View source for Dan Tawfik lab: Directed evolution

<StructureSection load='Crdes1.pdb' size='450' frame='true' side='right' scene='3iio/Int/1' >
== 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). 

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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. 

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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>.  
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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.   
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== 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. 

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<scene name='3gkl/Align/2'>Structural alignment</scene> of the immunity protein 9 (Im9, [[1bxi]], <span style="color:yellow;background-color:black;font-weight:bold;">colored yellow</span>), <span style="color:lime;background-color:black;font-weight:bold;">evolved variant R12-2 (lime)</span>, and <font color='blue'><b>immunity protein 7 (Im7, colored blue</b></font>, [[7cei]]) reveals their structural identity. However, when the immunity proteins-bound <scene name='3gkl/Align/3'>colicins within their complexes were aligned</scene>, they demonstrate somewhat different picture. The Im9 and Im7 are differ more in their binding configurations (19°, with Tyr54-Tyr55 as the pivot), while the variant R12-2 is in an intermediate configuration between Im9 and Im7. Of note, in the variant R12-2 (3gkl) and Im9 ([[1bxi]]) there are Tyr54 and Tyr55, while in the Im7 ([[7cei]]) Tyr55 and Tyr56 are homologous to them. The most <scene name='3gkl/Align/4'>prominent differences</scene> are in the loop between helices α1 and α2 in Im9 (yellow, labeled in black) and <span style="color:lime;background-color:black;font-weight:bold;">evolved variant R12-2 (lime, labeled in black)</span>. This loop consists of three mutations: N24D, T27A, and S28T in variant R12-2. We can see the deviations in the relative position of helices α1 and α2, in the loop's backbone and in the side chains of residues 24, 26 and 28.

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Comparison of the different Im-colicin complexes reveals changes in the binding configuration of the evolved variants which increase affinity toward ColE7 by re-aligning pre-existing Im9 residues. Glu30 of Im9 ([[1bxi]], <span style="color:yellow;background-color:black;font-weight:bold;">colored yellow</span>) forms <scene name='3gkl/Active_site/3'>double salt bridge</scene> with Arg54 of <span style="color:orange;background-color:black;font-weight:bold;">ColE9 (orange)</span>, whereas Asp51 have not direct side chain–side chain interactions. <font color='blue'><b>Asp31 of Im7 (blue)</b></font> which is corresponding to Im9 Glu30 is involved in <scene name='3gkl/Active_site/6'>cluster of salt bridge bonds</scene> to <font color='darkmagenta'><b>Arg520 and Lys525 of ColE7 (darkmagenta)</b></font>, while <font color='blue'><b>Asp52 of Im7</b></font> (corresponding to Im9 Asp51) is within hydrogen bond distance to <font color='darkmagenta'><b>Thr531 and Arg530 of ColE7</b></font> ([[7cei]]). <span style="color:lime;background-color:black;font-weight:bold;">Glu30 in the variant R12-2 (lime)</span> is shifted and forms a <scene name='3gkl/Active_site/8'>double salt bridge</scene> to <font color='magenta'><b>Arg520 of ColE7 (magenta)</b></font>. <span style="color:lime;background-color:black;font-weight:bold;">Asp51</span> is within hydrogen bond distance to <font color='magenta'><b>Thr531 of ColE7</b></font> ([[3gkl]]). However, the side chains of <font color='magenta'><b>Lys525 and Arg530</b></font>, which are very important in salt bridge contacts with Glu30 and Asp51, respectively, in the structure of the ColE7–Im7 complex have a different conformation that eliminates these contacts in evolved variant R12-2.
 
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In the <scene name='3gkl/Mut/2'>Im9</scene> <font color='magenta'><b>Val37 (colored magenta)</b></font> forms stabilizing hydrogen bond with Leu33. In the <scene name='3gkl/Mut/3'>evolved variant R12-2</scene>, <font color='darkmagenta'><b>Ile37 (colored darkmagenta)</b></font> interacts with two additional residues, Tyr54 and Ser50. Moreover, <font color='darkmagenta'><b>Ile37</b></font> also forms additional hydrogen bond with Gly41 and can thereby have enabled the appearance of the selectivity mutation E41G.
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In contrast to the <span style="color:lime;background-color:black;font-weight:bold;">evolved variant R12-2</span> ([[3gkl]]), the <span style="color:cyan;background-color:black;font-weight:bold;">evolved variant R12-13</span> ([[3gjn]]) carries the <scene name='3gkl/Align/10'>Tyr55Trp mutation</scene> in the conserved region. Both <span style="color:lime;background-color:black;font-weight:bold;">Tyr55 in R12-2</span> and <span style="color:cyan;background-color:black;font-weight:bold;">Trp55 in R12-13</span> could sustain the
hydrophobic core and create a <scene name='3gkl/Align/11'>hydrogen bond</scene> to Lys528 backbone ([[3gkl]] colicin residues are colored in <font color='magenta'><b>magenta</b></font>, 3gjn colicin residues are colored <font color='blueviolet'><b>blueviolet</b></font>). However, the additional bulkiness of the Trp contributes in expanding its  <scene name='3gkl/Align/9'>hydrophobic interactions</scene> to Phe541 and Phe513 also leading to the small shift in the alkyl chain of Arg530. 
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The <scene name='3gkl/Ali/1'>overall conformation</scene> of the two evolved variants <span style="color:lime;background-color:black;font-weight:bold;">R12-2</span> ([[3gkl]]) and <span style="color:cyan;background-color:black;font-weight:bold;">R12-13</span> ([[3gjn]]) is very similar. The variant <span style="color:lime;background-color:black;font-weight:bold;">R12-2</span> carries <scene name='3gkl/Ali/2'>mutation E41G</scene>. In the <span style="color:yellow;background-color:black;font-weight:bold;">bound wildtype Im9 (yellow)</span> Glu41 makes a <scene name='3gkl/Ali/3'>salt bridge</scene> with the <span style="color:orange;background-color:black;font-weight:bold;">ColE9’s</span> Lys97 ([[1bxi]]). While in the <font color='blueviolet'><b>R12-13</b></font>/<span style="color:cyan;background-color:black;font-weight:bold;">ColE7</span> complex the <font color='blueviolet'><b>closest ColE7 residues</b></font> <scene name='3gkl/Ali/4'>contacting</scene> <span style="color:cyan;background-color:black;font-weight:bold;">R12-13 Glu41</span> are <font color='blueviolet'><b>Thr531 (3.37Å) and Lys528 (8.85Å)</b></font> ([[3gjn]]). In the <span style="color:lime;background-color:black;font-weight:bold;">R12-2</span>/<font color='magenta'><b>ColE7</b></font> complex the <scene name='3gkl/Ali/5'>closest</scene> <font color='magenta'><b>ColE7 residue</b></font> to <span style="color:lime;background-color:black;font-weight:bold;">R12-2 Gly41</span> is <font color='magenta'><b>Thr531 (9.48Å)</b></font> ([[3gkl]]). 
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</StructureSection>

==References==
<ref group="xtra">PMID:18354394</ref> <ref group='xtra'>PMID:19749752</ref> <ref group='xtra'>PMID:20036254</ref>  <references group="xtra"/>


[[Category: Escherichia coli]]
[[Category: Dym, O.]]
[[Category: Khersonsky, O.]]
[[Category: Tawfik, D S.]]
[[Category: Beta barrel]]
[[Category: Lyase]]
[[Category: Albeck, S.]]
[[Category: ISPC, Israel Structural Proteomics Center.]]
[[Category: Alpha-beta barrel]]
[[Category: Amino-acid biosynthesis]]
[[Category: Cytoplasm]]
[[Category: Histidine biosynthesis]]
[[Category: ISPC]]
[[Category: Israel Structural Proteomics Center]]
[[Category: Lyase]]
[[Category: Structural genomic]]
[[Category: Antibiotic]]
[[Category: Antimicrobial]]
[[Category: Bacteriocin]]
[[Category: Bacteriocin immunity]]
[[Category: Endonuclease]]
[[Category: Hydrolase]]
[[Category: Metal-binding]]
[[Category: Nuclease]]
[[Category: Plasmid]]
[[Category: Protein-protein complex]]
[[Category: Zinc]]