Structural Basis for Allosteric Control of the SERCA-Phospholamban Membrane Complex by Ca 2+ and cAMP-dependent Phosphorylation

  1. Daniel K. Weber
  2. Máximo Sanz-Hernández
  3. U. Venkateswara Reddy
  4. Songlin Wang
  5. Erik K. Larsen
  6. Tata Gopinath
  7. Martin Gustavsson
  8. Razvan L. Cornea
  9. David D. Thomas
  10. Alfonso De Simone
  11. Gianluigi Veglia

  • Curated by eLife

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    Evaluation Summary:

    There are many of membrane-embedded mini-proteins, which fulfill a large range of regulatory functions. One of them is phospholamban, a single transmembrane helix protein that regulates the sarcoplasmic reticulum Ca2+-ATPase by binding in the membrane. The work presented here combines new experiments with computer simulations with the aim of arriving at a more definitive answer to the long-standing mechanistic question of how exactly phosphorylation of phospholamban modulates its regulatory behavior. In this manuscript, an allosteric mechanism is presented, which could be of general importance for the whole family of these mini-proteins.

    (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 #1 agreed to share their name with the authors.)

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Abstract

Phospholamban (PLN) is a mini-membrane protein that directly controls the cardiac Ca 2+ -transport response to β-adrenergic stimulation, thus modulating cardiac output during the fight- or-flight response. In the sarcoplasmic reticulum membrane, PLN binds to the sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA), keeping this enzyme’s function within a narrow physiological window. PLN phosphorylation by cAMP-dependent protein kinase A or increase in Ca 2+ concentration reverses the inhibitory effects through an unknown mechanism. Using oriented-sample solid-state NMR spectroscopy and replica-averaged NMR-restrained structural refinement, we reveal that phosphorylation of PLN’s cytoplasmic regulatory domain signals the disruption of several inhibitory contacts at the transmembrane binding interface of the SERCA-PLN complex that are propagated to the enzyme’s active site, augmenting Ca 2+ transport. Our findings address long-standing questions about SERCA regulation, epitomizing a signal transduction mechanism operated by posttranslationally-modified bitopic membrane proteins.

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  1. eLife

    Reviewer #3 (Public Review):

    The regulation of the calcium pump SERCA by phospholamban has been studied extensively over many years as this system has become a focus of many biophysical approaches to study the interplay between protein dynamics, the biological function of calcium transport, and its regulation via protein-protein interactions, all of which are occurring within the environment of the sarcoplasmic membrane of heart muscle.

    The authors themselves have a long track record with working on this system and the specific focus here is on the detailed mechanism of how phosphorylation of phospholamban leads to a release of its inhibitory function when bound to SERCA. Much effort has been spent on this question in the past, and the field has progressed over the years by deriving increasingly detailed structural models for SERCA-phospholamban interactions. There is now a structure from crystallography showing the interaction of the phospholamban TM domain with the SERCA TM helices and there is additional data from various biophysical methods that partially describe the conformational ensemble of the extramembrane N-terminal region of phospholamban and its interaction with SERCA. Some of that insight has distinguished between phosphorylated and unphosphorylated phospholamban, but despite much data and many simulation efforts, the exact mechanism for how phosphorylation of phospholamban alters its interaction with SERCA and thereby modulates its inhibitory functions has so far not been clearly described. This is the main goal of the present work.

    There is new experimental data presented here from oriented-sample solid-state NMR experiments with the main finding of orientational shifts of the phospholamban TM helix upon binding to SERCA and upon phosphorylation. Taking advantage of this data, the main part of the study is concerned with results from computer simulations that were restrained by experimental data to develop conformational ensembles of the SERCA-phospholamban complex with and without phosphorylated phospholamban. From that, new mechanistic hypotheses are developed. While the direction of the work proposed here is promising, there are concerns about the overall approach and - as a consequence - the significance of the reported findings:

    1. A main concern is the treatment of the extramembrane portion of phospholamban, which includes the serine that is being phosphorylated to relieve the inhibitory effect. Previous studies have described a helical conformation for the N-terminal segment that may be in equilibrium with a less-ordered/less-helical structure upon binding to SERCA. It is largely still not clear, however, how exactly that part of phospholamban would interact with SERCA. The idea put forth here is that a largely disordered conformation would interact with SERCA. That may be so, but it is unclear how much of that is a direct result of experimental constraints and how much could simply be a consequence of inadequate sampling. It seems that helical conformations for the N-terminal segment of phospholamban were not considered, while there is not enough discussion of why such conformations would be ruled out based on the experimental data.

    2. The simulations are probably too short to fully explore the full conformational landscape of a (partially) disordered N-terminal phospholamban and it is unclear how much the experimental constraints are really limiting the conformational space in that region.

    3. It is not completely clear how the present work relates to the crystal structure of the SERCA-phospholamban complex. Why were the starting structures for the SERCA-phospholamban complex initially taken from the available crystal structure (at least with respect to the TM domain of phospholamban) but then subsequently refined using much lower-resolution cross-linking data before initiating the simualtions? Is the crystal structure in significant disagreement with other experimental data considered here? More discussion and explanation is needed.

    4. The main focus of the analysis of the simulation results is on the impact of phosphorylated phospholamban on the conformational sampling of SERCA. That is the key step for developing new mechanistic hypotheses. However, given that the SERCA-phospholamban complex is very large and flexible and based on the results presented, it appears that the length of the simulations may not be sufficient to fully characterize the shift in the conformational ensemble of SERCA as a function of phospholamban phosphorylation. At the minimum, some time of convergence analysis is needed to establish confidence that the difference in conformational ensembles shown most prominently in Figure 2 are indeed significant. Moreover, related to Figure 2, it is unclear whether the projection of the conformational sampling onto just two principal coordinates is sufficient for a full characterization of the conformational dynamics. It is also unclear whether the principal coordinates are the same when projecting the sampling for PLN and pPLN, if not, the comparison between the two would be further complicated.

  2. eLife

    Reviewer #2 (Public Review):

    In this paper, the authors present an extensive ssNMR study on the mini-membrane protein phospholamban (PLN), which regulates the Ca2+ ATPase SERCA. PLN stabilizes the low-affinity Ca2+ state of SERCA, which can be reversed by phosphorylation or increase in [Ca2+]. Despite extensive, studies this mechanism is still unknown: Although interaction sites within the membrane have been identified, not structural changes within PLN have been detected. In the paper, the authors address this question by oriented ssNMR, an approach which is highly suited to map topological changes of membrane embedded peptides and proteins. While oriented ssNMR is conceptionally very appealing, it has been hampered by sample preparation restrictions preventing its widespread use on more complex samples. A breakthrough has been magnetic alignment of membrane proteins embedded in bicelles as demonstrated here. The presented spectra represent in principle a projection of labelled transmembrane helices onto a spectroscopic plane by which re-orientations of these helices can be elegantly visualized. Based on high quality data, the authors are able to convincingly demonstrate that PLN is in a topological equilibrium, which shifts upon phosphorylation at Ser60. In complex with SERCA, phosphorylation or Ca2+ binding triggers a topological change of the whole PLN transmembrane domain, which then act as a 'switch' on SERCA.

    All presented data are of high quality and data interpretation is convincing. The paper addresses a complex and relevant biomolecular question by very advanced methodology.

    The authors have identified a topological allostery for PLN connecting a posttranslational modification at the cytoplasmic site with signal transduction across the membrane. They argue that the underlying mechanism might be of general relevance for the regulatory role fulfilled by miniproteins.

  3. eLife

    Reviewer #1 (Public Review):

    The regulation of highly dynamic interactions is for many biological processes of great importance. The authors study the regulatory interaction of the single transmembrane helix protein Phospholamban with the P-type ATPase SERCA which is responsible for removing calcium ions from the sarcoplasm and restoring its concentrations in the sarcoplasmic reticulum. The inhibitory interaction between both proteins is relieved by phosphorylation of a single residue in the cytoplasmic domain of Phospholamban. The authors show by a combination of solid state NMR as well as MD simulations that phosphorylation results in a order to disorder transition in the cytoplasmic part which leads to an re-arrangement of electrostatic networks which is propagated into weakened hydrophobic interactions between the transmembrane parts, thus activating SERCA. Phospholamban has been studied extensively by solid state NMR, liquid state NMR or hybrid methods. For example the phosphorylated form was studied previously, showing that it interacts differently with lipids (doi: 10.1021/bi0614028) and that Ser-16 phosphorylation alters the structural properties of the cytoplasmic domain with respect to the lipid bilayers (doi:10.1016/j.bbamem.2009.12.020). There have also been EPR and other studies, in principle showing the same effect. The current paper adds to this a new solid state method that shows additional details that could not be investigated previously. The work confirms less well determined previous models. The major new aspect is an MD simulation that provides a more detailed view than what was previously possible.

  4. eLife

    Evaluation Summary:

    There are many of membrane-embedded mini-proteins, which fulfill a large range of regulatory functions. One of them is phospholamban, a single transmembrane helix protein that regulates the sarcoplasmic reticulum Ca2+-ATPase by binding in the membrane. The work presented here combines new experiments with computer simulations with the aim of arriving at a more definitive answer to the long-standing mechanistic question of how exactly phosphorylation of phospholamban modulates its regulatory behavior. In this manuscript, an allosteric mechanism is presented, which could be of general importance for the whole family of these mini-proteins.

    (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 #1 agreed to share their name with the authors.)

  5. Version published to 10.1101/2020.08.28.271940v2 on bioRxiv
  6. Version published to 10.1101/2020.08.28.271940v1 on bioRxiv