Computational structure-based redesign of enzyme activity

Abstract

We report a computational, structure-based redesign of the phenylalanine adenylation domain of the nonribosomal peptide synthetase enzyme gramicidin S synthetase A (GrsA-PheA) for a set of noncognate substrates for which the wild-type enzyme has little or virtually no specificity. Experimental validation of a set of top-ranked computationally predicted enzyme mutants shows significant improvement in the specificity for the target substrates. We further present enhancements to the methodology for computational enzyme redesign that are experimentally shown to result in significant additional improvements in the target substrate specificity. The mutant with the highest activity for a noncognate substrate exhibits 1/6 of the wild-type enzyme/wild-type substrate activity, further confirming the feasibility of our computational approach. Our results suggest that structure-based protein design can identify active mutants different from those selected by evolution.

Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

“…A high-quality crystal structure of wild-type LplA in complex with lipoyl-AMP is available (16) and permitted us to explore the use of Rosetta (17) to computationally expand our search space for a resorufin ligase. In previous work, computation was used to alter the specificity of a deaminase enzyme for ammelide (a one-ring structure) instead of guanine (two rings) (18), a DNA endonuclease for altered sequence recognition (19), and an amino acid adenylation domain for leucine instead of phenylalanine (20). Our problem of engineering LplA to ligate resorufin instead of lipoic acid presents a significantly greater challenge owing to the large size and shape difference between the original and new substrates.…”

“…The cells were then rinsed in cold cacodylate buffer and postfixed with 1% osmium tetroxide on ice. After rinses in cold water, cells were either stained with 2% (wt/vol) aqueous uranyl acetate at 4°C overnight en bloc or directly dehydrated in cold ethanol series [20,50,70,90, and 100% (vol/vol)] for 3 min each on ice, followed by a room temperature rinse in 100% ethanol. Cells were embedded in Durcupan ACM resin (Electron Microscopy Sciences).…”

BWM*: A Novel, Provable, Ensemble-Based Dynamic Programming Algorithm for Sparse Approximations of Computational Protein Design

“…However, protein design is NP-hard (Kingsford et al, 2005), making algorithms that guarantee optimality expensive for larger designs where many residues are allowed to mutate simultaneously. Therefore, researchers have developed tractable approximations of the protein design problem to obtain provably good approximate solutions (Leach and Lemon, 1998;Roberts et al, 2012;Chen et al, 2009;Lilien et al, 2005;Georgiev and Donald, 2007;Donald, 2011, Smadbeck et al, 2014, or employed heuristic approaches to rapidly generate candidate solutions (Lee and Subbiah, 1991;Kuhlman and Baker, 2000;Jones, 1994;Desjarlais and Handel, 1995;Koehl and Delarue, 1994;Jiang et al, 2000;Donald, 2011). Heuristic sampling of sequences quickly generates locally optimal candidate sequences, whereas provable algorithms are guaranteed to return the global minimum energy conformation (GMEC).…”

“…Heuristic sampling of sequences quickly generates locally optimal candidate sequences, whereas provable algorithms are guaranteed to return the global minimum energy conformation (GMEC). However, algorithms that compute only the GMEC have been shown to overlook sequences with better binding affinity, because proteins exist as a thermodynamic ensemble and not just as a single lowenergy conformation Lilien et al, 2005;Chen et al, 2009). Provable, ensemble-based algorithms ameliorate this issue Lilien et al, 2005;Silver et al, 2013).…”

“…By explicitly modeling proteins as a thermodynamic ensemble of molecular conformations, osprey/K* has successfully designed sequences that have performed well both in vitro Chen et al, 2009;Frey et al, 2010;Rudicell et al, 2014;Georgiev et al, 2012;Gorczynski et al, 2007;Stevens et al, 2006) and in vivo Gorczynski et al, 2007;Rudicell et al, 2014;Frey et al, 2010), as well as in non-human primates . K* accomplishes this by using dead end elimination followed by A* (DEE/A*) (Leach and Lemon, 1998;Goldstein, 1994) to provably compute a gap-free list of conformations within an energy window E w of the GMEC, and provably approximate partition functions over molecular ensembles.…”