Murray DT, Kato M, Lin Y, Thurber KR, Hung I, McKnight SL, Tycko R.
Structure of FUS Protein Fibrils and Its Relevance to Self-Assembly and Phase Separation of Low-Complexity Domains.
Cell. 2017 Sep 16;
PubMed.
This paper describes an elegant solid-state NMR structural characterization of a cross-β conformation within an ordered 57-residue element of the FUS low-complexity (LC) region. It provides an important piece to the puzzle of what interactions drive protein phase separation, an area that is currently being studied intensely due to its critical role in biological compartmentalization and biomaterial formation.
The conformational landscape of the FUS-LC region is complex. Previous solution NMR studies, as well as data from this current work, demonstrate dynamic disorder, but pelleting fibrils for study by solid-state NMR enables signal to emerge from the equilibrium sampling of more ordered conformers. While it has previously been shown that the FUS-LC in a liquid phase-separated state can remain disordered, avoiding significant population of β structure, this data demonstrating cross-β structure that can assemble into fibrils is consistent with the observation of FUS fibers in hydrogels.
It is not clear yet how these different conformational states fit into the physiological function of FUS or the pathogenesis in ALS and FTLD, but it is obvious that the physical chemistry enables such β structures to be sampled. Transient β interactions could certainly contribute to the mix of multivalent interactions needed to drive liquid phase separation, along with pi-pi, cation-pi, electrostatic, and hydrophobic interactions that have been discussed in the literature.
That this structure is significantly less stable than many cross- β fibril states, enabling their dissolution by phosphorylation, argues for the role of such transient interactions within a physiologically relevant and dynamically regulated equilibrium.
This is an important study that will stimulate a lot of additional work in the field. The observation that the structure is distinct from disease amyloid fibrils is of high interest for understanding assembly of not only FUS but many other low-complexity and "prion-like" domains. The existence of ALS-associated mutations (e.g. S57del, S96del) that map to the core region of the fibril structure presented by Murray et al. brings up the question if the mutations inappropriately over-stabilize this structure (or a related structure) leading to aggregation observed previously (Murakami et al., 2015).
Regarding the structure in the liquid forms, in our previous work (Burke et al., 2015) we examined a liquid-liquid phase separated (LLPS or "droplet") state formed by residues 1-163 of FUS via solution NMR. There we saw predominant disorder across the domain and very intense signals, suggesting we were observing the majority of the conformations. However, 1) those solution NMR techniques are not sensitive to small populations, and 2) large, rigid structures such as polymeric β-sheet assemblies would be invisible to our techniques. Furthermore, our results did not identify the molecular details of the contacts that mediate the liquid form. The molecular details of phase separation can indeed involve secondary structure—for example, in collaboration with Jeetain Mittal (Lehigh) we recently suggested that α-helical packing contributes to phase separation of TDP-43 (Conicella et al., 2016).
Going forward, it will be exciting to directly probe these contacts in LLPS states and to measure the amount of β-sheet (and α-helix) structure in the liquid forms both in vitro and in vivo, not only of FUS but of other phase-separated assemblies. Recent solution NMR work by Simon Sharpe as well as Julie Forman-Kay and Lewis Kay on phase separation is starting to directly probe the interactions that stabilize phase separation of other sequences (Reichheld et al., 2017; Brady et al.; 2017).
The observation that phosphosites that map to the core fibril region have the biggest contribution to droplet formation is exciting. The effect of phosphorylation in general is highly complementary to our recent study (in collaboration with Frank Shewmaker, USUHS) showing that phosphorylation and mimicking phosphorylation with charged amino acid substitutions can disrupt phase separation, aggregation, and cellular toxicity of FUS (Monahan et al., 2017), which was inspired by the seminal work from McKnight and coworkers on the effect of phosphorylation on FUS assembly (Han et al., 2012; Kato et al., 2012; Kwon et al., 2013).
In fact, I first heard about FUS when Rob Tycko invited Steven McKnight to speak about his work on FUS in 2012, before these papers were published. It was right before I started my own lab and was such a great area that he opened up that I decided to write my first independent grant applications on this topic. Steven McKnight was kind enough to support my applications and he and Masato Kato provided the initial plasmids.
References:
Murakami T, Qamar S, Lin JQ, Schierle GS, Rees E, Miyashita A, Costa AR, Dodd RB, Chan FT, Michel CH, Kronenberg-Versteeg D, Li Y, Yang SP, Wakutani Y, Meadows W, Ferry RR, Dong L, Tartaglia GG, Favrin G, Lin WL, Dickson DW, Zhen M, Ron D, Schmitt-Ulms G, Fraser PE, Shneider NA, Holt C, Vendruscolo M, Kaminski CF, St George-Hyslop P.
ALS/FTD Mutation-Induced Phase Transition of FUS Liquid Droplets and Reversible Hydrogels into Irreversible Hydrogels Impairs RNP Granule Function.
Neuron. 2015 Nov 18;88(4):678-90. Epub 2015 Oct 29
PubMed.
Burke KA, Janke AM, Rhine CL, Fawzi NL.
Residue-by-Residue View of In Vitro FUS Granules that Bind the C-Terminal Domain of RNA Polymerase II.
Mol Cell. 2015 Oct 15;60(2):231-41. Epub 2015 Oct 8
PubMed.
Conicella AE, Zerze GH, Mittal J, Fawzi NL.
ALS Mutations Disrupt Phase Separation Mediated by α-Helical Structure in the TDP-43 Low-Complexity C-Terminal Domain.
Structure. 2016 Sep 6;24(9):1537-49. Epub 2016 Aug 18
PubMed.
Reichheld SE, Muiznieks LD, Keeley FW, Sharpe S.
Direct observation of structure and dynamics during phase separation of an elastomeric protein.
Proc Natl Acad Sci U S A. 2017 May 30;114(22):E4408-E4415. Epub 2017 May 15
PubMed.
Brady JP, Farber PJ, Sekhar A, Lin YH, Huang R, Bah A, Nott TJ, Chan HS, Baldwin AJ, Forman-Kay JD, Kay LE.
Structural and hydrodynamic properties of an intrinsically disordered region of a germ cell-specific protein on phase separation.
Proc Natl Acad Sci U S A. 2017 Sep 11;
PubMed.
Monahan Z, Ryan VH, Janke AM, Burke KA, Rhoads SN, Zerze GH, O'Meally R, Dignon GL, Conicella AE, Zheng W, Best RB, Cole RN, Mittal J, Shewmaker F, Fawzi NL.
Phosphorylation of the FUS low-complexity domain disrupts phase separation, aggregation, and toxicity.
EMBO J. 2017 Oct 16;36(20):2951-2967. Epub 2017 Aug 8
PubMed.
Han TW, Kato M, Xie S, Wu LC, Mirzaei H, Pei J, Chen M, Xie Y, Allen J, Xiao G, McKnight SL.
Cell-free formation of RNA granules: bound RNAs identify features and components of cellular assemblies.
Cell. 2012 May 11;149(4):768-79.
PubMed.
Kato M, Han TW, Xie S, Shi K, Du X, Wu LC, Mirzaei H, Goldsmith EJ, Longgood J, Pei J, Grishin NV, Frantz DE, Schneider JW, Chen S, Li L, Sawaya MR, Eisenberg D, Tycko R, McKnight SL.
Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels.
Cell. 2012 May 11;149(4):753-67.
PubMed.
Kwon I, Kato M, Xiang S, Wu L, Theodoropoulos P, Mirzaei H, Han T, Xie S, Corden JL, McKnight SL.
Phosphorylation-regulated binding of RNA polymerase II to fibrous polymers of low-complexity domains.
Cell. 2013 Nov 21;155(5):1049-1060.
PubMed.
Comments
Hospital for Sick Children/ University of Toronto
This paper describes an elegant solid-state NMR structural characterization of a cross-β conformation within an ordered 57-residue element of the FUS low-complexity (LC) region. It provides an important piece to the puzzle of what interactions drive protein phase separation, an area that is currently being studied intensely due to its critical role in biological compartmentalization and biomaterial formation.
The conformational landscape of the FUS-LC region is complex. Previous solution NMR studies, as well as data from this current work, demonstrate dynamic disorder, but pelleting fibrils for study by solid-state NMR enables signal to emerge from the equilibrium sampling of more ordered conformers. While it has previously been shown that the FUS-LC in a liquid phase-separated state can remain disordered, avoiding significant population of β structure, this data demonstrating cross-β structure that can assemble into fibrils is consistent with the observation of FUS fibers in hydrogels.
It is not clear yet how these different conformational states fit into the physiological function of FUS or the pathogenesis in ALS and FTLD, but it is obvious that the physical chemistry enables such β structures to be sampled. Transient β interactions could certainly contribute to the mix of multivalent interactions needed to drive liquid phase separation, along with pi-pi, cation-pi, electrostatic, and hydrophobic interactions that have been discussed in the literature.
That this structure is significantly less stable than many cross- β fibril states, enabling their dissolution by phosphorylation, argues for the role of such transient interactions within a physiologically relevant and dynamically regulated equilibrium.
View all comments by Julie Forman-KayBrown University
This is an important study that will stimulate a lot of additional work in the field. The observation that the structure is distinct from disease amyloid fibrils is of high interest for understanding assembly of not only FUS but many other low-complexity and "prion-like" domains. The existence of ALS-associated mutations (e.g. S57del, S96del) that map to the core region of the fibril structure presented by Murray et al. brings up the question if the mutations inappropriately over-stabilize this structure (or a related structure) leading to aggregation observed previously (Murakami et al., 2015).
Regarding the structure in the liquid forms, in our previous work (Burke et al., 2015) we examined a liquid-liquid phase separated (LLPS or "droplet") state formed by residues 1-163 of FUS via solution NMR. There we saw predominant disorder across the domain and very intense signals, suggesting we were observing the majority of the conformations. However, 1) those solution NMR techniques are not sensitive to small populations, and 2) large, rigid structures such as polymeric β-sheet assemblies would be invisible to our techniques. Furthermore, our results did not identify the molecular details of the contacts that mediate the liquid form. The molecular details of phase separation can indeed involve secondary structure—for example, in collaboration with Jeetain Mittal (Lehigh) we recently suggested that α-helical packing contributes to phase separation of TDP-43 (Conicella et al., 2016).
Going forward, it will be exciting to directly probe these contacts in LLPS states and to measure the amount of β-sheet (and α-helix) structure in the liquid forms both in vitro and in vivo, not only of FUS but of other phase-separated assemblies. Recent solution NMR work by Simon Sharpe as well as Julie Forman-Kay and Lewis Kay on phase separation is starting to directly probe the interactions that stabilize phase separation of other sequences (Reichheld et al., 2017; Brady et al.; 2017).
The observation that phosphosites that map to the core fibril region have the biggest contribution to droplet formation is exciting. The effect of phosphorylation in general is highly complementary to our recent study (in collaboration with Frank Shewmaker, USUHS) showing that phosphorylation and mimicking phosphorylation with charged amino acid substitutions can disrupt phase separation, aggregation, and cellular toxicity of FUS (Monahan et al., 2017), which was inspired by the seminal work from McKnight and coworkers on the effect of phosphorylation on FUS assembly (Han et al., 2012; Kato et al., 2012; Kwon et al., 2013).
In fact, I first heard about FUS when Rob Tycko invited Steven McKnight to speak about his work on FUS in 2012, before these papers were published. It was right before I started my own lab and was such a great area that he opened up that I decided to write my first independent grant applications on this topic. Steven McKnight was kind enough to support my applications and he and Masato Kato provided the initial plasmids.
References:
Murakami T, Qamar S, Lin JQ, Schierle GS, Rees E, Miyashita A, Costa AR, Dodd RB, Chan FT, Michel CH, Kronenberg-Versteeg D, Li Y, Yang SP, Wakutani Y, Meadows W, Ferry RR, Dong L, Tartaglia GG, Favrin G, Lin WL, Dickson DW, Zhen M, Ron D, Schmitt-Ulms G, Fraser PE, Shneider NA, Holt C, Vendruscolo M, Kaminski CF, St George-Hyslop P. ALS/FTD Mutation-Induced Phase Transition of FUS Liquid Droplets and Reversible Hydrogels into Irreversible Hydrogels Impairs RNP Granule Function. Neuron. 2015 Nov 18;88(4):678-90. Epub 2015 Oct 29 PubMed.
Burke KA, Janke AM, Rhine CL, Fawzi NL. Residue-by-Residue View of In Vitro FUS Granules that Bind the C-Terminal Domain of RNA Polymerase II. Mol Cell. 2015 Oct 15;60(2):231-41. Epub 2015 Oct 8 PubMed.
Conicella AE, Zerze GH, Mittal J, Fawzi NL. ALS Mutations Disrupt Phase Separation Mediated by α-Helical Structure in the TDP-43 Low-Complexity C-Terminal Domain. Structure. 2016 Sep 6;24(9):1537-49. Epub 2016 Aug 18 PubMed.
Reichheld SE, Muiznieks LD, Keeley FW, Sharpe S. Direct observation of structure and dynamics during phase separation of an elastomeric protein. Proc Natl Acad Sci U S A. 2017 May 30;114(22):E4408-E4415. Epub 2017 May 15 PubMed.
Brady JP, Farber PJ, Sekhar A, Lin YH, Huang R, Bah A, Nott TJ, Chan HS, Baldwin AJ, Forman-Kay JD, Kay LE. Structural and hydrodynamic properties of an intrinsically disordered region of a germ cell-specific protein on phase separation. Proc Natl Acad Sci U S A. 2017 Sep 11; PubMed.
Monahan Z, Ryan VH, Janke AM, Burke KA, Rhoads SN, Zerze GH, O'Meally R, Dignon GL, Conicella AE, Zheng W, Best RB, Cole RN, Mittal J, Shewmaker F, Fawzi NL. Phosphorylation of the FUS low-complexity domain disrupts phase separation, aggregation, and toxicity. EMBO J. 2017 Oct 16;36(20):2951-2967. Epub 2017 Aug 8 PubMed.
Han TW, Kato M, Xie S, Wu LC, Mirzaei H, Pei J, Chen M, Xie Y, Allen J, Xiao G, McKnight SL. Cell-free formation of RNA granules: bound RNAs identify features and components of cellular assemblies. Cell. 2012 May 11;149(4):768-79. PubMed.
Kato M, Han TW, Xie S, Shi K, Du X, Wu LC, Mirzaei H, Goldsmith EJ, Longgood J, Pei J, Grishin NV, Frantz DE, Schneider JW, Chen S, Li L, Sawaya MR, Eisenberg D, Tycko R, McKnight SL. Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels. Cell. 2012 May 11;149(4):753-67. PubMed.
Kwon I, Kato M, Xiang S, Wu L, Theodoropoulos P, Mirzaei H, Han T, Xie S, Corden JL, McKnight SL. Phosphorylation-regulated binding of RNA polymerase II to fibrous polymers of low-complexity domains. Cell. 2013 Nov 21;155(5):1049-1060. PubMed.
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