Designing catalysts that achieve the rates and selectivities of natural enzymes is a long-standing goal in protein chemistry. Here, we show that an ultrahigh-throughput droplet-based microfluidic ...screening platform can be used to improve a previously optimized artificial aldolase by an additional factor of 30 to give a >10
rate enhancement that rivals the efficiency of class I aldolases. The resulting enzyme catalyses a reversible aldol reaction with high stereoselectivity and tolerates a broad range of substrates. Biochemical and structural studies show that catalysis depends on a Lys-Tyr-Asn-Tyr tetrad that emerged adjacent to a computationally designed hydrophobic pocket during directed evolution. This constellation of residues is poised to activate the substrate by Schiff base formation, promote mechanistically important proton transfers and stabilize multiple transition states along a complex reaction coordinate. The emergence of such a sophisticated catalytic centre shows that there is nothing magical about the catalytic activities or mechanisms of naturally occurring enzymes, or the evolutionary process that gave rise to them.
Primordial sequence signatures in modern proteins imply ancestral origins tracing back to simple peptides. Although short peptides seldom adopt unique folds, metal ions might have templated their ...assembly into higher-order structures in early evolution and imparted useful chemical reactivity. Recapitulating such a biogenetic scenario, we have combined design and laboratory evolution to transform a zinc-binding peptide into a globular enzyme capable of accelerating ester cleavage with exacting enantiospecificity and high catalytic efficiency (
/
~ 10
M
s
). The simultaneous optimization of structure and function in a naïve peptide scaffold not only illustrates a plausible enzyme evolutionary pathway from the distant past to the present but also proffers exciting future opportunities for enzyme design and engineering.
Enzymes rely on complex interactions between precisely positioned active site residues as a mechanism to compensate for the limited functionality contained within the genetic code. Heme enzymes ...provide a striking example of this complexity, whereby the electronic properties of reactive ferryl intermediates are finely tuned through hydrogen bonding interactions between proximal ligands and neighboring amino acids. Here, we show that introduction of a chemically programmed proximal Nδ-methyl histidine (NMH) ligand into an engineered ascorbate peroxidase (APX2) overcomes the reliance on the conserved Asp-His hydrogen bonding interaction, leading to a catalytically modified enzyme (APX2 NMH), which is able to achieve a significantly higher number of turnovers compared with APX2 without compromising catalytic efficiency. Structural, spectroscopic and kinetic characterization of APX2 NMH and several active site variants provides valuable insights into the role of the Asp-His-Fe triad of heme peroxidases. More significantly, simplification of catalytic mechanisms through the incorporation of chemically optimized ligands may facilitate efforts to create and evolve new active site heme environments within proteins.
Temperature influences the reaction kinetics and evolvability of all enzymes. To understand how evolution shapes the thermodynamic drivers of catalysis, we optimized the modest activity of a ...computationally designed enzyme for an elementary proton-transfer reaction by nearly 4 orders of magnitude over 9 rounds of mutagenesis and screening. As theorized for primordial enzymes, the catalytic effects of the original design were almost entirely enthalpic in origin, as were the rate enhancements achieved by laboratory evolution. However, the large reductions in Δ
were partially offset by a decrease in
Δ
and unexpectedly accompanied by a negative activation heat capacity, signaling strong adaptation to the operating temperature. These findings echo reports of temperature-dependent activation parameters for highly evolved natural enzymes and are relevant to explanations of enzymatic catalysis and adaptation to changing thermal environments.
Linus Pauling established the conceptual framework for understanding and mimicking enzymes more than six decades ago. The notion that enzymes selectively stabilize the rate-limiting transition state ...of the catalysed reaction relative to the bound ground state reduces the problem of design to one of molecular recognition. Nevertheless, past attempts to capitalize on this idea, for example by using transition state analogues to elicit antibodies with catalytic activities, have generally failed to deliver true enzymatic rates. The advent of computational design approaches, combined with directed evolution, has provided an opportunity to revisit this problem. Starting from a computationally designed catalyst for the Kemp elimination--a well-studied model system for proton transfer from carbon--we show that an artificial enzyme can be evolved that accelerates an elementary chemical reaction 6 × 10(8)-fold, approaching the exceptional efficiency of highly optimized natural enzymes such as triosephosphate isomerase. A 1.09 Å resolution crystal structure of the evolved enzyme indicates that familiar catalytic strategies such as shape complementarity and precisely placed catalytic groups can be successfully harnessed to afford such high rate accelerations, making us optimistic about the prospects of designing more sophisticated catalysts.
We have selected designed ankyrin repeat proteins (DARPins) from a synthetic library by using ribosome display that selectively bind to the mitogen-activated protein kinase ERK2 (extracellular ...signal-regulated kinase 2) in either its nonphosphorylated (inactive) or doubly phosphorylated (active) form. They do not bind to other kinases tested. Crystal structures of complexes with two DARPins, each specific for one of the kinase forms, were obtained. The two DARPins bind to essentially the same region of the kinase, but recognize the conformational change within the activation loop and an adjacent area, which is the key structural difference that occurs upon activation. Whereas the rigid phosphorylated activation loop remains in the same form when bound by the DARPin, the more mobile unphosphorylated loop is pushed to a new position. The DARPins can be used to selectively precipitate the cognate form of the kinases from cell lysates. They can also specifically recognize the modification status of the kinase inside the cell. By fusing the kinase with Renilla luciferase and the DARPin to GFP, an energy transfer from luciferase to GFP can be observed in COS-7 cells upon intracellular complex formation. Phosphorylated ERK2 is seen to increase by incubation of the COS-7 cells with FBS and to decrease upon adding the ERK pathway inhibitor PD98509. Furthermore, the anti-ERK2 DARPin is seen to inhibit ERK phosphorylation as it blocks the target inside the cell. This strategy of creating activation-state–specific sensors and kinase-specific inhibitors may add to the repertoire to investigate intracellular signaling in real time.
Abstract
α-adrenergic receptors (αARs) are G protein-coupled receptors that regulate vital functions of the cardiovascular and nervous systems. The therapeutic potential of αARs, however, is largely ...unexploited and hampered by the scarcity of subtype-selective ligands. Moreover, several aminergic drugs either show off-target binding to αARs or fail to interact with the desired subtype. Here, we report the crystal structure of human α
1B
AR bound to the inverse agonist (+)-cyclazosin, enabled by the fusion to a DARPin crystallization chaperone. The α
1B
AR structure allows the identification of two unique secondary binding pockets. By structural comparison of α
1B
AR with α
2
ARs, and by constructing α
1B
AR-α
2C
AR chimeras, we identify residues 3.29 and 6.55 as key determinants of ligand selectivity. Our findings provide a basis for discovery of α
1B
AR-selective ligands and may guide the optimization of aminergic drugs to prevent off-target binding to αARs, or to elicit a selective interaction with the desired subtype.
The discovery of the key role of Toll-like receptors (TLRs) in initiating innate immune responses and modulating adaptive immunity has revolutionized our understanding of vertebrate defence against ...pathogens. Yet, despite their central role in pathogen recognition and defence initiation, there is little information on how variation in TLRs influences disease susceptibility in natural populations. Here, we assessed the extent of naturally occurring polymorphisms at TLR2 in wild bank voles (Myodes glareolus) and tested for associations between TLR2 variants and infection with Borrelia afzelii, a common tick-transmitted pathogen in rodents and one of the causative agents of human Lyme disease. Bank voles in our population had 15 different TLR2 haplotypes (10 different haplotypes at the amino acid level), which grouped in three well-separated clusters. In a large-scale capture–mark–recapture study, we show that voles carrying TLR2 haplotypes of one particular cluster (TLR2c2) were almost three times less likely to be Borrelia infected than animals carrying other haplotypes. Moreover, neutrality tests suggested that TLR2 has been under positive selection. This is, to our knowledge, the first demonstration of an association between TLR polymorphism and parasitism in wildlife, and a striking example that genetic variation at innate immune receptors can have a large impact on host resistance.
While native scaffolds offer a large diversity of shapes and topologies for enzyme engineering, their often unpredictable behavior in response to sequence modification makes de novo generated ...scaffolds an exciting alternative. Here we explore the customization of the backbone and sequence of a de novo designed eight stranded β‐barrel protein to create catalysts for a retro‐aldolase model reaction. We show that active and specific catalysts can be designed in this fold and use directed evolution to further optimize activity and stereoselectivity. Our results support previous suggestions that different folds have different inherent amenability to evolution and this property could account, in part, for the distribution of natural enzymes among different folds.
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