Science_-_7_February_2020_UserUpload.Net

from different proteins that are known drivers
of LLPS. We identified a strong bias toward
uniform distribution of aromatic stickers
along linear sequences for the PLDs of RNA-
binding proteins such as FUS, TAF15, EWSR1,
hnRNPA2B1, and hnRNPA3 (Fig. 4E) in which
the patterning of aromatic residues is highly
conserved despite very low levels of sequence
conservation (fig. S15, A to C). A proteome-wide
analysis of the patterning of aromatic residues
within disordered PLDs showed a similar bias
toward nonrandom, uniform patterning of aro-
matic stickers in PLDs from an assortment of
dissimilar proteins that are known drivers of
LLPS ( 3 , 21 )(Fig.4Eandfig.S15,DandE).We
also identified disordered regions from proteins
involved in vesicular trafficking and signal trans-
duction that have a similar nonrandom bias
toward uniform patterning of aromatic resi-
dues (Fig. 4E). Intriguingly, the PLD of Xvelo,
a protein that drives the formation of solid-like
Balbiani bodies ( 44 ), is a prominent outlier in
terms of its large value ofWaro.
We propose that the uniform spacing of
aromatic stickers along the linear sequences
of LCDs ensures that strong interactions among
aromatic residues are weakened by the favor-
able solvation of the spacers. The preferential
solvation of spacers likely dilutes the effects
of aromatic stickers. Similar considerations are
likely to apply to other nonaromatic stickers,
such as hydrophobic motifs or those that con-
tribute to cation-pinteractions and/or comple-
mentary electrostatic interactions ( 41 ). Our
findings point to interactions encoded in PLDs
that are strong enough to drive LLPS and yet
weak enough to suppress aggregation—abal-
ance that is likely disrupted through mutations
that (i) increase the valence of aromatic or other
stickers; (ii) disrupt the nonrandom, well-mixed
patterning of aromatic stickers; and/or (iii) add
other cohesive interactions through mutations
to spacers that weaken their preference for being
well solvated.
We have demonstrated the applicability of the
stickers-and-spacers framework for quantitative
descriptions of sequence-binodal relationships
of archetypal PLDs. We have converged on a
transferable protocol (fig. S16) for identifying
stickers; quantifying the relative strengths of
sticker-sticker, sticker-spacer, and spacer-spacer

interactions as a function of temperature or other

equivalent control parameters; and using this

information to generate sequence-to-binodal

relationships. These methods will help in map-

ping cellular concentrations for PLDs to posi-

tions relative to their measured or calculated

binodals, thus allowing the prediction of how

condensates spontaneously form and dissolve

in response to changes in protein concentra-

tion and cellular conditions ( 1 ).

REFERENCES AND NOTES

1. T. M. Franzmannet al.,Science 359 , eaao5654 (2018).


2. J. A. Ribacket al.,Cell 168 , 1028–1040.e19 (2017).


3. A. Molliexet al.,Cell 163 , 123–133 (2015).


4. A. Wanget al.,EMBO J. 37 , e97452133 (2018).


5. J. B. Woodruff,J. Mol. Biol. 430 , 4762–4772 (2018).


6. A. G. Larsonet al.,Nature 547 , 236–240 (2017).


7. A. R. Stromet al.,Nature 547 , 241–245 (2017).


8. S. Banjade, M. K. Rosen,eLife 3 , e04123 (2014).


9. S. F. Banani, H. O. Lee, A. A. Hyman, M. K. Rosen,Nat. Rev.
   Mol. Cell Biol. 18 , 285–298 (2017).


10. Y. Shin, C. P. Brangwynne,Science 357 , eaaf4382 (2017).


11. A. Patelet al.,Cell 162 , 1066–1077 (2015).


12. C. P. Brangwynne, P. S. Tompa, R. V. Pappu,Nat. Phys. 11 ,
    899 – 904 (2015).


13. R. M. Vernon, J. D. Forman-Kay,Curr. Opin. Struct. Biol. 58 ,
    88 – 96 (2019).


14. T. J. Nottet al.,Mol. Cell 57 , 936–947 (2015).


15. X. Guiet al.,Nat. Commun. 10 , 2006 (2019).


16. M. P. Hugheset al.,Science 359 , 698 – 701 (2018).


17. M. Katoet al.,Cell 149 , 753–767 (2012).


18. J. P. Bradyet al.,Proc. Natl. Acad. Sci. U.S.A. 114 ,
    E8194–E8203 (2017).


19. K. A. Burke, A. M. Janke, C. L. Rhine, N. L. Fawzi,Mol. Cell 60 ,
    231 – 241 (2015).


20. M. Rubinstein, A. V. Dobrynin,Trends Polym. Sci. 5 , 181– 186
    (1997).


21. J. Wanget al.,Cell 174 , 688–699.e16 (2018).


22. A. N. Semenov, M. Rubinstein,Macromolecules 31 , 1373– 1385
    (1998).


23. T. S. Harmon, A. S. Holehouse, M. K. Rosen, R. V. Pappu,eLife
    6 , e30294 (2017).


24. P. J. Flory,J. Am. Chem. Soc. 63 , 3083–3090 (1941).


25. A. E. Posey, A. S. Holehouse, R. V. Pappu, inMethods in
    Enzymology, E. Rhoades, Ed. (Academic Press, 2018),
    vol. 611, pp. 1–30.


26. H. J. Kimet al.,Nature 495 , 467–473 (2013).


27. A. Vitalis, R. V. Pappu,J. Comput. Chem. 30 ,673– 699
    (2009).


28. A. S. Holehouse, R. V. Pappu,Annu. Rev. Biophys. 47 ,19– 39
    (2018).


29. G. Raos, G. Allegra,J. Chem. Phys. 107 , 6479–6490 (1997).


30. J. A. Ribacket al.,Science 358 , 238– 241 (2017).


31. J. Klein-Seetharamanet al.,Science 295 ,1719– 1722
    (2002).


32. P. A. Evans, K. D. Topping, D. N. Woolfson, C. M. Dobson,
    Proteins 9 , 248–266 (1991).


33. D. Neri, M. Billeter, G. Wider, K. Wüthrich,Science 257 ,
    1559 – 1563 (1992).


34. G. L. Dignon, W. Zheng, R. B. Best, Y. C. Kim, J. Mittal,
    Proc. Natl. Acad. Sci. U.S.A. 115 , 9929–9934 (2018).


35. M. T. Weiet al.,Nat. Chem. 9 , 1118–1125 (2017).
    36. M. Rubinstein, R. H. Colby,Polymer Physics(Oxford Univ. Press,
    2003).
    37. F. Luoet al.,Nat. Struct. Mol. Biol. 25 , 341–346 (2018).
    38. Z. Monahanet al.,EMBO J. 36 , 2951–2967 (2017).
    39. A. E. Conicella, G. H. Zerze, J. Mittal, N. L. Fawzi,Structure 24 ,
    1537 – 1549 (2016).
    40. R. K. Das, R. V. Pappu,Proc. Natl. Acad. Sci. U.S.A. 110 ,
    13392 – 13397 (2013).
    41. C. W. Paket al.,Mol. Cell 63 ,72–85 (2016).
    42. E. W. Martinet al.,J. Am. Chem. Soc. 138 , 15323– 15335
    (2016).
    43. F. Tanaka, M. Ishida,Macromolecules 30 ,1836– 1844
    (1997).
    44. E. Bokeet al.,Cell 166 , 637– 650 (2016).
    45. A. Holehouse, R. V. Pappu, PIMMS (0.24 pre-beta), Zenodo
    (2019); doi:10.5281/zenodo.3588456

ACKNOWLEDGMENTS

We thank J.-M. Choi, A. A. Hyman, and J. P. Taylor for insightful

discussions; M. Stuchell-Brereton and N. Milkovic for technical

help; S. Chakravarthy, J. Hopkins, and all BioCAT beamline

staff at the Advanced Photon Source for assistance with SAXS

measurements; C. Liu for providing the raw data associated with

the FUS measurements; and anonymous reviewers for constructive

criticisms that helped us immensely with our narrative. Microscopy

images were acquired at the Cell & Tissue Imaging Center, which is

supported by SJCRH and NCI (grant P30 CA021765). NMR

assignments are available from the BMRB at accession code ID

50017.Funding:This work was funded by the St. Jude Children’s

Research Hospital Research Collaborative on Membrane-less

Organelles in Health and Disease (to T.M. and R.V.P.), the U.S.

National Science Foundation (MCB1614766 to R.V.P.), the Human

Frontier Science Program (RGP0034/2017 to R.V.P), the American

Federation for Aging Research (to A.S.), and the American

Lebanese Syrian Associated Charities (to T.M.). Use of the

Advanced Photon Source was supported by the U.S. Department

of Energy under contract DE-AC02-06CH11357.Author

contributions:Conceptualization: E.W.M., A.S.H., R.V.P., and

T.M.; Methodology: E.W.M., I.P., A.S.H., A.S., R.V.P., and T.M.;

Investigation: E.W.M., I.P., A.S.H., M.F., J.J.I., A.B., C.R.G., A.S.,

R.V.P., and T.M.; Resources: A.S., R.V.P., and T.M.; Writing–

original draft: E.W.M., A.S.H., R.V.P., and T.M.; Writing–reviewing

and editing: all authors; Visualization: E.W.M., I.P., A.S.H., and

A.S.; Funding acquisition: R.V.P. and T.M.Competing interests:

R.V.P. is a member of the scientific advisory board of DewpointX.

This work was not funded or influenced in any way by this

affiliation. The remaining authors declare no competing interests.

Data and materials availability:Code needed to reproduce

the results is available at Zenodo ( 45 ). All other data are available

in the manuscript or the supplementary materials. All expression

plasmids are available from T.M. under a material transfer

agreement with St. Jude Children’s Hospital.

SUPPLEMENTARY MATERIALS

science.sciencemag.org/content/367/6478/694/suppl/DC1

Materials and Methods

Figs. S1 to S16

Tables S1 to S3

References ( 46 – 70 )

Movies S1 to S6

View/request a protocol for this paper fromBio-protocol.

16 February 2019; resubmitted 30 August 2019

Accepted 6 January 2020

10.1126/science.aaw8653

Martinet al.,Science 367 , 694–699 (2020) 7 February 2020 6of6

RESEARCH | REPORT