SummaryUpon 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 ∼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 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 http://pross.weizmann.ac.il.

As a result of an author oversight in the originally published version of this article, the Rosetta script file design.xml provided in Data S1 was missing the res_type_constraint value, causing code crash. The value appears in the script filterscan.xml that is used just before design.xml. We updated the file design.xml, which now includes the res_type_constraint value reported in the main text (0.4). In addition, a file called design_text had no use and was omitted in this update. The authors apologize for the error and any inconvenience that may have resulted.

Degradation of mutant Ndc10 is mediated by the E3 ligase Doa10 at the endoplasmic reticulum/nuclear envelope membrane. An autonomous degradation motif was localized to the C-terminal region of Ndc10. The motif is composed of two indispensable elements: a hydrophobic surface of an amphipathic helix and a loosely structured, hydrophobic C-terminal tail.

Mammalian SIRT6 is a well-studied histone deacetylase that was recently shown to exhibit high protein deacylation activity enabling the removal of long chain fatty acyl groups from proteins. SIRT6 was shown to play key roles in cellular homeostasis by regulating a variety of cellular processes including DNA repair and glucose metabolism. However, the link between SIRT6 enzymatic activities and its cellular functions is not clear. Here, we utilized a directed enzyme evolution approach to generate SIRT6 mutants with improved deacylation activity. We found that while two mutants show increased deacylation activity at high substrate concentration and improved glucose metabolism they exhibit no improvement and even abolished deacetylation activity on H3K9Ac and H3K56Ac in cells. Our results demonstrate the separation of function between SIRT6 catalytic activities and suggest that SIRT6 deacylation activity in cells is important for glucose metabolism and can be mediated by still unknown acylated cellular proteins.

No abstract