Dan Tawfik lab: Directed evolution: Difference between revisions
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<StructureSection load='Crdes1.pdb' size='450' frame='true' side='right' scene='3iio/Int/1' > | |||
== I) Kemp eliminase == | == I) Kemp eliminase == | ||
A series of [http://en.wikipedia.org/wiki/Protein_design computationally designed enzymes] that catalyze the Kemp elimination have described. Kemp eliminase (KE07) has <scene name='3iio/Int/10'>TIM barrel scaffold</scene>. The Kemp elimination of <scene name='3iio/Int/4'>5-nitrobenzisoxazole</scene> was chosen as a model reaction for [http://en.wikipedia.org/wiki/Proton#Hydrogen_as_proton proton (H)] transfer from [http://en.wikipedia.org/wiki/Carbon carbon], simultaneously with the cut of the [http://en.wikipedia.org/wiki/Nitrogen nitrogen]–[http://en.wikipedia.org/wiki/Oxygen oxygen] (N-O) bond, resulting in <scene name='3iio/Int/8'>cyanophenol product</scene>. Such reaction is a critical step in many [http://en.wikipedia.org/wiki/Enzyme enzymatic reactions]. The [http://en.wikipedia.org/wiki/Enzyme_catalysis catalytic] base (E101), the general acid/[http://en.wikipedia.org/wiki/Hydrogen_bond H-bond] donor (K222), and the stacking residue (W50) make interactions with the 5-nitrobenzisoxazole at the <scene name='3iio/Int/9'>active site of KE07</scene>. [http://en.wikipedia.org/wiki/Directed_evolution Directed evolution] can significantly improve the stability, expression and activity of enzymes. In the catalytically improved directed evolutionary variants of KE07 containing the <scene name='3iio/Ali/7'>Ile7Asp mutation</scene>, Asp7 breaks the Glu101–Lys222 salt bridge (for example [[3iiv]], chain A is shown). | A series of [http://en.wikipedia.org/wiki/Protein_design computationally designed enzymes] that catalyze the Kemp elimination have described. Kemp eliminase (KE07) has <scene name='3iio/Int/10'>TIM barrel scaffold</scene>. The Kemp elimination of <scene name='3iio/Int/4'>5-nitrobenzisoxazole</scene> was chosen as a model reaction for [http://en.wikipedia.org/wiki/Proton#Hydrogen_as_proton proton (H)] transfer from [http://en.wikipedia.org/wiki/Carbon carbon], simultaneously with the cut of the [http://en.wikipedia.org/wiki/Nitrogen nitrogen]–[http://en.wikipedia.org/wiki/Oxygen oxygen] (N-O) bond, resulting in <scene name='3iio/Int/8'>cyanophenol product</scene>. Such reaction is a critical step in many [http://en.wikipedia.org/wiki/Enzyme enzymatic reactions]. The [http://en.wikipedia.org/wiki/Enzyme_catalysis catalytic] base (E101), the general acid/[http://en.wikipedia.org/wiki/Hydrogen_bond H-bond] donor (K222), and the stacking residue (W50) make interactions with the 5-nitrobenzisoxazole at the <scene name='3iio/Int/9'>active site of KE07</scene>. [http://en.wikipedia.org/wiki/Directed_evolution Directed evolution] can significantly improve the stability, expression and activity of enzymes. In the catalytically improved directed evolutionary variants of KE07 containing the <scene name='3iio/Ali/7'>Ile7Asp mutation</scene>, Asp7 breaks the Glu101–Lys222 salt bridge (for example [[3iiv]], chain A is shown). | ||
{{Clear}} | {{Clear}} | ||
The comparison of the <scene name='3iio/Ali/3'>designed structure</scene> <span style="color:orange;background-color:black;font-weight:bold;">(colored orange)</span> modelled in the presence of the <span style="color:cyan;background-color:black;font-weight:bold;">5-nitrobenzisoxazole</span> and <scene name='3iio/Ali/4'>unbound crystal structure</scene> ([[2rkx]], <span style="color:lime;background-color:black;font-weight:bold;">(colored lime)</span> of KE07 shows only limited conformational changes. In the <scene name='3iio/Ali/5'>designed structure</scene>, the amino group of Lys222 is ~4 Å away from the transition state phenolic oxygen, to stabilize the negative charge of the product phenoxide. However, Lys222 can also form a weak salt bridge with the catalytic Glu101 with a distance of ~3.6 Å. In the <scene name='3iio/Ali/6'>crystal structure</scene> of the KE07 without ligand ([[2rkx]]), the Glu101–Lys222 distance is 2.84 Å, ''i.g.'' within [http://en.wikipedia.org/wiki/Salt_bridge_(protein) salt bridge] distance. This is probably a unique feature of the unbound conformation of KE07. As was mentioned above, in the catalytically improved directed evolutionary mutants of KE07 bearing the <scene name='3iio/Ali/7'>Ile7Asp mutation</scene>, Asp7 breaks the Glu101–Lys222 salt bridge (in the evolved mutants, the Nε<sub>Lys222</sub>–Oγ<sub>Glu101</sub> distance is 3.3–5.7 Å), in some cases directly (as in the present case [[3iiv]], chain A) interacting with Lys222 (the Nε<sub>Lys222</sub>–Oβ<sub>Asp7</sub> distance is 2.8–5.7 Å). <scene name='3iio/Ali/9'>An overlap</scene> of the structures of the <span style="color:lime;background-color:black;font-weight:bold;">wildtype (lime) KE07)</span> and the its <span style="color:lightskyblue;background-color:black;font-weight:bold;">evolved Ile7Asp mutant</span> reveals how the Ile7Asp mutation causes the shift of the Lys222 side chain away from Glu101. | The comparison of the <scene name='3iio/Ali/3'>designed structure</scene> <span style="color:orange;background-color:black;font-weight:bold;">(colored orange)</span> modelled in the presence of the <span style="color:cyan;background-color:black;font-weight:bold;">5-nitrobenzisoxazole</span> and <scene name='3iio/Ali/4'>unbound crystal structure</scene> ([[2rkx]], <span style="color:lime;background-color:black;font-weight:bold;">(colored lime)</span>) of KE07 shows only limited conformational changes. In the <scene name='3iio/Ali/5'>designed structure</scene>, the amino group of Lys222 is ~4 Å away from the transition state phenolic oxygen, to stabilize the negative charge of the product phenoxide. However, Lys222 can also form a weak salt bridge with the catalytic Glu101 with a distance of ~3.6 Å. In the <scene name='3iio/Ali/6'>crystal structure</scene> of the KE07 without ligand ([[2rkx]]), the Glu101–Lys222 distance is 2.84 Å, ''i.g.'' within [http://en.wikipedia.org/wiki/Salt_bridge_(protein) salt bridge] distance. This is probably a unique feature of the unbound conformation of KE07. As was mentioned above, in the catalytically improved directed evolutionary mutants of KE07 bearing the <scene name='3iio/Ali/7'>Ile7Asp mutation</scene>, Asp7 breaks the Glu101–Lys222 salt bridge (in the evolved mutants, the Nε<sub>Lys222</sub>–Oγ<sub>Glu101</sub> distance is 3.3–5.7 Å), in some cases directly (as in the present case [[3iiv]], chain A) interacting with Lys222 (the Nε<sub>Lys222</sub>–Oβ<sub>Asp7</sub> distance is 2.8–5.7 Å). <scene name='3iio/Ali/9'>An overlap</scene> of the structures of the <span style="color:lime;background-color:black;font-weight:bold;">wildtype (lime) KE07)</span> and the its <span style="color:lightskyblue;background-color:black;font-weight:bold;">evolved Ile7Asp mutant</span> reveals how the Ile7Asp mutation causes the shift of the Lys222 side chain away from Glu101. | ||
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The crystal structures of the catalytically improved directed evolutionary KE07 mutants also demonstrate that replacement of side chains via mutations, combined with minor backbone changes, could allowed the new enzyme–substrate interactions. For example, <scene name='3iio/Ali1/5'>superposition</scene> of the structures of the <span style="color:orange;background-color:black;font-weight:bold;">KE07 design</span> and evolved <font color='darkmagenta'><b>KE07 round 4 1E/11H chain A</b></font> ([[3iio]]) reveals that the mutation Gly202Arg caused a shift of the adjacent loop (residues 175–177) and could introduce a new interaction with the nitro group of the 5-nitrobenzisoxazole. The directed evolution also creates new interaction networks of charged surface residues at the upper part of the active site. In the <scene name='3iio/Ali1/6'>wildtype KE07</scene> ([[2rkx]], <span style="color:lime;background-color:black;font-weight:bold;">colored lime</span>), Gly is in the position 202, Asn is in the position 224, and distance between Asn224 O and His201 N is 7.9 Å. In the evolved variants, following the Gly202Arg and Asn224Asp mutations, Asp224 and His201 gradually became closer, with distances between Asn224 O and His201 N of 4.6 Å in the <scene name='3iio/Ali1/7'>round 4 variant</scene> ([[3iio]], <font color='darkmagenta'><b>colored darkmagenta</b></font>) and 3.6 Å in the <scene name='3iio/Ali1/8'>round 7 variant, chain B</scene> ([[3iiv]], <span style="color:tan;background-color:black;font-weight:bold;">colored tan</span>). In rounds 6-7 variants, Asp224 can potentially interact with Arg202 and with His201. This network of Arg202–Asp224–His201 also brings His201 closer to the substrate (not shown). Interestingly, the <scene name='3iio/Ali1/9'>conformation of Trp50</scene> at the active site in <span style="color:lightskyblue;background-color:black;font-weight:bold;">chain A of round 7 1/3H variant</span> ([[3iiv]]) significantly differs from those in all other structures, including <span style="color:tan;background-color:black;font-weight:bold;">chain B</span>) within the asymmetric unit of round 7 1/3H. Of note, that Trp50 of chain A overlaps the substrate. | The crystal structures of the catalytically improved directed evolutionary KE07 mutants also demonstrate that replacement of side chains via mutations, combined with minor backbone changes, could allowed the new enzyme–substrate interactions. For example, <scene name='3iio/Ali1/5'>superposition</scene> of the structures of the <span style="color:orange;background-color:black;font-weight:bold;">KE07 design</span> and evolved <font color='darkmagenta'><b>KE07 round 4 1E/11H chain A</b></font> ([[3iio]]) reveals that the mutation Gly202Arg caused a shift of the adjacent loop (residues 175–177) and could introduce a new interaction with the nitro group of the 5-nitrobenzisoxazole. The directed evolution also creates new interaction networks of charged surface residues at the upper part of the active site. In the <scene name='3iio/Ali1/6'>wildtype KE07</scene> ([[2rkx]], <span style="color:lime;background-color:black;font-weight:bold;">colored lime</span>), Gly is in the position 202, Asn is in the position 224, and distance between Asn224 O and His201 N is 7.9 Å. In the evolved variants, following the Gly202Arg and Asn224Asp mutations, Asp224 and His201 gradually became closer, with distances between Asn224 O and His201 N of 4.6 Å in the <scene name='3iio/Ali1/7'>round 4 variant</scene> ([[3iio]], <font color='darkmagenta'><b>colored darkmagenta</b></font>) and 3.6 Å in the <scene name='3iio/Ali1/8'>round 7 variant, chain B</scene> ([[3iiv]], <span style="color:tan;background-color:black;font-weight:bold;">colored tan</span>). In rounds 6-7 variants, Asp224 can potentially interact with Arg202 and with His201. This network of Arg202–Asp224–His201 also brings His201 closer to the substrate (not shown). Interestingly, the <scene name='3iio/Ali1/9'>conformation of Trp50</scene> at the active site in <span style="color:lightskyblue;background-color:black;font-weight:bold;">chain A of round 7 1/3H variant</span> ([[3iiv]]) significantly differs from those in all other structures, including <span style="color:tan;background-color:black;font-weight:bold;">chain B</span>) within the asymmetric unit of round 7 1/3H. Of note, that Trp50 of chain A overlaps the substrate. | ||
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== II) Colicin7 and Immunity proteins == | == II) Colicin7 and Immunity proteins == | ||
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Iterative rounds of random mutagenesis and selection of <span style="color:yellow;background-color:black;font-weight:bold;">immunity protein 9 (colored yellow)</span> toward higher affinity for ColE7, and selectivity (against ColE9 inhibition), led to significant increase in affinity and selectivity. Several evolved variants were obtained. The crystal structures of the two final generation <scene name='3gkl/Al/3'>variants</scene> <span style="color:lime;background-color:black;font-weight:bold;">R12-2</span> ('''3gkl'''; T20A, N24D, T27A, S28T, V34D, V37J, E41G, and K57E) and <font color='darkred'><b>R12-13</b></font> ([[3gjn]]; N24D, D25E, T27A, S28T, V34D, V37J, and Y55W) in complex with ColE7 were solved. | Iterative rounds of random mutagenesis and selection of <span style="color:yellow;background-color:black;font-weight:bold;">immunity protein 9 (colored yellow)</span> toward higher affinity for ColE7, and selectivity (against ColE9 inhibition), led to significant increase in affinity and selectivity. Several evolved variants were obtained. The crystal structures of the two final generation <scene name='3gkl/Al/3'>variants</scene> <span style="color:lime;background-color:black;font-weight:bold;">R12-2</span> ('''3gkl'''; T20A, N24D, T27A, S28T, V34D, V37J, E41G, and K57E) and <font color='darkred'><b>R12-13</b></font> ([[3gjn]]; N24D, D25E, T27A, S28T, V34D, V37J, and Y55W) in complex with ColE7 were solved. | ||