+GROUP'>VX</scene></td></tr>
+</td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 2.6&#8491;</td></tr>
-<tr id='gene'><td class="sblockLbl"><b>[[Gene|Gene:]]</b></td><td class="sblockDat">ACHE ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=9606 HUMAN])</td></tr>
+<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=MES:2-(N-MORPHOLINO)-ETHANESULFONIC+ACID'>MES</scene>, <scene name='pdbligand=VX:O-ETHYLMETHYLPHOSPHONIC+ACID+ESTER+GROUP'>VX</scene></td></tr>
-<tr id='activity'><td class="sblockLbl"><b>Activity:</b></td><td class="sblockDat"><span class='plainlinks'>[http://en.wikipedia.org/wiki/Acetylcholinesterase Acetylcholinesterase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.1.7 3.1.1.7] </span></td></tr>
+<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=5hq3 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=5hq3 OCA], [https://pdbe.org/5hq3 PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=5hq3 RCSB], [https://www.ebi.ac.uk/pdbsum/5hq3 PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=5hq3 ProSAT]</span></td></tr>
-<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=5hq3 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=5hq3 OCA], [http://pdbe.org/5hq3 PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=5hq3 RCSB], [http://www.ebi.ac.uk/pdbsum/5hq3 PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=5hq3 ProSAT]</span></td></tr>
 </table>
 == Function ==
-[[http://www.uniprot.org/uniprot/ACES_HUMAN ACES_HUMAN]] Terminates signal transduction at the neuromuscular junction by rapid hydrolysis of the acetylcholine released into the synaptic cleft. Role in neuronal apoptosis.<ref>PMID:2714437</ref> <ref>PMID:1748670</ref> <ref>PMID:1517212</ref> <ref>PMID:11985878</ref>
+[https://www.uniprot.org/uniprot/ACES_HUMAN ACES_HUMAN] Terminates signal transduction at the neuromuscular junction by rapid hydrolysis of the acetylcholine released into the synaptic cleft. Role in neuronal apoptosis.<ref>PMID:2714437</ref> <ref>PMID:1748670</ref> <ref>PMID:1517212</ref> <ref>PMID:11985878</ref>
 <div style="background-color:#fffaf0;">
 == Publication Abstract from PubMed ==
@@ Line 23: / Line 22: @@
 ==See Also==
 *[[Acetylcholinesterase 3D structures|Acetylcholinesterase 3D structures]]
-*[[Journal:Molecular Cell:1|Journal:Molecular Cell:1]]
+*[[Human Acetylcholinesterase|Human Acetylcholinesterase]]
 == References ==
 <references/>
 __TOC__
 </StructureSection>
-[[Category: Acetylcholinesterase]]
+[[Category: Homo sapiens]]
-[[Category: Human]]
+[[Category: Large Structures]]
-[[Category: Aharoni, A]]
+[[Category: Aharoni A]]
-[[Category: Albeck, S]]
+[[Category: Albeck S]]
-[[Category: Ashani, Y]]
+[[Category: Ashani Y]]
-[[Category: Dym, O]]
+[[Category: Dym O]]
-[[Category: Fleishman, S J]]
+[[Category: Fleishman SJ]]
-[[Category: Gertman, O]]
+[[Category: Gertman O]]
-[[Category: Goldenzweig, A]]
+[[Category: Goldenzweig A]]
-[[Category: Goldsmith, M]]
+[[Category: Goldsmith M]]
-[[Category: Hill, S E]]
+[[Category: Hill SE]]
-[[Category: Laurino, P]]
+[[Category: Laurino P]]
-[[Category: Lieberman, R L]]
+[[Category: Lieberman RL]]
-[[Category: Prilusky, J]]
+[[Category: Prilusky J]]
-[[Category: Silman, I]]
+[[Category: Silman I]]
-[[Category: Sussman, J L]]
+[[Category: Sussman JL]]
-[[Category: Tawfik, D S]]
+[[Category: Tawfik DS]]
-[[Category: Unger, T]]
+[[Category: Unger T]]
-[[Category: De novo protein]]
-[[Category: Design]]
-[[Category: Hydrolase]]

5hq3: Difference between revisions

Latest revision as of 10:50, 16 August 2023

Stable, high-expression variant of human acetylcholinesterase

Structural highlights

Function

Publication Abstract from PubMed

See Also

References

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