Non‐allosteric enzyme switches possess larger effector‐induced changes in thermodynamic stability than their non‐switch analogs

Choi, Jay H.; San, Angela; Ostermeier, Marc

doi:10.1002/pro.2234

Cited by 15 publications

(30 citation statements)

References 33 publications

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“…Previous efforts to develop switchable proteins by combining sensor and effector domains (39,40) have yielded both ‘true’ allosteric molecules as well as ‘phenotypic’ constructs. Activity differences of the latter can often be attributed to increased cellular protein accumulation in the ‘on’ condition of the switch (40). …”

Section: Resultsmentioning

confidence: 99%

Engineering of temperature- and light-switchable Cas9 variants

Richter

Fonfara

Bouazza

et al. 2013

Nucleic Acids Research

114

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Sensory photoreceptors have enabled non-invasive and spatiotemporal control of numerous biological processes. Photoreceptor engineering has expanded the repertoire beyond natural receptors, but to date no generally applicable strategy exists towards constructing light-regulated protein actuators of arbitrary function. We hence explored whether the homodimeric Rhodobacter sphaeroides light-oxygen-voltage (LOV) domain (RsLOV) that dissociates upon blue-light exposure can confer light sensitivity onto effector proteins, via a mechanism of light-induced functional site release. We chose the RNA-guided programmable DNA endonuclease Cas9 as proof-of-principle effector, and constructed a comprehensive library of RsLOV inserted throughout the Cas9 protein. Screening with a high-throughput assay based on transcriptional repression in Escherichia coli yielded paRC9, a moderately light-activatable variant. As domain insertion can lead to protein destabilization, we also screened the library for temperature-sensitive variants and isolated tsRC9, a variant with robust activity at 29°C but negligible activity at 37°C. Biochemical assays confirmed temperature-dependent DNA cleavage and binding for tsRC9, but indicated that the light sensitivity of paRC9 is specific to the cellular setting. Using tsRC9, the first temperature-sensitive Cas9 variant, we demonstrate temperature-dependent transcriptional control over ectopic and endogenous genetic loci. Taken together, RsLOV can confer light sensitivity onto an unrelated effector; unexpectedly, the same LOV domain can also impart strong temperature sensitivity.

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Section: Resultsmentioning

confidence: 99%

Engineering of temperature- and light-switchable Cas9 variants

Richter

Fonfara

Bouazza

et al. 2013

Nucleic Acids Research

114

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“…There are specific design advantages associated with using each distinct mechanism: these may include intrinsic differences in dynamic range 52 , selectivity 53,54 , kinetics 51,55 , and the ability to modulate signals by altering cellular accumulation through resistance to proteolysis 50,56 . The precise functional requirements associated with responding to a particular stimulus can therefore be met, in part, by selecting a recognition mechanism that will confer the desired kinetics, sensitivity, and dynamic range 7 .…”

Section: Discussionmentioning

confidence: 99%

The Designability of Protein Switches by Chemical Rescue of Structure: Mechanisms of Inactivation and Reactivation

Xia¹,

DiPrimio

Keppel³

et al. 2013

J. Am. Chem. Soc.

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The ability to selectively activate function of particular proteins via pharmacological agents is a longstanding goal in chemical biology. Recently, we reported an approach for designing a de novo allosteric effector site directly into the catalytic domain of an enzyme. This approach is distinct from traditional chemical rescue of enzymes in that it relies on disruption and restoration of structure, rather than active site chemistry, as a means to achieve modulate function. However, rationally identifying analogous de novo binding sites in other enzymes represents a key challenge for extending this approach to introduce allosteric control into other enzymes. Here we show that mutation sites leading to protein inactivation via tryptophan-to-glycine substitution and allowing (partial) reactivation by the subsequent addition of indole are remarkably frequent. Through a suite of methods including a cell-based reporter assay, computational structure prediction and energetic analysis, fluorescence studies, enzymology, pulse proteolysis, x-ray crystallography and hydrogen-deuterium mass spectrometry we find that these switchable proteins are most commonly modulated indirectly, through control of protein stability. Addition of indole in these cases rescues activity not by reverting a discrete conformational change, as we had observed in the sole previously reported example, but rather rescues activity by restoring protein stability. This important finding will dramatically impact the design of future switches and sensors built by this approach, since evaluating stability differences associated with cavity-forming mutations is a far more tractable task than predicting allosteric conformational changes. By analogy to natural signaling systems, the insights from this study further raise the exciting prospect of modulating stability to design optimal recognition properties into future de novo switches and sensors built through chemical rescue of structure.

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“…This phenomenon along with MBP317‐347's allosteric properties contributes to the maltose‐dependent ampicillin resistance phenotype. We have shown that an increase in abundance can result from maltose‐binding increasing the cellular stability (thermodynamic and proteolytic) of the switch …”

Section: Resultsmentioning

confidence: 99%

“…We subsequently developed media and growth conditions that allowed substantial improvements in mutant protein expression in the absence of maltose. The key attributes of the process were M9 salts in the growth media with fructose and glycerol as carbon sources . Additionally, we used image analysis of SDS‐PAGE gels to quantify the yield of protein and thus could normalize the enzymatic rates on a protein basis.…”

Section: Resultsmentioning

confidence: 99%

“…We have shown that an increase in abundance can result from maltose-binding increasing the cellular stability (thermodynamic and proteolytic) of the switch. 31 For each member of our focused library, we examined switch abundance for cells grown in the presence and absence of maltose by a western dot blot analysis on lysates of overnight cultures [ Fig. 4(A,B)].…”

Section: Mgc Mal Provides Reasonable Estimates Of Ligand Binding Affimentioning

confidence: 99%

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The interplay between effector binding and allostery in an engineered protein switch

2016

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The protein design rules for engineering allosteric regulation are not well understood. A fundamental understanding of the determinants of ligand binding in an allosteric context could facilitate the design and construction of versatile protein switches and biosensors. Here, we conducted extensive in vitro and in vivo characterization of the effects of 285 unique point mutations at 15 residues in the maltose-binding pocket of the maltose-activated b-lactamase MBP317-347. MBP317-347 is an allosteric enzyme formed by the insertion of TEM-1 b-lactamase into the E. coli maltose binding protein (MBP). We find that the maltose-dependent resistance to ampicillin conferred to the cells by the MBP317-347 switch gene (the switch phenotype) is very robust to mutations, with most mutations slightly improving the switch phenotype. We identified 15 mutations that improved switch performance from twofold to 22-fold, primarily by decreasing the catalytic activity in the absence of maltose, perhaps by disrupting interactions that cause a small fraction of MBP in solution to exist in a partially closed state in the absence of maltose. Other notable mutations include K15D and K15H that increased maltose affinity 30-fold and Y155K and Y155R that compromised switching by diminishing the ability of maltose to increase catalytic activity. The data also provided insights into normal MBP physiology, as select mutations at D14, W62, and F156 retained high maltose affinity but abolished the switch's ability to substitute for MBP in the transport of maltose into the cell. The results reveal the complex relationship between ligand binding and allostery in this engineered switch.

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Non‐allosteric enzyme switches possess larger effector‐induced changes in thermodynamic stability than their non‐switch analogs

Cited by 15 publications

References 33 publications

Engineering of temperature- and light-switchable Cas9 variants

Engineering of temperature- and light-switchable Cas9 variants

The Designability of Protein Switches by Chemical Rescue of Structure: Mechanisms of Inactivation and Reactivation

The interplay between effector binding and allostery in an engineered protein switch

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