Computational design is a test of our understanding of enzyme catalysis and a means of engineering novel, tailor-made enzymes. While the de novo computational design of catalytically efficient ...enzymes remains a challenge, designed enzymes may comprise unique starting points for further optimization by directed evolution. Directed evolution of two computationally designed Kemp eliminases, KE07 and KE70, led to low to moderately efficient enzymes (k cₐₜ/ K ₘ values of ≤ 5 10 ⁴ M ⁻¹s ⁻¹). Here we describe the optimization of a third design, KE59. Although KE59 was the most catalytically efficient Kemp eliminase from this design series (by k cₐₜ/ K ₘ, and by catalyzing the elimination of nonactivated benzisoxazoles), its impaired stability prevented its evolutionary optimization. To boost KE59’s evolvability, stabilizing consensus mutations were included in the libraries throughout the directed evolution process. The libraries were also screened with less activated substrates. Sixteen rounds of mutation and selection led to > 2,000-fold increase in catalytic efficiency, mainly via higher k cₐₜ values. The best KE59 variants exhibited k cₐₜ/ K ₘ values up to 0.6 10 ⁶ M ⁻¹s ⁻¹, and k cₐₜ/ k ᵤₙcₐₜ values of ≤ 10 ⁷ almost regardless of substrate reactivity. Biochemical, structural, and molecular dynamics (MD) simulation studies provided insights regarding the optimization of KE59. Overall, the directed evolution of three different designed Kemp eliminases, KE07, KE70, and KE59, demonstrates that computational designs are highly evolvable and can be optimized to high catalytic efficiencies.
Evaluation and ranking of enzyme designs Kiss, Gert; Röthlisberger, Daniela; Baker, David ...
Protein science,
September 2010, Letnik:
19, Številka:
9
Journal Article
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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unpublished inactive designs to determine the sources of activity or lack thereof, and to predict which of the designed structures are most likely to be catalytic.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.
Interactive View Figure 1; Interactive View Figure 2; Interactive View Figure 3; Interactive View Figure 5
The creation of novel enzymes capable of catalyzing any desired chemical reaction is a grand challenge for computational protein design. Here we describe two new algorithms for enzyme design that ...employ hashing techniques to allow searching through large numbers of protein scaffolds for optimal catalytic site placement. We also describe an in silico benchmark, based on the recapitulation of the active sites of native enzymes, that allows rapid evaluation and testing of enzyme design methodologies. In the benchmark test, which consists of designing sites for each of 10 different chemical reactions in backbone scaffolds derived from 10 enzymes catalyzing the reactions, the new methods succeed in identifying the native site in the native scaffold and ranking it within the top five designs for six of the 10 reactions. The new methods can be directly applied to the design of new enzymes, and the benchmark provides a powerful in silico test for guiding improvements in computational enzyme design.
A series of enzymes for Kemp elimination of 5-nitrobenzisoxazole has been recently designed and tested. In conjunction with the design process, extensive computational analyses were carried out to ...evaluate the potential performance of four of the designs, as presented here. The enzyme-catalyzed reactions were modeled using mixed quantum and molecular mechanics (QM/MM) calculations in the context of Monte Carlo (MC) statistical mechanics simulations. Free-energy perturbation (FEP) calculations were used to characterize the free-energy surfaces for the catalyzed reactions as well as for reference processes in water. The simulations yielded detailed information about the catalytic mechanisms, activation barriers, and structural evolution of the active sites over the course of the reactions. The catalytic mechanism for the designed enzymes KE07, KE10(V131N), and KE15 was found to be concerted with proton transfer, generally more advanced in the transition state than breaking of the isoxazolyl N−O bond. On the basis of the free-energy results, all three enzymes were anticipated to be active. Ideas for further improvement of the enzyme designs also emerged. On the technical side, the synergy of parallel QM/MM and experimental efforts in the design of artificial enzymes is well illustrated.
De Novo Computational Design of Retro-Aldol Enzymes Jiang, Lin; Althoff, Eric A.; Clemente, Fernando R. ...
Science (American Association for the Advancement of Science),
03/2008, Letnik:
319, Številka:
5868
Journal Article
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The creation of enzymes capable of catalyzing any desired chemical reaction is a grand challenge for computational protein design. Using new algorithms that rely on hashing techniques to construct ...active sites for multistep reactions, we designed retro-aldolases that use four different catalytic motifs to catalyze the breaking of a carbon-carbon bond in a nonnatural substrate. Of the 72 designs that were experimentally characterized, 32, spanning a range of protein folds, had detectable retroaldolase activity. Designs that used an explicit water molecule to mediate proton shuffling were significantly more successful, with rate accelerations of up to four orders of magnitude and multiple turnovers, than those involving charged side-chain networks. The atomic accuracy of the design process was confirmed by the x-ray crystal structure of active designs embedded in two protein scaffolds, both of which were nearly superimposable on the design model.
Antibody variable domains differ considerably in stability. Single-chain Fv (scFv) fragments derived from natural repertoires frequently lack the high stability needed for therapeutic application, ...necessitating reengineering not only to humanize their sequence, but also to improve their biophysical properties. The human VH3 domain has been identified as having the best biophysical properties among human subtypes. However, complementarity determining region (CDR) grafts from highly divergent VH domains to huVH3 frequently fail to reach its superior stability. In previous experiments involving a CDR graft from a murine VH9 domain of very poor stability to huVH3, a hybrid VH framework was obtained which combines the lower core residues of muVH9 with the surface residues of huVH3. It resulted in a scFv with far better biophysical properties than the corresponding grafts to the consensus huVH3 framework. To better understand the origin of the superior properties of the hybrid framework, we constructed further hybrids, but now in the context of the consensus CDR-H1 and -H2 of the original human VH3 domain. The new hybrids included elements from either murine VH9, human VH1 or human VH5 domains. From guanidinium chloride-induced equilibrium denaturation measurements, kinetic denaturation experiments, measurements of heat-induced aggregation and comparison of soluble expression yield in Escherichia coli, we conclude that the optimal VH framework is CDR-dependent. The present work pinpoints structural features responsible for this dependency and helps to explain why the immune system uses more than one framework with different structural subtypes in framework 1 to optimally support widely different CDRs.
The determination of 3D structures of membrane proteins is still extremely difficult. The co-crystallization with specific binding proteins may be an important aid in this process, as these proteins ...provide rigid, hydrophilic surfaces for stable protein–protein contacts. Also, the conformational homogeneity of the membrane protein may be increased to obtain crystals suitable for high resolution structures. Here, we describe the efficient generation and characterization of Designed Ankyrin Repeat Proteins (DARPins) as specific binding molecules for membrane proteins. We used both phage display and ribosome display to select DARPins
in vitro that are specific for the detergent-solubilized Na
+-citrate symporter CitS of
Klebsiella pneumoniae. Compared to classical hybridoma technology, the
in vitro selection systems allow a much better control of the structural integrity of the target protein and allow the use of other protein classes in addition to recombinant antibodies. We also compared the selected DARPins to a Fab fragment previously selected by phage display and demonstrate that different epitopes are recognized, unique to each class of binding molecules. Therefore, the use of several classes of binding molecules will make suitable crystal formation and the determination of their 3D structure more likely.
Understanding enzyme catalysis through the analysis of natural enzymes is a daunting challenge—their active sites are complex and combine numerous interactions and catalytic forces that are finely ...coordinated. Study of more rudimentary (wo)man-made enzymes provides a unique opportunity for better understanding of enzymatic catalysis. KE07, a computationally designed Kemp eliminase that employs a glutamate side chain as the catalytic base for the critical proton abstraction step and an apolar binding site to guide substrate binding, was optimized by seven rounds of random mutagenesis and selection, resulting in a >200-fold increase in catalytic efficiency. Here, we describe the directed evolution process in detail and the biophysical and crystallographic studies of the designed KE07 and its evolved variants. The optimization of KE07's activity to give a kcat/KM value of ∼2600 s−1 M−1 and an ∼106-fold rate acceleration (kcat/kuncat) involved the incorporation of up to eight mutations. These mutations led to a marked decrease in the overall thermodynamic stability of the evolved KE07s and in the configurational stability of their active sites. We identified two primary contributions of the mutations to KE07's improved activity: (i) the introduction of new salt bridges to correct a mistake in the original design that placed a lysine for leaving-group protonation without consideration of its “quenching” interactions with the catalytic glutamate, and (ii) the tuning of the environment, the pKa of the catalytic base, and its interactions with the substrate through the evolution of a network of hydrogen bonds consisting of several charged residues surrounding the active site.
The design of new enzymes for reactions not catalysed by naturally occurring biocatalysts is a challenge for protein engineering and is a critical test of our understanding of enzyme catalysis. Here ...we describe the computational design of eight enzymes that use two different catalytic motifs to catalyse the Kemp elimination-a model reaction for proton transfer from carbon-with measured rate enhancements of up to 10(5) and multiple turnovers. Mutational analysis confirms that catalysis depends on the computationally designed active sites, and a high-resolution crystal structure suggests that the designs have close to atomic accuracy. Application of in vitro evolution to enhance the computational designs produced a >200-fold increase in k(cat)/K(m) (k(cat)/K(m) of 2,600 M(-1)s(-1) and k(cat)/k(uncat) of >10(6)). These results demonstrate the power of combining computational protein design with directed evolution for creating new enzymes, and we anticipate the creation of a wide range of useful new catalysts in the future.
Co-crystallization of membrane proteins with antibody fragments may emerge as a general tool to facilitate crystal growth and improve crystal quality. The bound antibody fragment enlarges the ...hydrophilic part of the mostly hydrophobic membrane protein, thereby increasing the interaction area for possible protein–protein contacts in the crystal. Additionally, it may restrain flexible parts or lock the membrane protein in a defined conformational state. For successful co-crystallization trials, the antibody fragments must be stable in detergents during the extended period of crystal growth and must be easily produced in amounts necessary for crystallography. Therefore, we constructed a library of antibody Fab fragments from a framework subset of the HuCAL GOLD® library (Morphosys, Munich, Germany). By combining the most stable and well expressed frameworks, VH3 and Vκ3, with the further stabilizing constant domains, a Fab library with the desired properties was obtained in a standard phage display format. As a proof of principle, we selected binders with phage display against the detergent-solubilized citrate transporter CitS of Klebsiella pneumoniae. We describe efficient methods for the immobilization of the membrane protein during selection, for ELISA screening, and for BIAcore evaluation. We demonstrate that the selected Fab fragments form stable complexes with native CitS and recognize conformational epitopes with affinities in the low nanomolar range.
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