Dan Tawfik lab: Directed evolution: Difference between revisions

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Alexander Berchansky, Karsten Theis

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+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> <font color='orange'><b>(colored orange)</b></font> modelled in the presence of the <font color='cyan'><b>5-nitrobenzisoxazole</b></font> and <scene name='3iio/Ali/4'>unbound crystal structure</scene> ([[2rkx]], <font color='lime'><b>colored lime)</b></font> 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 <font color='lime'><b>wildtype (lime) KE07</b></font> and the its <font color='lightskyblue'><b>evolved Ile7Asp mutant</b></font> reveals how the Ile7Asp mutation causes the shift of the Lys222 side chain away from Glu101.
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+The residues Arg5, Glu46, Lys99, and Glu167 of <span style="color:lime;background-color:black;font-weight:bold;">KE07 unbound wildtype crystal structure</span> ([[2rkx]]) form <scene name='3iio/Ali1/1'>electrostatic network</scene> at the bottom of the active site. In this case Lys222, of course, is not involved in this network, because it could not form electrostatic interaction with Ile7. Ile7Asp mutation in the evolved mutants introduces Lys222 to this electrostatic network or <scene name='3iio/Ali1/3'>directly</scene> as <span style="color:lightskyblue;background-color:black;font-weight:bold;">in case of</span> [[3iiv]]<span style="color:lightskyblue;background-color:black;font-weight:bold;">, chain A</span>, or <scene name='3iio/Ali1/4'>via water molecule</scene> ([[3iiv]], <span style="color:tan;background-color:black;font-weight:bold;">chain B</span>.
-The residues Arg5, Glu46, Lys99, and Glu167 of <font color='lime'><b>KE07 unbound wildtype crystal structure</b></font> ([[2rkx]]) form <scene name='3iio/Ali1/1'>electrostatic network</scene> at the bottom of the active site. In this case Lys222, of course, is not involved in this network, because it could not form electrostatic interaction with Ile7. Ile7Asp mutation in the evolved mutants introduces Lys222 to this electrostatic network or <scene name='3iio/Ali1/3'>directly</scene> as <font color='lightskyblue'><b>in case of</b></font> [[3iiv]]<font color='lightskyblue'><b>, chain A</b></font>, or <scene name='3iio/Ali1/4'>via water molecule</scene> ([[3iiv]], <font color='tan'><b>chain B</b></font>).
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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 <font color='orange'><b>KE07 design</b></font> 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]], <font color='lime'><b>colored lime</b></font>), 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]], <font color='tan'><b>colored tan</b></font>). 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 <font color='lightskyblue'><b>chain A of round 7 1/3H variant</b></font> ([[3iiv]]) significantly differs from those in all other structures, including <font color='tan'><b>chain B</b></font> within the asymmetric unit of round 7 1/3H. Of note, that Trp50 of chain A overlaps the substrate.
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