Control of protein signaling using a computationally designed GTPase/GEF orthogonal pair

Kapp, Gregory T.; Liu, Sen; Stein, Amelie; Wong, Derek; Reményi, Attila; Yeh, Brian J.; Fraser, James S.; Taunton, Jack; Lim, Wendell A.; Kortemme, Tanja

doi:10.1073/pnas.1114487109

Cited by 76 publications

(74 citation statements)

References 38 publications

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“…Here, a "computational second-site suppressor" simulation aims to predict mutations in both partners of a protein-protein interface that would be destabilizing when only one of the partners is mutated, but stabilizing if both partners are changed simultaneously (8). This approach has previously been applied to redesign the specificity of a diverse set of interactions, including a DNase-inhibitor pair (8,9), a small GTPase and its guanine exchange factor (10), as well as the interaction between a ubiquitin ligase and a ubiquitin-conjugating enzyme (11). Despite these success cases, the extent to which a designed specificity switch is transferrable between homologous domains with structurally conserved interaction sites is unknown.…”

mentioning

confidence: 99%

Quantification of the transferability of a designed protein specificity switch reveals extensive epistasis in molecular recognition

Melero

Ollikainen

Harwood

et al. 2014

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

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Reengineering protein-protein recognition is an important route to dissecting and controlling complex interaction networks. Experimental approaches have used the strategy of "second-site suppressors," where a functional interaction is inferred between two proteins if a mutation in one protein can be compensated by a mutation in the second. Mimicking this strategy, computational design has been applied successfully to change protein recognition specificity by predicting such sets of compensatory mutations in protein-protein interfaces. To extend this approach, it would be advantageous to be able to "transplant" existing engineered and experimentally validated specificity changes to other homologous protein-protein complexes. Here, we test this strategy by designing a pair of mutations that modulates peptide recognition specificity in the Syntrophin PDZ domain, confirming the designed interaction biochemically and structurally, and then transplanting the mutations into the context of five related PDZ domain-peptide complexes. We find a wide range of energetic effects of identical mutations in structurally similar positions, revealing a dramatic context dependence (epistasis) of designed mutations in homologous protein-protein interactions. To better understand the structural basis of this context dependence, we apply a structure-based computational model that recapitulates these energetic effects and we use this model to make and validate forward predictions. Although the context dependence of these mutations is captured by computational predictions, our results both highlight the considerable difficulties in designing protein-protein interactions and provide challenging benchmark cases for the development of improved protein modeling and design methods that accurately account for the context. computational design | recognition specificity | promiscuity | protein interaction domains | interface evolution M any protein-protein interactions are mediated by small modular protein recognition domains (1). These interaction modules, such as PDZ, SH3, and WW domains, generally recognize their protein partners using a structurally conserved binding site, and there are often tens or even hundreds of proteins containing a given type of recognition domain expressed simultaneously in a cell or organism (2). This repeated use of recognition modules with conserved structures poses the following question: how do cells maintain the specificity of binding interactions when so many members of the same domain family are present? Moreover, to what extent do different domain family members in fact have distinct or overlapping preferences for binding their partners? Addressing these questions is of considerable importance, because a significant fraction of protein interactions in a cell is mediated a limited number of protein interaction domain families (1). Despite a large amount of information on the biochemical recognition preferences of many domain members in vitro (3-5), much less is known about the actual extent of specificity an...

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mentioning

confidence: 99%

Quantification of the transferability of a designed protein specificity switch reveals extensive epistasis in molecular recognition

Melero

Ollikainen

Harwood

et al. 2014

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

Self Cite

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“…The programmable aspect of these synthetic GEFs was the ability to tie precise cytoskeletal output responses with previously unconnected signaling pathways (Yeh et al 2007). Kapp et al (2012) improved upon the idea of integrating synthetic components into pre-existing signaling pathways and engineered a protein-protein pair that remained orthogonal to their wild-type counterparts, i.e., created an orthogonal protein interaction. Using computational methods, they engineered a GTPase Cdc42-Intersectin, inducible with a small molecule, that can interface only with each other and function in their normal capacity in the host signaling cascade (Kapp et al 2012).…”

Section: Mammalian Cellsmentioning

confidence: 98%

“…Kapp et al (2012) improved upon the idea of integrating synthetic components into pre-existing signaling pathways and engineered a protein-protein pair that remained orthogonal to their wild-type counterparts, i.e., created an orthogonal protein interaction. Using computational methods, they engineered a GTPase Cdc42-Intersectin, inducible with a small molecule, that can interface only with each other and function in their normal capacity in the host signaling cascade (Kapp et al 2012). Designer protein-protein interactions represent an important approach in synthetic signaling.…”

Section: Mammalian Cellsmentioning

confidence: 98%

Synthetic biology of cell signaling

Hansen

Benenson

2015

Nat Comput

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Synthetic biology often takes cues from complex natural networks and pathways to create novel biological systems. The design modalities used in synthetic systems generally follow the different classes of gene regulation in cells such as transcriptional, post-transcriptional and post-translational regulation. The latter class is most prominent in cell signaling pathways that operate via multitude of protein-protein interactions. Engineering signaling pathways is a relatively unexplored area. This review discussed the work toward signaling pathway engineering in diverse biological contexts including bacteria, yeast, vertebrate, and plant cells. While creating synthetic signaling cascades is perhaps one of the most challenging tasks in synthetic biology, potential implications can be far-reaching and include new tools for programming cells and tissues, artificial developmental processes, and therapeutic tools.

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“…Since engineered GPCRs activating non-native pathways may interfere with other native important G protein or beta—arrestin dependent signaling functions in the cell, there is growing interest in creating minimal orthogonal signaling systems [62•]. In such approaches, a designed receptor will only signal through a designed effector avoiding cross-reactivity with native cellular effectors.…”

Section: Manipulating Receptor Signaling Through Design Of Intrinsmentioning

confidence: 99%

Reprogramming cellular functions with engineered membrane proteins

Arber

Young

Barth

2017

Current Opinion in Biotechnology

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Taking inspiration from Nature, synthetic biology utilizes and modifies biological components to expand the range of biological functions for engineering new practical devices and therapeutics. While early breakthroughs mainly concerned the design of gene circuits, recent efforts have focused on engineering signaling pathways to reprogram cellular functions. Since signal transduction across cell membranes initiates and controls intracellular signaling, membrane receptors have been targeted by diverse protein engineering approaches despite limited mechanistic understanding of their function. The modular architecture of several receptor families has enabled the empirical construction of chimeric receptors combining domains from distinct native receptors which have found successful immunotherapeutic applications. Meanwhile, progress in membrane protein structure determination, computational modeling and rational design promise to foster the engineering of a broader range of membrane receptor functions. Marrying empirical and rational membrane protein engineering approaches should enable the reprogramming of cells with widely diverse fine-tuned functions.

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Control of protein signaling using a computationally designed GTPase/GEF orthogonal pair

Cited by 76 publications

References 38 publications

Quantification of the transferability of a designed protein specificity switch reveals extensive epistasis in molecular recognition

Quantification of the transferability of a designed protein specificity switch reveals extensive epistasis in molecular recognition

Synthetic biology of cell signaling

Reprogramming cellular functions with engineered membrane proteins

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