Systematic control of protein interaction using a modular ER/K α-helix linker

Abstract

Cellular functions of proteins are strongly influenced by their interactions with other proteins. The frequency of protein interactions is a function of the local concentration of two proteins and their affinity for one another. When two proteins are tethered together, the link between them influences their effective concentrations and therefore the frequency of their interaction. Currently no methods exist to systematically vary the effective concentration within this intramolecular interaction. Here we outline a modular, genetically encoded linker, namely, an ER∕K [genetically encoded polypeptide motif based on alternating sequence of approximately four glutamic acid (E) followed by approximately four arginine (R) or lysine (K) residues] single α-helix that can be used to regulate the frequency of interaction between two proteins, or between a protein and a peptide, one at each end. We exploit the wide range of interaction affinities between calmodulin and its binding peptides, combined with FRET to determine the effect of the ER∕K α-helix in regulating protein interactions. We find that increasing the length of the ER∕K α-helix reduces the on rate of the intramolecular interaction without significantly affecting the off rate, regardless of the affinity of the bimolecular interaction. We outline a genetically encoded approach to determine the dissociation constant for both moderate (micromolar K d ) and strong (nanomolar K d ) protein interactions. Our studies demonstrate the use of the ER∕K α-helix to systematically engineer FRET biosensors that detect changes in concentration or affinity of interacting proteins, and modulate enzyme autoinhibition. Our findings are consistent with the ER∕K α-helix as a worm-like chain with rare, stochastic breaks in the helix backbone that may account for the behavior of myosin VI stepping along actin.systematic protein affinity strength | modulation | cell signaling | biochemistry I nteractions between proteins are essential for cellular function. The association of proteins facilitates their localization to, and therefore function in, specific subcellular compartments (1). Proteins often associate to form a macromolecular complex that can perform functions that the individual components cannot (2). Brief bimolecular interactions among a series of proteins are characteristic of signaling cascades that are essential to propagate and amplify an extracellular signal across the cell and elicit an appropriate response (3). Interactions between proteins are dictated by their binding affinity and the local cellular concentrations of the proteins involved. In addition to bimolecular interactions between proteins, many proteins have intramolecular interactions between distinct domains that regulate their function (4). Such intramolecular interactions within a protein in turn are regulated by the free solution binding affinity of the two domains and the effective concentration experienced by the two domains within the protein.Regulation of intermolecular and intramolecular protein int...

Substrate Affinity Differentially Influences Protein Kinase C Regulation and Inhibitor Potency

“…This finding is consistent with the dominant role of the pseudosubstrate in PKC autoinhibition (20). (14,15), which separates the catalytic domain of PKC␣ and a variety of short peptides Յ14 residues in length (Fig. 2).…”

“…2). The linker itself contains an ER/K ␣-helix, ϳ10 nm in length, flanked by a FRET donor (mCerulean or GFP) and a FRET acceptor (mCitrine or mCherry, respectively) (14). The ER/K FRET linker also contains an N-terminal tobacco etch virus (TEV) 3 protease cleavage site, and each discrete unit of this ER/K linker is separated by (Gly-Ser-Gly) 2-4 linkers (GSG) to provide rotational freedom.…”

“…In one set, we made a sensor where peptide number 14 was separated from the catalytic domain by either a 10-or 30-nm ER/K linker. By increasing the linker length, we are changing the effective concentration of the peptide for the catalytic domain from ϳ10 M to 100 nM (14). Reducing the effective concentration should weaken the apparent peptide affinity, as observed by a decrease in the FRET ratio, and result in a higher basal activity of this sensor.…”

“…Consequently, only a few crystal structures have thus far been reported for the kinase catalytic domain bound to substrate (4). Here, we instead use a recently developed approach to compare weak ( M) interactions between any two proteins or protein domains (14,15). We apply this approach to directly compare the binding affinities of a kinase catalytic domain for a variety of weak binding substrate peptides.…”

Visualizing and Manipulating Focal Adhesion Kinase Regulation in Live Cells

“…In this study, we derive insights into these important questions with a toolbox of FAK FRET sensors engineered using a new technique, systematic protein affinity strength modulation (SPASM), that probes specifically for changes in the affinity of interaction between two proteins or protein domains in live cells (28). The SPASM sensors retain all domains of native FAK and control the interaction between the FERM and kinase domains with a genetically encoded ER/K linker, the length of which controls the strength of interaction between the FERM and kinase domains.…”