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. 2010 Sep;19(9):1760-73.
doi: 10.1002/pro.462.

Evaluation and ranking of enzyme designs

Gert Kiss  1 , Daniela Röthlisberger, David Baker, K N Houk
Affiliations

Evaluation and ranking of enzyme designs

Gert Kiss et al. Protein Sci. 2010 Sep.

Abstract

In 2008, a successful computational design procedure was reported that yielded active enzyme catalysts for the Kemp elimination. Here, we studied these proteins together with a set of previously unpublished inactive designs to determine the sources of activity or lack thereof, and to predict which of the designed structures are most likely to be catalytic. Methods that range from quantum mechanics (QM) on truncated model systems to the treatment of the full protein with ONIOM QM/MM and AMBER molecular dynamics (MD) were explored. The most effective procedure involved molecular dynamics, and a general MD protocol was established. Substantial deviations from the ideal catalytic geometries were observed for a number of designs. Penetration of water into the catalytic site and insufficient residue-packing around the active site are the main factors that can cause enzyme designs to be inactive. Where in the past, computational evaluations of designed enzymes were too time-extensive for practical considerations, it has now become feasible to rank and refine candidates computationally prior to and in conjunction with experimentation, thus markedly increasing the efficiency of the enzyme design process.

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Figures

Scheme 1
Scheme 1
The base-catalyzed Kemp elimination of 5-nitrobenzisoxazole, depicted with hydrogen-bond donor for stabilization of the developing negative charge in the transition state.
Figure 1
Figure 1
(a) TS in cluster model 1 (CM1). (b) Cluster model 2 (CM2); sticks computed with DFT, lines computed with PM3MM. Both (a) and (b) of KE07. Backbone heavy atoms (circled in CM1) were constrained. An interactive view is available in the electronic version of the article. PRO462 Figure 1
Figure 2
Figure 2
Full enzyme QM/MM from 2ns MD (here: KE07); explicit waters within 10 Å of substrate were retained; QM layer (sticks in inset) was treated with DFT; MM layer with the AMBER force field. A movie of the computed reaction path is available as part of the SI. An interactive view is available in the electronic version of the article. PRO462 Figure 2
Scheme 2
Scheme 2
Schematic representation of the enzyme design process in terms of potential energy and conformational space.
Figure 3
Figure 3
Overlay of representative MD geometry (blue) and crystal structure (yellow). (a) Active site of antibody 34E4 with backbone RMSD of 0.74 Å (σ = 0.30 Å) and all atom RMSD of 1.26 Å (σ = 0.25 Å). (b) Active site of design KE07/1thf with backbone RMSD of 0.67 Å (σ = 0.17 Å) and all atom RMSD of 1.15 Å (σ = 0.16 Å). The positions of two ordered active site water molecules that were co-crystallized in 2RKX (spheres) are also very well reproduced with MD. An interactive view is available in the electronic version of the article. PRO462 Figure 3
Figure 4
Figure 4
Angle versus distance scatter plots. (a) Cys–His contact and (b) His–Asn contact of the naturally evolved cathepsin K catalytic triad; (c) substrate-His contact and (d) His–Asp contact of the active design KE70; (e) substrate-His contact and (f) His–Glu contact of the inactive design KE38. Data points are from 20 ns MD. The individual distributions are projected onto the axes. The three hydrogen bond categories of Table II are outlined with dashes.
Figure 5
Figure 5
Design versus MD. Schematic representation of the catalytic unit (a and c) and representative MD geometry (blue) over Rosetta design geometry (black with orange substrate) (b and d). Bond labels in (a) and (c) are maxima of distance distributions with FWHMs in parentheses. All values in Å. The backbone RMSD of the catalytic unit of KE70 and KE38 is 0.57 and 0.76 Å; the sidechain RMSD is 0.95 and 2.24 Å, respectively. The inset in (d) shows Glu170 in direct contact with seven water molecules. An interactive view is available in the electronic version of the article. PRO462 Figure 5
Figure 6
Figure 6
Water coordination numbers from MD at d < 3.2 Å. Asn182 in the naturally evolved cathepsin K, GluH50 in the catalytic antibody 34E4, Asp44 in the active KE70/1jcl, and Glu170 in the inactive KE38/1lbm. Note: histograms are scaled to the same height.
Figure 7
Figure 7
The entire dataset. (a) Sidechain versus backbone RMSDs of active sites. (b) Angles versus distances of the catalytic H-bond contacts.
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