Directed evolution of O6-alkylguanine-DNA alkyltransferase for applications in protein labeling

Gronemeyer, Thomas; Chidley, Christopher; Juillerat, Alexandre; Heinis, Christian; Johnsson, Kai

doi:10.1093/protein/gzl014

Cited by 139 publications

(118 citation statements)

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“…Moreover, the binding mechanism for bGua in the absence of DNA, as in ref. 30, could be very different from that in its presence.…”

Section: Discussionmentioning

confidence: 83%

“…The simulations also point to the importance of the active-site loop (Gly-153 to Gly-160). In support of this idea, mutants of many of these residues were recently obtained during directed evolution of AGT to maximize activity toward O 6 -benzylguanine derivatives (bGua) (30). In particular, A154T by itself was found to give a 4-fold increase in activity; this is of interest because Ala-154 interacts directly with the methyl group of mGua in the extrahelical intermediate in the simulations.…”

Section: Discussionmentioning

confidence: 95%

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A two-step nucleotide-flipping mechanism enables kinetic discrimination of DNA lesions by AGT

Dinner

2008

Proc. Natl. Acad. Sci. U.S.A.

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O 6 -alkylguanine-DNA alkyltransferase (AGT) repairs damage to the human genome by flipping guanine and thymine bases into its active site for irreversible transfer of alkyl lesions to Cys-145, but how the protein identifies its targets has remained unknown. Understanding molecular recognition in this system, which can serve as a paradigm for the many nucleotide-flipping proteins that regulate genes and repair DNA in all kingdoms of life, is particularly important given that inhibitors are in clinical trials as anticancer therapeutics. Computational approaches introduced recently for harvesting and statistically characterizing trajectories of molecularly rare events now enable us to elucidate a pathway for nucleotide flipping by AGT and the forces that promote it. In contrast to previously proposed flipping mechanisms, we observe a two-step process that promotes a kinetic, rather than a thermodynamic, gate-keeping strategy for lesion discrimination. Connection is made to recent single-molecule studies of DNA-repair proteins sliding on DNA to understand how they sense subtle chemical differences between bases efficiently. commitment probabilities ͉ DNA repair ͉ reaction coordinates ͉ transition path sampling

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“…Moreover, the binding mechanism for bGua in the absence of DNA, as in ref. 30, could be very different from that in its presence.…”

Section: Discussionmentioning

confidence: 83%

Section: Discussionmentioning

confidence: 95%

A two-step nucleotide-flipping mechanism enables kinetic discrimination of DNA lesions by AGT

Dinner

2008

Proc. Natl. Acad. Sci. U.S.A.

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“…The SNAP tag represents an engineered version of the human O 6 -Alkylguanine-DNA-Alkyltransferase (Gronemeyer et al, 2006).…”

Section: Statistical Testsmentioning

confidence: 99%

Septin rings act as template for myosin higher-order structures and inhibit redundant polarity establishment

Schneider

Grois

Renz

et al. 2013

Journal of Cell Science

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SummaryThe mechanisms of the coordinated assembly and disassembly of the septin/myosin ring is central for the understanding of polar growth and cytokinesis in yeast and other organisms. The septin-and myosin-binding protein Bni5p provides a dual function during the formation and disassembly of septin/myosin rings. Early in the cell cycle, Bni5p captures Myo1p at the incipient bud site and actively transforms it into higher-order structures. Additionally, Bni5p stabilizes the septin/myosin ring and is released from the septins shortly before the onset of cytokinesis. If this Bni5p dissociation from the septins is artificially prevented, ring disassembly is impaired and the untimely appearance of septin/myosin ring is induced. The prematurely formed septin/myosin rings delay the establishment of a new polarity axis and the progression into a new cell cycle. This observation suggests a negative feedback between septin/myosin ring formation and polarity establishment that might help to guarantee the singular assembly of this structure and the synchronization of its formation with the cell cycle.

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“…To rescue the activity of M AGT against BG derivatives, an additional round of saturation mutagenesis (residues 150− 154 and 32 and 33) followed by phage display was performed, resulting in SNAP-tag. 10 Relative to hAGT, SNAP-tag possesses a 52-fold higher reactivity toward BG derivatives, does not bind to DNA, and is expressed as well in cells as on cell surfaces. However, our understanding of the structure−function relationship of SNAP-tag and how the introduced mutations affect activity is poor.…”

mentioning

confidence: 99%

Directed Evolution of the Suicide Protein O⁶-Alkylguanine-DNA Alkyltransferase for Increased Reactivity Results in an Alkylated Protein with Exceptional Stability

et al. 2012

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Here we present a biophysical, structural, and computational analysis of the directed evolution of the human DNA repair protein O 6 -alkylguanine-DNA alkyltransferase (hAGT) into SNAP-tag, a self-labeling protein tag. Evolution of hAGT led not only to increased protein activity but also to higher stability, especially of the alkylated protein, suggesting that the reactivity of the suicide enzyme can be influenced by stabilizing the product of the irreversible reaction. Whereas wild-type hAGT is rapidly degraded in cells after alkyl transfer, the high stability of benzylated SNAP-tag prevents proteolytic degradation. Our data indicate that the intrinstic stability of a key α helix is an important factor in triggering the unfolding and degradation of wild-type hAGT upon alkyl transfer, providing new insights into the structure−function relationship of the DNA repair protein.T he specific labeling of proteins with synthetic probes is a powerful approach for studying protein function. One way to achieve such a specific labeling is based on so-called selflabeling protein tags.1 In this approach, the protein of interest is expressed as a fusion protein with a peptide or protein (i.e., tag) whose role is to specifically bind to a synthetic probe in vitro or in vivo. A well-established example of a self-labeling protein tag is SNAP-tag.2 SNAP-tag specifically reacts with substituted O 6 -benzylguanine derivatives and thereby permits the labeling of SNAP-tag fusion proteins with a wide variety of different synthetic probes. Recent applications include its use for the analysis of protein complexes, 3 super-resolution microscopy, 4 the identification of protein−protein interactions, 5 drug target identification, 6 and the determination of protein half-life in animals. 7 The appeal of self-labeling tags such as SNAP-tag is the ease with which fusion proteins can be labeled with synthetic probes even in living cells. A conceptual limitation of the approach is the fact that the tag can affect the properties of its fusion partner. It is therefore important that the properties of the tag be as thoroughly characterized as possible.SNAP-tag was generated in a stepwise manner from human O 6 -alkylguanine-DNA alkyltransferase (hAGT) by introduction of a total of 19 point mutations (Figure 1) and deletion of 25 C-terminal residues. Saturation mutagenesis of four active-site residues followed by phage display and selection for activity against BG derivatives resulted in GE AGT, a mutant with 20-fold increased activity toward such substrates ( Figure 1B). 8Subsequent saturation mutagenesis of four additional residues involved in substrate binding followed by phage selection resulted in AGT-54, a mutant with 1.5-fold higher activity than GE AGT. To further optimize the protein for applications in protein labeling, mutations were introduced to suppress DNA binding and reactivity toward nucleosides, to remove nonessential cysteines, and to truncate the last 25 residues. 9The resulting mutant M AGT displayed relatively low activ...

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Directed evolution of O6-alkylguanine-DNA alkyltransferase for applications in protein labeling

Cited by 139 publications

References 20 publications

A two-step nucleotide-flipping mechanism enables kinetic discrimination of DNA lesions by AGT

A two-step nucleotide-flipping mechanism enables kinetic discrimination of DNA lesions by AGT

Septin rings act as template for myosin higher-order structures and inhibit redundant polarity establishment

Directed Evolution of the Suicide Protein O⁶-Alkylguanine-DNA Alkyltransferase for Increased Reactivity Results in an Alkylated Protein with Exceptional Stability

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Directed evolution of O6-alkylguanine-DNA alkyltransferase for applications in protein labeling

Cited by 139 publications

References 20 publications

A two-step nucleotide-flipping mechanism enables kinetic discrimination of DNA lesions by AGT

A two-step nucleotide-flipping mechanism enables kinetic discrimination of DNA lesions by AGT

Septin rings act as template for myosin higher-order structures and inhibit redundant polarity establishment

Directed Evolution of the Suicide Protein O6-Alkylguanine-DNA Alkyltransferase for Increased Reactivity Results in an Alkylated Protein with Exceptional Stability

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Directed Evolution of the Suicide Protein O⁶-Alkylguanine-DNA Alkyltransferase for Increased Reactivity Results in an Alkylated Protein with Exceptional Stability