Structures of PKA–phospholamban complexes reveal a mechanism of familial dilated cardiomyopathy
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Curated by eLife
Evaluation Summary:
Protein kinase A phosphorylation of phospholamban (PLN) is part of the "fight or flight" response, which ultimately increases the force of cardiac contraction. Mutations in PLN have been linked to familial dilated cardiomyopathy (DCM). Crystal structures of wild-type and mutant PLN in complex with the PKA catalytic domain provide insights into both the nature of the complex, and potential mechanisms by which DCM mutations may cause disease. This paper is of interest to scientists interested in the mechanism of substrate recruitment by protein kinases, and particularly those who have an interest in understanding the mechanism of mutations associated with dilated cardiomyopathy.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Abstract
Several mutations identified in phospholamban (PLN) have been linked to familial dilated cardiomyopathy (DCM) and heart failure, yet the underlying molecular mechanism remains controversial. PLN interacts with sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) and regulates calcium uptake, which is modulated by the protein kinase A (PKA)-dependent phosphorylation of PLN during the fight-or-flight response. Here, we present the crystal structures of the catalytic domain of mouse PKA in complex with wild-type and DCM-mutant PLNs. Our structures, combined with the results from other biophysical and biochemical assays, reveal a common disease mechanism: the mutations in PLN reduce its phosphorylation level by changing its conformation and weakening its interactions with PKA. In addition, we demonstrate that another more ubiquitous SERCA-regulatory peptide, called another-regulin (ALN), shares a similar mechanism mediated by PKA in regulating SERCA activity.
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Author Response:
Reviewer #1 (Public Review):
In this paper, Qin et al. investigated the molecular mechanism of phospholamban (PLN) linked dilated cardiomyopathy (DCM), using structural approaches combined with biophysical measurements. Structures of the catalytic domain of protein kinase A (PKAc) in complex with PLN peptides (both wild-type and the R9C and A11E DCM mutants) provide insights into the mechanism of substrate recruitment and how it is perturbed in the disease state. Qin et al. show convincingly that the mutant peptides all have lower affinity for PKA than the wild-type peptide, suggesting models in which heterozygous DCM mutations act via sequestering PKA and thereby preventing phosphorylation of the wild-type peptide may be incorrect.
The authors highlight significant differences between their structure of the …
Author Response:
Reviewer #1 (Public Review):
In this paper, Qin et al. investigated the molecular mechanism of phospholamban (PLN) linked dilated cardiomyopathy (DCM), using structural approaches combined with biophysical measurements. Structures of the catalytic domain of protein kinase A (PKAc) in complex with PLN peptides (both wild-type and the R9C and A11E DCM mutants) provide insights into the mechanism of substrate recruitment and how it is perturbed in the disease state. Qin et al. show convincingly that the mutant peptides all have lower affinity for PKA than the wild-type peptide, suggesting models in which heterozygous DCM mutations act via sequestering PKA and thereby preventing phosphorylation of the wild-type peptide may be incorrect.
The authors highlight significant differences between their structure of the WT-PLN:PKAc complex, which has a 1:1 stoichiometry, and a previous structure of the complex (PDB 3O7L), which has 1 PLN bound between two PKAc monomers (a 1:2 complex). The authors posit that the stoichiometry observed in 3O7L is an artifact of the crystal lattice, and does not occur in solution, supporting this with analysis of the elution volumes of the peptide complexes on size exclusion chromatography compared to PKAc alone. They further suggest that the AMP-PNP ligand included in the 3O7L structure is not bound, based on analysis of Fo-Fc maps calculated from the deposited coordinates. Inspecting 3O7L I am not convinced of this last point - it seems more likely that a technical error was made in assigning or refining the B-factor of the ligand in 3O7L, because there is clearly density present in SA-omit maps for the nucleotide.
Taking these results together, the authors suggest a mechanism for DCM, whereby mutations in PLN result in lower affinity for PKA, and consequently reduced phosphorylation. This seems plausible and well supported by the data, although in the ADP-Glo assay used here, the reductions in phosphorylation observed for some of the mutant peptides are rather modest. However, as the authors state, it is plausible that even relatively subtle changes in PLN phosphorylation could have substantial effects on Ca2+ homeostasis via increasing SERCA inhibition.
We thank the reviewer for the appreciation of our work.
Reviewer #2 (Public Review):
Strengths:
The authors presented new high-resolution 3D crystal structures of the PKA catalytic domain (PKAc) in complex with PLN WT or mutant peptides (residues 8-22) containing the DCM-associated PLN mutations (R9C or A11E). These are novel and important data given that the present structures are dramatically different from those reported previously. The authors made convincing argument that the 3D model reported previously may result from a crystallization artifact.
By characterizing the interactions between the PKAc domain and PLN WT or DCM-associated mutant peptides using surface plasmon resonance (SPR) analysis, the authors convincingly showed that the DCM-associated PLN mutations at positions 9, 14, and 18 alter the conformation of the PLN peptide and reduce the binding affinity of the PLN peptide with PKAc. These data provide an explanation how some DCM-associated PLN mutations at these positions reduce the level of PKA-dependent phosphorylation of PLN.
The authors also performed nuclear magnetic resonance (NMR) to determine the structural dynamics of PLN WT, R9C, P-Ser16, and P-Thr-17 peptides. These NMR structures combined with the SPR analysis also support their conclusion that PLN phosphorylation and DCM-associated PLN mutations have an impact on its conformation.
We thank the reviewer for the comments.
Weakness:
The present study used PLN-derived peptides (aa 8-22). Although technically challenging, it is important to consider if the full-length WT or mutant PLN will behave the same as those observed with the peptides. This is especially crucial in light of the prior work showing substantially different structures using a different segment of PLN.
We are fully aware of the potential risk to draw conclusion from an isolated peptide instead of the full-length PLN as a transmembrane protein. In the previous study, people showed that the PLN peptide could be used as a good model substrate that gets phosphorylated as efficiently as the full-length PLN protein (L. R. Masterson et al., Dynamics connect substrate recognition to catalysis in protein kinase A. Nat Chem Biol 6, 821-828 (2010); D. K. Ceholski, C. A. Trieber, C. F. Holmes, H. S. Young, Lethal, hereditary mutants of phospholamban elude phosphorylation by protein kinase A. The Journal of biological chemistry 287, 26596-26605 (2012)). These results together with our biochemistry results suggest the tail peptides are indeed active substrates of PKA. Due to the technical difficulty, we were not able to crystallize PKAc in complex with the full-length PLN. To explain the potential difference between the peptides and the full-length PLNs, we added more text in the discussion section “Additionally, the trend of the reduced phosphorylation by DCM mutations can be significantly affected by the oligomerization state of PLN. Ceholski et al. showed that R9C severely inhibits PKA phosphorylation in the context of full-length pentameric PLN, but has a much milder effect in the context of full-length monomeric PLN or an isolated tail peptide [41].”
Although it is convincing that DCM-associated PLN mutations likely reduce the interaction between PKAc and PLN (assuming that the peptides behave the same as the full-length PLN with respect to interaction with PKA) and, as a result, the PKA dependent phosphorylation of the mutant PLN, it is unclear how this impaired interaction between PKA and PLN mutant could explain the effects of the DCM-associated PLN mutations on SERCA function (either reduced or enhanced PLN-dependent inhibition of SERCA, as proposed previously). In this regard, can the authors predict if the DCM-associated PLN R9C mutation reduces or increases SERCA inhibition based on the results of their present study?
It is indeed controversial how PLN mutations cause DCM. Previous studies have shown that the DCM mutations in PLN might change this regulation in either a phosphorylation-dependent or phosphorylation-independent manner. Our results show that the mutations may act through both manners: 1) the mutations reduce the phosphorylation level of PLN, which has been shown to enhance the inhibition of SERCA and inhibit the uptake of Ca2+; 2) the mutations change the conformation of PLN before binding to PKA or SERCA, which could have additional consequences, such as altered assembly state of PLN, phosphorylation of PLN by CaMKII, or changes in interactions of PLN with the lipid membrane. This could impact in either directions, reducing or increasing SERCA inhibition, which is difficult to predict based on our data. We added the explanation in the discussion “While decreased PLN phosphorylation is likely an important contributor to the physiological dysfunction associated with familial DCM, disease-causing mutations in PLN may have additional consequences, such as altered assembly state of PLN, phosphorylation of PLN by CaMKII, or changes in interactions of PLN with the lipid membrane. The influence of such factors on SERCA inhibition are unclear. In principle, they might further increase inhibition of SERCA and act in conjunction with lower PKA-mediated phosphorylation to manifest the disease symptoms. Conversely, it is possible that these factors could decrease the inhibition of SERCA, partially compensating for the decreased phosphorylation level, and mitigating the symptoms.”
It is also unclear how reduced PKA phosphorylation of mutant PLN could lead to DCM. PLN is unlikely to be significantly phosphorylated by PKA at rest (in other words, PLN is likely to be phosphorylated by PKA during stress, i.e. during the adrenergic fight-or-flight response). Therefore, it is puzzling how such reduced PKA-dependent phosphorylation of PLN would significantly affect the PLN function during the absence of flight-or-flight response.
As explained above, we think that this regulation could be through both phosphorylation-dependent and phosphorylation-independent manner. Even only considering the phosphorylation-dependent manner, the DCM phenotype could be due to an accumulation of the Ca2+ imbalance in the cell over repeated cycles of cardiac muscle contraction upon chronic accumulation of the sporadic phosphorylation events. It is also possible that the mutations affect the CaMKII-dependent regulation of PLN, which leads to DCM.
Given that the DCM-associated PLN mutations have significant effects on the conformation of PLN itself, at least in the form of short-peptides, it is possible that these mutations could affect the folding, oligomerization, trafficking, degradation, etc., in addition to PKA-dependent phosphorylation. The relevance and contribution of reduced PKA-dependent PLN phosphorylation to DCM remain unresolved.
We agree with the reviewers that both phosphorylation-dependent and phosphorylation-independent manners could contribute to the DCM disease phenotype. It remains unresolved which factor is the major contributor. We have added a statement in the discussion (see point above).
Reviewer #3 (Public Review):
This manuscript describes an elegant study utilizing the crystal structures for the elucidation of the disease mechanism of familial dilated cardiomyopathy. It has been known for decades that the mutations in PLN are associated with DCM, but the underlying mechanism remains controversial. In my opinion, Prof Yuchi and co-authors did excellent job on revealing the high-resolution crystal structures of PKA-phospholamban complexes, representing both the native and diseased states. Combined with various of biophysical and biochemical methods, including SPR, ADP-glo, thermal melts, NMR, etc, the authors systematically investigated the correlations between the PLN conformation, the binding affinity, and the phosphorylation level. The mechanism of PKA phosphorylation on another related substrate, ALN, was also convincingly revealed. The results are very helpful for understanding the pathological mechanism of PLN-related DCM. More importantly, the atomic structures of PKA-phospholamban complexes lay a solid foundation for the structure-based rational design of therapeutic molecules that can reverse the effects of the DCM-causing mutations in the future, e.g. by stabilizing the interactions between PLN and PKA.
We thank the reviewer for the appreciation of our work.
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Evaluation Summary:
Protein kinase A phosphorylation of phospholamban (PLN) is part of the "fight or flight" response, which ultimately increases the force of cardiac contraction. Mutations in PLN have been linked to familial dilated cardiomyopathy (DCM). Crystal structures of wild-type and mutant PLN in complex with the PKA catalytic domain provide insights into both the nature of the complex, and potential mechanisms by which DCM mutations may cause disease. This paper is of interest to scientists interested in the mechanism of substrate recruitment by protein kinases, and particularly those who have an interest in understanding the mechanism of mutations associated with dilated cardiomyopathy.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback …
Evaluation Summary:
Protein kinase A phosphorylation of phospholamban (PLN) is part of the "fight or flight" response, which ultimately increases the force of cardiac contraction. Mutations in PLN have been linked to familial dilated cardiomyopathy (DCM). Crystal structures of wild-type and mutant PLN in complex with the PKA catalytic domain provide insights into both the nature of the complex, and potential mechanisms by which DCM mutations may cause disease. This paper is of interest to scientists interested in the mechanism of substrate recruitment by protein kinases, and particularly those who have an interest in understanding the mechanism of mutations associated with dilated cardiomyopathy.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In this paper, Qin et al. investigated the molecular mechanism of phospholamban (PLN) linked dilated cardiomyopathy (DCM), using structural approaches combined with biophysical measurements. Structures of the catalytic domain of protein kinase A (PKAc) in complex with PLN peptides (both wild-type and the R9C and A11E DCM mutants) provide insights into the mechanism of substrate recruitment and how it is perturbed in the disease state. Qin et al. show convincingly that the mutant peptides all have lower affinity for PKA than the wild-type peptide, suggesting models in which heterozygous DCM mutations act via sequestering PKA and thereby preventing phosphorylation of the wild-type peptide may be incorrect.
The authors highlight significant differences between their structure of the WT-PLN:PKAc complex, which …
Reviewer #1 (Public Review):
In this paper, Qin et al. investigated the molecular mechanism of phospholamban (PLN) linked dilated cardiomyopathy (DCM), using structural approaches combined with biophysical measurements. Structures of the catalytic domain of protein kinase A (PKAc) in complex with PLN peptides (both wild-type and the R9C and A11E DCM mutants) provide insights into the mechanism of substrate recruitment and how it is perturbed in the disease state. Qin et al. show convincingly that the mutant peptides all have lower affinity for PKA than the wild-type peptide, suggesting models in which heterozygous DCM mutations act via sequestering PKA and thereby preventing phosphorylation of the wild-type peptide may be incorrect.
The authors highlight significant differences between their structure of the WT-PLN:PKAc complex, which has a 1:1 stoichiometry, and a previous structure of the complex (PDB 3O7L), which has 1 PLN bound between two PKAc monomers (a 1:2 complex). The authors posit that the stoichiometry observed in 3O7L is an artifact of the crystal lattice, and does not occur in solution, supporting this with analysis of the elution volumes of the peptide complexes on size exclusion chromatography compared to PKAc alone. They further suggest that the AMP-PNP ligand included in the 3O7L structure is not bound, based on analysis of Fo-Fc maps calculated from the deposited coordinates. Inspecting 3O7L I am not convinced of this last point - it seems more likely that a technical error was made in assigning or refining the B-factor of the ligand in 3O7L, because there is clearly density present in SA-omit maps for the nucleotide.
Taking these results together, the authors suggest a mechanism for DCM, whereby mutations in PLN result in lower affinity for PKA, and consequently reduced phosphorylation. This seems plausible and well supported by the data, although in the ADP-Glo assay used here, the reductions in phosphorylation observed for some of the mutant peptides are rather modest. However, as the authors state, it is plausible that even relatively subtle changes in PLN phosphorylation could have substantial effects on Ca2+ homeostasis via increasing SERCA inhibition.
-
Reviewer #2 (Public Review):
Strengths:
The authors presented new high-resolution 3D crystal structures of the PKA catalytic domain (PKAc) in complex with PLN WT or mutant peptides (residues 8-22) containing the DCM-associated PLN mutations (R9C or A11E). These are novel and important data given that the present structures are dramatically different from those reported previously. The authors made convincing argument that the 3D model reported previously may result from a crystallization artifact.
By characterizing the interactions between the PKAc domain and PLN WT or DCM-associated mutant peptides using surface plasmon resonance (SPR) analysis, the authors convincingly showed that the DCM-associated PLN mutations at positions 9, 14, and 18 alter the conformation of the PLN peptide and reduce the binding affinity of the PLN peptide …
Reviewer #2 (Public Review):
Strengths:
The authors presented new high-resolution 3D crystal structures of the PKA catalytic domain (PKAc) in complex with PLN WT or mutant peptides (residues 8-22) containing the DCM-associated PLN mutations (R9C or A11E). These are novel and important data given that the present structures are dramatically different from those reported previously. The authors made convincing argument that the 3D model reported previously may result from a crystallization artifact.
By characterizing the interactions between the PKAc domain and PLN WT or DCM-associated mutant peptides using surface plasmon resonance (SPR) analysis, the authors convincingly showed that the DCM-associated PLN mutations at positions 9, 14, and 18 alter the conformation of the PLN peptide and reduce the binding affinity of the PLN peptide with PKAc. These data provide an explanation how some DCM-associated PLN mutations at these positions reduce the level of PKA-dependent phosphorylation of PLN.
The authors also performed nuclear magnetic resonance (NMR) to determine the structural dynamics of PLN WT, R9C, P-Ser16, and P-Thr-17 peptides. These NMR structures combined with the SPR analysis also support their conclusion that PLN phosphorylation and DCM-associated PLN mutations have an impact on its conformation.
Weakness:
The present study used PLN-derived peptides (aa 8-22). Although technically challenging, it is important to consider if the full-length WT or mutant PLN will behave the same as those observed with the peptides. This is especially crucial in light of the prior work showing substantially different structures using a different segment of PLN.
Although it is convincing that DCM-associated PLN mutations likely reduce the interaction between PKAc and PLN (assuming that the peptides behave the same as the full-length PLN with respect to interaction with PKA) and, as a result, the PKA dependent phosphorylation of the mutant PLN, it is unclear how this impaired interaction between PKA and PLN mutant could explain the effects of the DCM-associated PLN mutations on SERCA function (either reduced or enhanced PLN-dependent inhibition of SERCA, as proposed previously). In this regard, can the authors predict if the DCM-associated PLN R9C mutation reduces or increases SERCA inhibition based on the results of their present study?
It is also unclear how reduced PKA phosphorylation of mutant PLN could lead to DCM. PLN is unlikely to be significantly phosphorylated by PKA at rest (in other words, PLN is likely to be phosphorylated by PKA during stress, i.e. during the adrenergic fight-or-flight response). Therefore, it is puzzling how such reduced PKA-dependent phosphorylation of PLN would significantly affect the PLN function during the absence of flight-or-flight response.
Given that the DCM-associated PLN mutations have significant effects on the conformation of PLN itself, at least in the form of short-peptides, it is possible that these mutations could affect the folding, oligomerization, trafficking, degradation, etc., in addition to PKA-dependent phosphorylation. The relevance and contribution of reduced PKA-dependent PLN phosphorylation to DCM remain unresolved.
-
Reviewer #3 (Public Review):
This manuscript describes an elegant study utilizing the crystal structures for the elucidation of the disease mechanism of familial dilated cardiomyopathy. It has been known for decades that the mutations in PLN are associated with DCM, but the underlying mechanism remains controversial. In my opinion, Prof Yuchi and co-authors did excellent job on revealing the high-resolution crystal structures of PKA-phospholamban complexes, representing both the native and diseased states. Combined with various of biophysical and biochemical methods, including SPR, ADP-glo, thermal melts, NMR, etc, the authors systematically investigated the correlations between the PLN conformation, the binding affinity, and the phosphorylation level. The mechanism of PKA phosphorylation on another related substrate, ALN, was also …
Reviewer #3 (Public Review):
This manuscript describes an elegant study utilizing the crystal structures for the elucidation of the disease mechanism of familial dilated cardiomyopathy. It has been known for decades that the mutations in PLN are associated with DCM, but the underlying mechanism remains controversial. In my opinion, Prof Yuchi and co-authors did excellent job on revealing the high-resolution crystal structures of PKA-phospholamban complexes, representing both the native and diseased states. Combined with various of biophysical and biochemical methods, including SPR, ADP-glo, thermal melts, NMR, etc, the authors systematically investigated the correlations between the PLN conformation, the binding affinity, and the phosphorylation level. The mechanism of PKA phosphorylation on another related substrate, ALN, was also convincingly revealed. The results are very helpful for understanding the pathological mechanism of PLN-related DCM. More importantly, the atomic structures of PKA-phospholamban complexes lay a solid foundation for the structure-based rational design of therapeutic molecules that can reverse the effects of the DCM-causing mutations in the future, e.g. by stabilizing the interactions between PLN and PKA.
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