An evolution-based strategy for engineering allosteric regulation

Pincus, David; Resnekov, Orna; Reynolds, Kimberly A.

doi:10.1088/1478-3975/aa64a4

Cited by 13 publications

(11 citation statements)

References 29 publications

(39 reference statements)

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“…In allosteric regulation, protein activity is modulated by an input effector signal spatially removed from the active site. Allostery is a desirable engineering target because it can yield sensitive, reversible, and rapid control of protein activity in response to diverse inputs [1][2][3].…”

Section: Introductionmentioning

confidence: 99%

Structurally distributed surface sites tune allosteric regulation

McCormick

Russo

Thompson

et al. 2021

eLife

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Our ability to rationally optimize allosteric regulation is limited by incomplete knowledge of the mutations that tune allostery. Are these mutations few or abundant, structurally localized or distributed? To examine this, we conducted saturation mutagenesis of a synthetic allosteric switch in which Dihydrofolate reductase (DHFR) is regulated by a blue-light sensitive LOV2 domain. Using a high-throughput assay wherein DHFR catalytic activity is coupled to E. coli growth, we assessed the impact of 1548 viable DHFR single mutations on allostery. Despite most mutations being deleterious to activity, fewer than 5% of mutations had a statistically significant influence on allostery. Most allostery disrupting mutations were proximal to the LOV2 insertion site. In contrast, allostery enhancing mutations were structurally distributed and enriched on the protein surface. Combining several allostery enhancing mutations yielded near-additive improvements to dynamic range. Our results indicate a path towards optimizing allosteric function through variation at surface sites.

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Section: Introductionmentioning

confidence: 99%

Structurally distributed surface sites tune allosteric regulation

McCormick

Russo

Thompson

et al. 2021

eLife

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“…Moreover, in the case of Hog1, we found that more complex regulatory mechanisms can be instantiated by phosphorylation of sites at sector edges. Overall, these results suggest a general strategy for engineering new cell signaling pathways – in vivo phosphoregulation can in principle be introduced into any soluble protein by targeting negatively charged residues at sector-connected surfaces (35).…”

Section: Discussionmentioning

confidence: 94%

Engineering allosteric regulation in protein kinases

et al. 2018

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Phosphoregulation – in which the addition of a negatively charged phosphate group modulates protein activity – is a common feature of proteins that enables dynamic cellular responses. To understand how new phosphoregulation might be acquired, we mutationally scanned the surface of a prototypical yeast kinase (Kss1) to identify potential regulatory sites. The data reveal a set of spatially distributed “hotspots” that coevolve with the active site and preferentially modulate kinase activity. By engineering simple consensus phosphorylation sites at these hotspots we rewired cell signaling in yeast. Following the same approach in a homolog (Hog1), we introduced new phosphoregulation that modifies localization and signaling dynamics. Beyond synthetic biology, the hotspots are used by the diversity of natural allosteric regulatory mechanisms in the kinase family and exploited in human disease.

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“…Reynolds and coworkers [98] have used this computational tool for scanning domain insertion positions in the enzyme dihydrofolate reductase (DHFR) and inserted a light-sensitive domain (LOV2) into these “hotspots” positions, which allowed the identification of a chimeric protein regulated in vivo by light. Thus, linking of existing allosteric networks in the individual protein domains could lead to allosteric connections [99] (Figure 4(g)). The large data set generated by previous work on building protein switches could serve to expand this approach.…”

Section: Future Perspectivesmentioning

confidence: 99%

Converting a Periplasmic Binding Protein into a Synthetic Biosensing Switch through Domain Insertion

Ribeiro

Amarelle

Ribeiro

et al. 2019

BioMed Research International

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All biosensing platforms rest on two pillars: specific biochemical recognition of a particular analyte and transduction of that recognition into a readily detectable signal. Most existing biosensing technologies utilize proteins that passively bind to their analytes and therefore require wasteful washing steps, specialized reagents, and expensive instruments for detection. To overcome these limitations, protein engineering strategies have been applied to develop new classes of protein-based sensor/actuators, known as protein switches, responding to small molecules. Protein switches change their active state (output) in response to a binding event or physical signal (input) and therefore show a tremendous potential to work as a biosensor. Synthetic protein switches can be created by the fusion between two genes, one coding for a sensor protein (input domain) and the other coding for an actuator protein (output domain) by domain insertion. The binding of a signal molecule to the engineered protein will switch the protein function from an “off” to an “on” state (or vice versa) as desired. The molecular switch could, for example, sense the presence of a metabolite, pollutant, or a biomarker and trigger a cellular response. The potential sensing and response capabilities are enormous; however, the recognition repertoire of natural switches is limited. Thereby, bioengineers have been struggling to expand the toolkit of molecular switches recognition repertoire utilizing periplasmic binding proteins (PBPs) as protein-sensing components. PBPs are a superfamily of bacterial proteins that provide interesting features to engineer biosensors, for instance, immense ligand-binding diversity and high affinity, and undergo large conformational changes in response to ligand binding. The development of these protein switches has yielded insights into the design of protein-based biosensors, particularly in the area of allosteric domain fusions. Here, recent protein engineering approaches for expanding the versatility of protein switches are reviewed, with an emphasis on studies that used PBPs to generate novel switches through protein domain insertion.

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An evolution-based strategy for engineering allosteric regulation

Cited by 13 publications

References 29 publications

Structurally distributed surface sites tune allosteric regulation

Structurally distributed surface sites tune allosteric regulation

Engineering allosteric regulation in protein kinases

Converting a Periplasmic Binding Protein into a Synthetic Biosensing Switch through Domain Insertion

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