Creation of an Allosteric Enzyme by Domain Insertion

Guntas, Gurkan; Ostermeier, Marc

doi:10.1016/j.jmb.2003.12.016

Cited by 134 publications

(136 citation statements)

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“…We have previously described three switches created by recombining the bla and malE genes. Switches EE and T164-165 were isolated from a library in which bla was randomly inserted into malE (9) (Fig. 1, Library 1).…”

Section: Resultsmentioning

confidence: 99%

“…Ligand binding or covalent modifications at one site affects the output function at a distant site through a conformational change. This mechanism has inspired a modular design strategy for creating switches in which existing proteins with the prerequisite input and output functions are combined such that their functions are coupled (4)(5)(6)(7)(8)(9)(10)(11)(12)(13). Such switches have potential applications as molecular sensors, as tools for metabolic and cellular engineering, and as therapeutic proteins.…”

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confidence: 99%

“…We have recently demonstrated that a single round of nonhomologous recombination followed by genetic selection͞screening can create modest switches from genes coding for the desired input and output modules (9,12). We recombined the genes coding for Escherichia coli maltose binding protein (MBP) and TEM1 ␤-lactamase (BLA) to create ␤-lactamase enzymes whose catalytic activity was modulated by the presence of maltose.…”

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confidence: 99%

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Directed evolution of protein switches and their application to the creation of ligand-binding proteins

Guntas

Mansell

Kim

et al. 2005

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

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202

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We describe an iterative approach for creating protein switches involving the in vitro recombination of two nonhomologous genes. We demonstrate this approach by recombining the genes coding for TEM1 ␤-lactamase (BLA) and the Escherichia coli maltose binding protein (MBP) to create a family of MBP-BLA hybrids in which maltose is a positive or negative effector of ␤-lactam hydrolysis. Some of these MBP-BLA switches were effectively ''on-off'' in nature, with maltose altering catalytic activity by as much as 600-fold. The ability of these switches to confer an effector-dependent growth͞no growth phenotype to E. coli cells was exploited to rapidly identify, from a library of 4 ؋ 10 6 variants, MBP-BLA switch variants that respond to sucrose as the effector. The transplantation of these mutations into wild-type MBP converted MBP into a ''sucrose-binding protein,'' illustrating the switches potential as a tool to rapidly identify ligand-binding proteins.allostery ͉ ␤-lactamase ͉ maltose binding protein R egulation of protein activity is fundamental to cellular function.One of the mechanisms a cell uses to modulate the level of protein activity is regulation of the amount of a protein present in a cell. Accordingly, many strategies for engineering control of cellular protein activity have focused on modulation of protein production and degradation, often through small-moleculedependent switches that regulate transcription, translation, localization, degradation, or protein splicing (1) or through the engineering of artificial gene-regulatory networks (2). The key strength of many of these approaches is that they are easily generalized. For example, a switch that regulates transcription of one gene can easily be adapted to regulate transcription of an arbitrary gene. However, the significant limitation of these approaches is the slow dynamics stemming from the indirect nature of the regulation (i.e., protein activity is regulated by controlling the amount of protein and not by regulating the protein's specific activity directly). In addition, modulation is only feasible in the context of the cell and cannot be easily transferred to an in vitro setting.A more satisfying approach is to modulate the protein activity directly, but this presents a considerable design problem. Inhibitors, if they can be found, can only can be used to down-regulate activity, and such a strategy suffers from the fact that one is not free to choose the signal that modulates the activity: The signal must be an inhibitor of the protein. One clever way around this limitation is to engineer the inhibitor such that a third molecule can regulate the inhibitor's affinity for the regulated protein, as was demonstrated for RNA aptamer inhibitors that could be regulated by an organic small molecule (3). Natural allosteric proteins solve this problem by having spatially distinct regulatory and active sites. Ligand binding or covalent modifications at one site affects the output function at a distant site through a conformational change. This mechanism has ...

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Directed evolution of protein switches and their application to the creation of ligand-binding proteins

Guntas

Mansell

Kim

et al. 2005

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

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246

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“…Several steps have been made toward developing possible ''nanoallostery'' modules, many cases of which have used a protein template already known to change conformation upon ligand binding. The approaches involve either mutating residues so that one state is preferentially stable over others (3)(4)(5)(6)(7), manipulating the binding specificity so that unnatural ligands can bind (8)(9)(10), or fusing the template to another protein that can sense the signal (11)(12)(13). Such approaches are subject to the structural and functional limitations imposed on the template during evolution.…”

mentioning

confidence: 99%

Use of sequence duplication to engineer a ligand-triggered, long-distance molecular switch in T4 lysozyme

Yousef

Baase

Matthews

2004

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

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We have designed a molecular switch in a T4 lysozyme construct that controls a large-scale translation of a duplicated helix. As shown by crystal structures of the construct with the switch on and off, the conformational change is triggered by the binding of a ligand (guanidinium ion) to a site that in the wild-type protein was occupied by the guanidino head group of an Arg. In the design template, a duplicated helix is flanked by two loop regions of different stabilities. In the ''on'' state, the N-terminal loop is weakly structured, whereas the C-terminal loop has a well defined conformation that is stabilized by means of nonbonded interactions with the Arg head group. The truncation of the Arg to Ala destabilizes this loop and switches the protein to the ''off'' state, in which the duplicated helix is translocated Ϸ20 Å. Guanidinium binding restores the key interactions, restabilizes the C-terminal loop, and restores the ''on'' state. Thus, the presence of an external ligand, which is unrelated to the catalytic activity of the enzyme, triggers the inserted helix to translate 20 Å away from the binding site. The results illustrate a proposed mechanism for protein evolution in which sequence duplication followed by point mutation can lead to the establishment of new function.T he ability to create and manipulate ligand-induced conformational changes is one of the major challenges in protein engineering and biotechnology. This ability demands a detailed understanding of the interplay between binding, structure, dynamics and energetics (1, 2). Several steps have been made toward developing possible ''nanoallostery'' modules, many cases of which have used a protein template already known to change conformation upon ligand binding. The approaches involve either mutating residues so that one state is preferentially stable over others (3-7), manipulating the binding specificity so that unnatural ligands can bind (8-10), or fusing the template to another protein that can sense the signal (11-13). Such approaches are subject to the structural and functional limitations imposed on the template during evolution. In an experiment notable for the use of different templates (14), allosteric switching was observed when two proteins (ubiquitin and barnase), neither of which undergoes conformational changes in its native form, were fused in such a way that the folding of one protein unfolded the other. However, the lack of a regulatory site that bound ligand(s) limited the ability to switch between the two states.In the present report, a nanostructural module was added to T4 lysozyme. Part of the module, a duplicated secondary structure element, switches conformation upon binding a ligand, in this case guanidinium ion. The ligand is unrelated to the function of the protein, but its binding induces a large-scale conformational change.The reference protein on which the design is based is designated L20 and has been described in ref. 15. In this protein, residues 40-50, corresponding to the B helix of T4 lysozyme, are duplicated ...

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“…1,2 Proteins and peptides can undergo conformation and activity changes in response to changes in pH or temperature, small-molecule binding, 3,4 or enzymatic activity. 5 Such sophisticated input/output control has proven useful in the development of biosensors, 6,7 cell internalizing agents, 5 and selective in vivo diagnostics and therapeutics.…”

Section: Introductionmentioning

confidence: 99%

Proligands with protease‐regulated binding activity identified from cell‐displayed prodomain libraries

Thomas

Daugherty

2009

Protein Science

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A general method was developed for the discovery of protease-activated binding ligands, or proligands, from combinatorial prodomain libraries displayed on the surface of E. coli. Peptide libraries of candidate prodomains were fused with a matrix metalloprotease-2 substrate linker to a vascular endothelial growth factor-binding peptide and sorted using a two-stage flow cytometry screening procedure to isolate proligands that required protease treatment for binding activity. Prodomains that imparted protease-mediated switching activity were identified after three sorting cycles using two unique library design strategies. The best performing proligand exhibited a 100-fold improvement in apparent binding affinity after exposure to protease. This method may prove useful for developing therapeutic and diagnostic ligands with improved systemic targeting specificity.

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Creation of an Allosteric Enzyme by Domain Insertion

Cited by 134 publications

References 48 publications

Directed evolution of protein switches and their application to the creation of ligand-binding proteins

Directed evolution of protein switches and their application to the creation of ligand-binding proteins

Use of sequence duplication to engineer a ligand-triggered, long-distance molecular switch in T4 lysozyme

Proligands with protease‐regulated binding activity identified from cell‐displayed prodomain libraries

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