Automated computational design of human enzymes for high bacterial expression and stability
Adi Goldenzweig, Moshe Goldsmith, Shannon E Hill, Or Gertman, Paola Laurino, Yacov Ashani, Orly Dym, Tamar Unger, Shira Albeck, Jaime Prilusky, Raquel L Lieberman, Amir Aharoni, Israel Silman, Joel L Sussman, Dan S Tawfik and Sarel J Fleishman [1]
Molecular Tour
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. Using a novel computational strategy, we designed an AChE variant bearing 51 mutations that improved core packing, surface polarity, and backbone rigidity. This variant expressed at ~2,000-fold higher levels in E. coli compared to wild-type hAChE, and exhibited 20°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 protein in E. coli. Our algorithm requires only a 3D structure and several dozen sequences of naturally occurring homologues, and is available at http://pross.weizmann.ac.il.
. Wild type hAChE is shown in cyan and 51 mutated positions, which are distributed throughout dAChE4, are indicated by orange spheres.
Image:MC.png Eliminating potentially destabilizing mutations through homologous-sequence analysis and computational mutation scanning. Left: Sequence logo for hAChE position Gly416. The height of letters represents the respective amino acid’s frequency in an alignment of homologous AChE sequences. The evolutionarily ‘allowed’ sequence space (PSSM scores ≥0) at position 416 includes the 9 amino acids shown. Right: Structural models of mutations to the evolutionarily favored amino acid His, and to Gln, which is favored by Rosetta energy calculations. The His side chain is strained due to its proximity to the bulky Tyr504 aromatic ring, whereas the Gln side chain is relaxed and forms a favorable hydrogen bond with Tyr504 (dashed line)
Scenes highlight stabilizing effects of selected mutations (wild type hAChE is shown in cyan and mutant hAChE is in green.
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