Conformational regulation and target-myristoyl switch of calcineurin B homologous protein 3
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eLife assessment
In this work, the authors provide important mechanistic insights into how the intracellular effector protein Calcineurin B homologous protein 3 (CHP3) can be regulated in a calcium-independent manner to expose its lipid binding site. Compelling evidence demonstrates a binding partner protein (NHE1) triggers a conformation change and exposure of the myristoyl group in CHP3 resulting in membrane association. This provides mechanistic insight into the signalling mechanisms achieved by CHP3 in a target-dependent manner, which will be of broad scientific interest.
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Abstract
Calcineurin B homologous protein 3 (CHP3) is an EF-hand Ca 2+ -binding protein involved in regulation of cancerogenesis, cardiac hypertrophy, and neuronal development through interactions with sodium/proton exchangers (NHEs) and signalling proteins. While the importance of Ca 2+ binding and myristoylation for CHP3 function has been recognized, the underlying molecular mechanism remained elusive. In this study, we demonstrate that Ca 2+ binding and myristoylation independently affect the conformation and functions of human CHP3. Ca 2+ binding increased local flexibility and hydrophobicity of CHP3 indicative of an open conformation. The Ca 2+ -bound CHP3 exhibited a higher affinity for NHE1 and associated stronger with lipid membranes compared to the Mg 2+ -bound CHP3, which adopted a closed conformation. Myristoylation enhanced the local flexibility of CHP3 and decreased its affinity to NHE1 independently of the bound ion, but did not affect its binding to lipid membranes. The data exclude the proposed Ca 2+ -myristoyl switch for CHP3. Instead, a Ca 2+ -independent exposure of the myristoyl moiety is induced by binding of the target peptide to CHP3 enhancing its association to lipid membranes. We name this novel regulatory mechanism ‘target-myristoyl switch’. Collectively, the interplay of Ca 2+ binding, myristoylation, and target binding allows for a context-specific regulation of CHP3 functions.
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Author Response
eLife assessment
In this work, the authors provide important mechanistic insights into how the intracellular effector protein Calcineurin B homologous protein 3 (CHP3) can be regulated in a calcium-independent manner to expose its lipid binding site. Compelling evidence demonstrates a binding partner protein (NHE1) triggers a conformation change and exposure of the myristoyl group in CHP3 resulting in membrane association. This provides mechanistic insight into the signalling mechanisms achieved by CHP3 in a target-dependent manner, which will be of broad scientific interest.
Thank you for providing an accompanying eLife assessment. As we slightly modified the name of the novel mechanism to meet the suggestion of reviewer 2, and to emphasize the binding to a lipid membrane, we suggest the following update:
“In this …
Author Response
eLife assessment
In this work, the authors provide important mechanistic insights into how the intracellular effector protein Calcineurin B homologous protein 3 (CHP3) can be regulated in a calcium-independent manner to expose its lipid binding site. Compelling evidence demonstrates a binding partner protein (NHE1) triggers a conformation change and exposure of the myristoyl group in CHP3 resulting in membrane association. This provides mechanistic insight into the signalling mechanisms achieved by CHP3 in a target-dependent manner, which will be of broad scientific interest.
Thank you for providing an accompanying eLife assessment. As we slightly modified the name of the novel mechanism to meet the suggestion of reviewer 2, and to emphasize the binding to a lipid membrane, we suggest the following update:
“In this work, the authors provide important mechanistic insights into how the intracellular effector protein Calcineurin B homologous protein 3 (CHP3) can be regulated in a calcium-independent manner to expose its lipid membrane binding site. Compelling evidence demonstrates a binding partner protein (NHE1) triggers a conformation change and exposure of the myristoyl group in CHP3 resulting in membrane association. This provides mechanistic insight into the signalling mechanisms achieved by CHP3 in a target-binding dependent manner, which will be of broad scientific interest.
Reviewer #1 (Public Review):
This study examines the effects of Ca2+ and NHE1 peptide binding on the conformation of CHP3, one of three related calcineurin-homologous proteins. One question that is addressed is whether Ca2+ binding triggers membrane association of the myristoyl group, a so-called "Ca2+-myristoyl switch". This is convincingly demonstrated to not be the case by the experiment in Figure 6B: unlike myristoylated recoverin, mCHP3 does not show enhanced association with liposomes. In the presence of a target peptide, however, myristoylation enhances membrane association. Curiously, this interaction is not Ca2+ dependent, but the membrane association of the non-myristoylated CHP3 is Ca2+-dependent.
My concerns with this study relate to physiological relevance. First, it is unclear if Ca2+ binding has a regulatory function in any of the CHP proteins. The authors state that CHP1 and CHP2 have Ca2+ binding affinities <100 nM, so these proteins are likely saturated with Ca2+ under all physiological conditions. On the other hand, CHP3 binds Ca2+ with a Kd of 8 micromolar (in the presence of physiological concentrations of Mg2+) so it will be largely unbound under most normal cellular concentrations of Ca2+ which are in the submicromolar range. Free Ca2+ rarely reaches 1 micromolar under non-pathological concentrations, and if it does, the fraction of CHP3 bound to Ca2+ should be estimated for context. Given these caveats, I am not convinced that experiments done with millimolar concentrations of Ca2+ (e.g., Figures 2, 3, 6) are physiologically informative.
Precise knowledge on the distinct and isoform-specific molecular basis of the important physiological roles of calcineurin homologous proteins is only emerging. Here, we ruled out the suggested Ca2+-myristoyl switch and showed that instead, target-binding (NHE1-peptide) induces membrane association of myristoylated CHP3. In respect to Ca2+ response, we showed in this work and previous studies that all CHPs undergo Ca2+-induced conformational changes, a feature that is required for EFCaBPs to act as Ca2+ sensor. Millimolar Ca2+ concentrations are commonly used in this type of in vitro characterization to ensure uniform conformational states of the protein, thus we followed this approach. We agree that in future studies, the distinct molecular responses to Ca2+ signals have to be studied in cellular context. So far, one can state that for CHP1 and CHP2, affinities for Ca2+ were reported with Kd values of ~90 nM determined in vitro in the absence of Mg2+. This is close to the cellular Ca2+ concentration in the resting cell, but would not lead to saturation of all CHP1 or CHP2 molecules in the cell with Ca2+. The presence of Mg2+ in the cell may further attenuate the affinity of CHPs for Ca2+. One cannot exclude, that CHP1 and CHP2 could respond to Ca2+ signals in the cell. For target-free CHP3, a Kd of 3.5 µM for Ca2+ in the presence of Mg2+ was reported, so it is unlikely to respond to Ca2+-signals. However, target binding (at least for NHE1) does not require the presence of Ca2+ (as shown in the present study), and target binding can increase Ca2+-binding affinity of EFCaBPs up to 100 fold (reported 45-fold for CHP1 and 42-fold for CHP2). Target-bound CHP3 might have an affinity for Ca2+ that enables a response to Ca2+-signals.
Reviewer #2 (Public Review):
The manuscript by Becker and coworkers describes a target-binding myristoyl switch in the calcium-binding EF hand protein CHP3 using one of its targets, the NHE1. The work uses a suite of biophysical methods including SEC, nanoDSF, fluorescence, and native MS, to address conformations, ligand binding (Ca2+, Mg2+, NHE1), and liposome association, pinpointing a conformation switch which they term a target-dependent myristoyl switch. The strength of the manuscript is a convincing mapping of the different conformations and the conclusion that target binding, and not Ca2+ binding is necessary to expel the lipid from the protein, and that this jointly enhances membrane binding. It would have been even stronger if additional structural data had been included to address the properties of the different states and hence support if there indeed are changes in dynamics and flexibility.
We are thankful to Reviewer #2 for a number of valuable comments on our manuscript which we addressed systematically to enhance description and discussion of our results. Specifically, we clarified the use of conformation, flexibility, state, dynamics and now consistently refer to distinct states of the protein (Ca2+-, Mg2+- and apo-state) as well as defined conformations (open, closed and target-bound). We agree that structural characterization is important, yet, it is beyond the focus of the present biochemical and biophysical characterization and needs to be addressed in future studies.
Reviewer #3 (Public Review):
This work provides new insights into the regulation of the intracellular effector protein Calcineurin B homologous protein 3 (CHP3). The authors precisely delineate how intracellular calcium signals and myristoylation affect the binding of CHP3 to lipid membranes and the sodium/proton exchanger NHE1. Different mechanisms are known to trigger the exposure of the myristoyl-moiety in the calcium-binding protein family and CHP3 was proposed to use a "calcium-myristoyl switch", which leads to exposure of the myristoyl group due to conformational changes in the protein triggered by calcium-binding. Becker and Fuchs et al. now demonstrate that CHP3 uses a novel mechanism, in which not calcium-binding but binding to the target protein NHE1 triggers exposure of its myristoyl-group. This paper represents a detailed functional characterization of CHP3 and the maximum level of mechanistic interpretation that can be achieved without high-resolution structural information.
The conclusions of this paper are fully supported by the data.
Strengths
The protein biochemistry is of an exceptionally high level, both with respect to the quality of the material and the stringency with which the authors assess and assure the protein quality. The authors purify CHP3 without any affinity tags, and thus in its most representative relevant state. Their validations indicate that complete myristoylation of CHP3 is achieved and that all protein is functional with respect to calcium binding.
The authors go to extensive lengths to convince themselves of the quality of their data and their interpretation. They use an extensive amount of replicates, including both biological and technical replicates. Assays and experimental procedures are verified using model proteins, such as Recoverin. In addition, the authors employ an extensive set of complementary approaches to assure their observations are universal.
We highly appreciate the positive feedback of Reviewer #3 on our experimental design and quality of biochemical data.
Weaknesses
A small weakness is the fact that the interpretation in terms of mechanistic insights contributed by some of the assays employed is rather limited, resulting in comparably unprecise descriptions of the state of the protein such as "affects the conformation and/or flexibility of CHP3" or the "open" and "closed" conformations. As indicated by the authors, structural studies are required to precisely detail the conformational states and delineate their mechanism of action.
We updated the manuscript for a stringent use of the descriptions “conformation”, “state” and “flexibility” to match terminology commonly used for EFCaBPs.
The authors imply that the major form of CHP3 is the myristoylated state. However, it remains unclear whether the source of the biological material, which appears to be membrane-only, already implies a significant experimental bias that only allows (or highly favors) the identification of myristoylated CHP3. Without a calcium-signal, unmyristoylated CHP may not associate with membranes, or be less strong, resulting in its depletion upon isolation of the vesicles.
We agree that our data are based on membrane fractions, so referring to the “major form of CHP3” was misleading. We updated two sentences as follows: “Finally, we investigated the N-terminal myristoylation status of membrane associated CHP3 in vivo using liquid-chromatography coupled mass spectrometry (LC-MS/MS). ………Together, this suggests that myristoylated CHP3 is both NHE1-associated and membrane-anchored in agreement with a target-induced exposure and membrane integration of the N-terminal myristoyl moiety.”
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eLife assessment
In this work, the authors provide important mechanistic insights into how the intracellular effector protein Calcineurin B homologous protein 3 (CHP3) can be regulated in a calcium-independent manner to expose its lipid binding site. Compelling evidence demonstrates a binding partner protein (NHE1) triggers a conformation change and exposure of the myristoyl group in CHP3 resulting in membrane association. This provides mechanistic insight into the signalling mechanisms achieved by CHP3 in a target-dependent manner, which will be of broad scientific interest.
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Reviewer #1 (Public Review):
This study examines the effects of Ca2+ and NHE1 peptide binding on the conformation of CHP3, one of three related calcineurin-homologous proteins. One question that is addressed is whether Ca2+ binding triggers membrane association of the myristoyl group, a so-called "Ca2+-myristoyl switch". This is convincingly demonstrated to not be the case by the experiment in Figure 6B: unlike myristoylated recoverin, mCHP3 does not show enhanced association with liposomes. In the presence of a target peptide, however, myristoylation enhances membrane association. Curiously, this interaction is not Ca2+ dependent, but the membrane association of the non-myristoylated CHP3 is Ca2+-dependent.
My concerns with this study relate to physiological relevance. First, it is unclear if Ca2+ binding has a regulatory function in …
Reviewer #1 (Public Review):
This study examines the effects of Ca2+ and NHE1 peptide binding on the conformation of CHP3, one of three related calcineurin-homologous proteins. One question that is addressed is whether Ca2+ binding triggers membrane association of the myristoyl group, a so-called "Ca2+-myristoyl switch". This is convincingly demonstrated to not be the case by the experiment in Figure 6B: unlike myristoylated recoverin, mCHP3 does not show enhanced association with liposomes. In the presence of a target peptide, however, myristoylation enhances membrane association. Curiously, this interaction is not Ca2+ dependent, but the membrane association of the non-myristoylated CHP3 is Ca2+-dependent.
My concerns with this study relate to physiological relevance. First, it is unclear if Ca2+ binding has a regulatory function in any of the CHP proteins. The authors state that CHP1 and CHP2 have Ca2+ binding affinities <100 nM, so these proteins are likely saturated with Ca2+ under all physiological conditions. On the other hand, CHP3 binds Ca2+ with a Kd of 8 micromolar (in the presence of physiological concentrations of Mg2+) so it will be largely unbound under most normal cellular concentrations of Ca2+ which are in the submicromolar range. Free Ca2+ rarely reaches 1 micromolar under non-pathological concentrations, and if it does, the fraction of CHP3 bound to Ca2+ should be estimated for context. Given these caveats, I am not convinced that experiments done with millimolar concentrations of Ca2+ (e.g., Figures 2, 3, 6) are physiologically informative.
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Reviewer #2 (Public Review):
The manuscript by Becker and coworkers describes a target-binding myristoyl switch in the calcium-binding EF hand protein CHP3 using one of its targets, the NHE1. The work uses a suite of biophysical methods including SEC, nanoDSF, fluorescence, and native MS, to address conformations, ligand binding (Ca2+, Mg2+, NHE1), and liposome association, pinpointing a conformation switch which they term a target-dependent myristoyl switch. The strength of the manuscript is a convincing mapping of the different conformations and the conclusion that target binding, and not Ca2+ binding is necessary to expel the lipid from the protein, and that this jointly enhances membrane binding. It would have been even stronger if additional structural data had been included to address the properties of the different states and …
Reviewer #2 (Public Review):
The manuscript by Becker and coworkers describes a target-binding myristoyl switch in the calcium-binding EF hand protein CHP3 using one of its targets, the NHE1. The work uses a suite of biophysical methods including SEC, nanoDSF, fluorescence, and native MS, to address conformations, ligand binding (Ca2+, Mg2+, NHE1), and liposome association, pinpointing a conformation switch which they term a target-dependent myristoyl switch. The strength of the manuscript is a convincing mapping of the different conformations and the conclusion that target binding, and not Ca2+ binding is necessary to expel the lipid from the protein, and that this jointly enhances membrane binding. It would have been even stronger if additional structural data had been included to address the properties of the different states and hence support if there indeed are changes in dynamics and flexibility.
-
Reviewer #3 (Public Review):
This work provides new insights into the regulation of the intracellular effector protein Calcineurin B homologous protein 3 (CHP3). The authors precisely delineate how intracellular calcium signals and myristoylation affect the binding of CHP3 to lipid membranes and the sodium/proton exchanger NHE1. Different mechanisms are known to trigger the exposure of the myristoyl-moiety in the calcium-binding protein family and CHP3 was proposed to use a "calcium-myristoyl switch", which leads to exposure of the myristoyl group due to conformational changes in the protein triggered by calcium-binding. Becker and Fuchs et al. now demonstrate that CHP3 uses a novel mechanism, in which not calcium-binding but binding to the target protein NHE1 triggers exposure of its myristoyl-group. This paper represents a detailed …
Reviewer #3 (Public Review):
This work provides new insights into the regulation of the intracellular effector protein Calcineurin B homologous protein 3 (CHP3). The authors precisely delineate how intracellular calcium signals and myristoylation affect the binding of CHP3 to lipid membranes and the sodium/proton exchanger NHE1. Different mechanisms are known to trigger the exposure of the myristoyl-moiety in the calcium-binding protein family and CHP3 was proposed to use a "calcium-myristoyl switch", which leads to exposure of the myristoyl group due to conformational changes in the protein triggered by calcium-binding. Becker and Fuchs et al. now demonstrate that CHP3 uses a novel mechanism, in which not calcium-binding but binding to the target protein NHE1 triggers exposure of its myristoyl-group. This paper represents a detailed functional characterization of CHP3 and the maximum level of mechanistic interpretation that can be achieved without high-resolution structural information.
The conclusions of this paper are fully supported by the data.
Strengths
The protein biochemistry is of an exceptionally high level, both with respect to the quality of the material and the stringency with which the authors assess and assure the protein quality. The authors purify CHP3 without any affinity tags, and thus in its most representative relevant state. Their validations indicate that complete myristoylation of CHP3 is achieved and that all protein is functional with respect to calcium binding.The authors go to extensive lengths to convince themselves of the quality of their data and their interpretation. They use an extensive amount of replicates, including both biological and technical replicates. Assays and experimental procedures are verified using model proteins, such as Recoverin. In addition, the authors employ an extensive set of complementary approaches to assure their observations are universal.
Weaknesses
A small weakness is the fact that the interpretation in terms of mechanistic insights contributed by some of the assays employed is rather limited, resulting in comparably unprecise descriptions of the state of the protein such as "affects the conformation and/or flexibility of CHP3" or the "open" and "closed" conformations. As indicated by the authors, structural studies are required to precisely detail the conformational states and delineate their mechanism of action.The authors imply that the major form of CHP3 is the myristoylated state. However, it remains unclear whether the source of the biological material, which appears to be membrane-only, already implies a significant experimental bias that only allows (or highly favors) the identification of myristoylated CHP3. Without a calcium-signal, unmyristoylated CHP may not associate with membranes, or be less strong, resulting in its depletion upon isolation of the vesicles.
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