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Containing Catalytic Triads with Atomic-Level Accuracy

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Containing Catalytic Triads with Atomic-Level Accuracy ARTICLE PUBLISHED ONLINE: 6 APRIL 2014 | DOI: 10.1038/NCHEMBIO.1498 Design of activated serine–containing catalytic triads with atomic-level accuracy Sridharan Rajagopalan1,8, Chu Wang2,8, Kai Yu1, Alexandre P Kuzin3, Florian Richter1,4,7, Scott Lew3, Aleksandr E Miklos5, Megan L Matthews2, Jayaraman Seetharaman3, Min Su3, John F Hunt3, Benjamin F Cravatt2 & David Baker1,4,6* A challenge in the computational design of enzymes is that multiple properties, including substrate binding, transition state stabilization and product release, must be simultaneously optimized, and this has limited the absolute activity of successful designs. Here, we focus on a single critical property of many enzymes: the nucleophilicity of an active site residue that initiates catalysis. We design proteins with idealized serine-containing catalytic triads and assess their nucleophilicity directly in native biological systems using activity-based organophosphate probes. Crystal structures of the most successful designs show unprecedented agreement with computational models, including extensive hydrogen bonding networks between the catalytic triad (or quartet) residues, and mutagenesis experiments demonstrate that these networks are critical for serine activation and organophosphate reactivity. Following optimization by yeast display, the designs react with organophosphate probes at rates comparable to natural serine hydrolases. Co-crystal structures with diisopropyl fluorophosphate bound to the serine nucleo- phile suggest that the designs could provide the basis for a new class of organophosphate capture agents. erine hydrolases use a conserved nucleophilic serine to hydro- of the most intrinsically nucleophilic amino acids, and its inherent lyze ester, amide or thio-ester bonds in proteins and small reactivity can be enhanced without specific interactions with sup- molecules and constitute one of the largest enzyme families in porting residues in the enzyme’s active site. Compared to cysteine S 1 12 the human proteome . The canonical serine-histidine–aspartic acid and lysine, which was used in de novo designed retroaldolases , catalytic triad mechanism has been well studied: the serine Oγ is serine has a much higher pKa at ~13, and hence its nucleophilicity the nucleophile, the imidazole ring of the histidine acts as a general typically depends on interactions with activating residues such as acid/base, and the carboxylate of the aspartic acid orients the the histidine and the aspartate or glutamate of the catalytic triad. imidazole ring and neutralizes the charge developed on the histidine It is therefore methodologically more challenging to design serines in the transition state. In addition to the catalytic triad residues, a with heightened nucleophilicity: success most likely depends on Nature America, Inc. All rights reserved. America, Inc. Nature highly conserved oxyanion-binding site, commonly referred to as accurate designed hydrogen bonding interactions with an activating 4 the oxyanion hole, stabilizes the negative charge on the carbonyl residue (or residues). oxygen of the tetrahedral intermediate. The peptide backbone NH Here we take on the challenge of designing active sites with © 201 groups stabilize these high-energy species in most cases2–7. The serines with nucleophilicity approaching that found in the catalytic activated nucleophilic serine can be covalently modified by electro- triads of native hydrolases. De novo designed proteins with activated philic organophosphate compounds, resulting in the loss of catalytic serine nucleophiles could be useful in their own right as scavengers npg activity. This has inspired the development of fluorophosphonates of organophosphate nerve agents. We use computational methods as activity-based probes for serine hydrolases, which enable moni- to design proteins with idealized serine-containing catalytic triads toring of the activity of many serine hydrolases in parallel directly in and take advantage of activity-based fluorophosphonate probes13 to complex proteomes by the activity-based protein profiling (ABPP) screen and assess the serine nucleophilicity of these designs directly technology8,9. in native cellular systems. To make progress in enzyme design, it is useful to focus on critical aspects of catalysis (in this case, the active site nucleophile RESULTS that initiates catalysis) independent of full multiple turnover reaction Computational design of catalytic triad active sites cycles, whose optimization requires many complex tradeoffs; such a We selected a fluorophosphonate-reactive group (Fig. 1a) as the reduction in complexity can facilitate the solution of very challenging model organophosphate substrate because it provides (i) a suitably problems. Recently, we designed esterases with a cysteine-histidine tempered electrophile to test the serine nucleophilicity of protein dyad and an oxyanion hole in the active site10. Though these designs designs and (ii) a mimic of the tetrahedral transition state that occurs contained active site cysteines with heightened nucleophilicity11 and during serine hydrolase catalysis. From a technical perspective, were catalytically active, crystal structures revealed that the active reporter-tagged fluorophosphonates should provide a straightfor- site histidine residues, which were intended to activate the cysteine ward way to screen enzyme designs in complex proteomes, as we nucleophiles, were not properly positioned10. Catalytic activity was have shown for other ABPP probes11. Four transition state models most likely achieved because cysteine, with a pKa around 8, is one corresponding to syn and anti attack for two fluorophosphonate 1Department of Biochemistry, University of Washington, Seattle, Washington, United States. 2The Skaggs Institute for Chemical Biology and Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California, United States. 3Northeast Structural Genomics Consortium, Department of Biological Sciences, Columbia University, New York, New York, USA. 4Graduate Program in Biological Physics, Structure and Design, University of Washington, Seattle, Washington, USA. 5EXCET, Inc., Springfield, Virginia, USA. 6Howard Hughes Medical Institute, University of Washington, Seattle, Washington, USA. 7Present address: Institute of Biology, Humboldt-Universität zu Berlin, Germany. 8These authors contributed equally to this work. *e-mail: [email protected] 386 NATURE CHEMICAL BIOLOGY | VOL 10 | MAY 2014 | www.nature.com/naturechemicalbiology Nature cheMIcal BIOlOGY DOI: 10.1038/NCHEMBIO.1498 ARTICLE a b Figure 1 | Design strategy. (a) Transition state F F models. The fluorophosphonate (FP) ligand Oγ Ser O (ethyl ethylphosphonofluoridate) used in design O2 O1 O2 O1 calculations is in the middle panel flanked by Oγ N Oxyanion Ser H transition state models corresponding to syn and O2 hole FP1 FP3 anti attack by the serine (purple) nucleophile Oγ (S, syn) P (S, anti) O Ser on the two isomers (R and S) of the ligand with F O1 F F P O the leaving atom fluorine (F) in blue spheres. O O Ser O1 O1 F H (b) Theozyme geometry. Geometric parameters H O Oγ O2 NN were derived from the computed ideal active O2 Oγ Asp/Glu site geometries and native hydrolase statistics Ser FP2 previously reported30. Theozymes with the FP4 His (R, syn) (R, anti) histidine hydrogen bonding reversed were also considered during RosettaMatch (Online c d Methods). (c,d) Design models of OSH55 (c) and 1.9 OSH98 (d). Active site residues of OSH55 and 2.0 2.0 2.0 OSH98 are shown in orange and white sticks, 1.9 respectively. The modeled fluorophosphonate H146 3.0 2.9 S50 ligand is shown in yellow sticks. In both designs, H51 multiple backbone NHs form the oxyanion hole S151 2.9 2.9 to stabilize the transition state. (e,f) Selective D75 FP-Rh labeling of OSH55 (e) and OSH98 (f) E6 but not the active site serine knockouts. Serine reactivity was assessed by gel-based ABPP (top, in-gel fluorescence; bottom, Coomassie e f blue (CB) staining; full-size gel images are in OSH55 H146A S151A E6A OSH98 D75A S50A Supplementary Fig. 15). The experiments were H51A performed in duplicate with consistent results. FP-Rh FP-Rh CB isomers (R and S) were generated using CB bond angles and bond lengths obtained from quantum mechanics/molecular mechanics (QM/MM) (PDB) code 2DX6; Supplementary Fig. 3). Mutagenesis of the calculations (Fig. 1a and Supplementary Results, Supplementary designed active site serine and histidine residues to alanine indicated Table 1)14. Ideal active sites with the transition state model, a that probe labeling was specific to the designed serines (Fig. 1e,f). serine-histidine-aspartate/glutamate catalytic triad, and a backbone OSH55 was chosen for further characterization because of its small Nature America, Inc. All rights reserved. America, Inc. Nature oxyanion hole in orientations optimal for catalysis (Fig. 1b) were size (163 amino acids) and the stability of the starting scaffold. 4 built as described in the Online Methods. We used RosettaMatch to In the design model, OSH55 has two backbone amide protons search for the placement of these ‘theozymes’ in a set of 800 protein contributing to the oxyanion hole (Fig. 1c). © 201 scaffolds15, none of which contained an existing catalytic triad– based active site. The residues surrounding the matched sites Characterization and optimization of OSH55 were then optimized by RosettaDesign16 to stabilize the active site We solved the structure of OSH55 by crystallography at 2.3-Å reso- npg residues, increase affinity
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