Nicolas Fawzi - Residue-by-Residue View of the In Vitro FUS Granules

Nicolas Lux Fawzi

Department of Molecular Pharmacology, Physiology and Biotechnology

Brown University

Residue-by-Residue View of In Vitro FUS granules

that bind the C-terminal domain

of RNA polymerase II

Alzforum Webinar

October 30, 2015

Fused in Sarcoma (FUS)

• Aggregation-prone Low complexity (LC

aka SYGQ-rich) essential for aggregation

in ALS

• Mutations in NLS and LC regions have

been associated with amyotrophic lateral

sclerosis, leading to protein aggregation

• RNA binding

domains

• Low complexity

putatively

disordered domains

• Nuclear/cytoplasm

shuttling

• Cellular [FUS]

> 2 μM *

*Alberti and coworkers

Questions

What is the structure of the low complexity domain of FUS?

as a monomer? (Before it assembles)

self-assembled into phase separated liquid droplets?

How does FUS LC contribute to assembly into dynamic granules?

Do droplets of FUS LC localize other low complexity domain proteins?

FUS LC structure?LC or “prion-like” domain of FUS has

an unusual sequence

Rich in amino acids:

serine (S)

tyrosine (Y)

glycine (G)

glutamine (Q)

Lacking other amino acids

Positively charged (none present)

Negatively charged (only 2)

Aliphatic (e.g. leucine, isoleucine,

alanine almost entirely absent)

What structure does FUS LC form?

FUS LC structure?

Adapted from: Schmidt and Görlich, Elife 2015

FUS LC: a very unusual part of protein sequence universe

LC or “prion-like” domain of FUS has

an unusual sequence

Rich in amino acids:

serine (S)

tyrosine (Y)

glycine (G)

glutamine (Q)

Lacking other amino acids

Positively charged (none present)

Negatively charged (only 2)

Aliphatic (e.g. leucine, isoleucine,

alanine almost entirely absent)

What structure does FUS LC form?

FUS LC is unstructuredNMR spectrum of FUS LC

• Atomic resolution NMR

• Narrow range of amide 1H

shifts characteristic of

disordered protein

Translation

Each dot here is a “peak” that

represents a signal from a

particular position in the FUS LC

protein sequence

From the distribution of peak

positions, we see that FUS LC

does not fold into one structure50 μM FUS LC in 50 mM MES/BisTris 150mM NaCl pH 5.5 @ 25 °C

FUS LC is unstructuredNMR spectrum of FUS LC

• Atomic resolution NMR

• Narrow range of amide 1H

shifts characteristic of

disordered protein

Simulations can probe the vast

array of disordered structures

50 μM FUS LC in 50 mM MES/BisTris 150mM NaCl pH 5.5 @ 25 °C

Example FUS structure by Zerze and Mittal, @ Lehigh

277K

FUS LC phase separation25 °C

4 °C

FUS LC solution opalescent at

low temperature

Liquid-liquid phase separation?

277K


4 °C


low temperature


FUS LC opalescent at high

concentration

Liquid droplets observed by

DIC

277K


4 °C


low temperature



concentration


DIC

FUS LC droplets

277K


4 °C


low temperature



concentration


DIC

FUS LC droplets flow and fuse near the air/water interface

277K


4 °C


low temperature



concentration


DIC

FUS droplet formation is

reversible with temperature

277K


4 °C


low temperature



concentration


DIC



FUS full length forms droplets

when released from N-terminal

solublizing tag

277K


4 °C


low temperature



concentration


DIC



FUS full length forms droplets

when released from N-terminal

solublizing tag

FUS LC droplets bind the C-

terminal tail of RNA pol II

FUS LC phase separationWhat is the structure of FUS LC

inside a “droplet”?

50 μM FUS LC in 50 mM MES/BisTris 150mM NaCl pH 5.5 @ 25 °C

Dis

pe

rse

d

FUS LC phase separation

Clo

ud

y, p

ha

se

se

pa

rate

d

1 m

MF

US

LC

--

un

sta

ble

time

What is the structure of FUS LC


FUS LC phase separation

Clo

ud

y, p

ha

se

se

pa

rate

d

1 m

MF

US

LC

--

un

sta

ble

Clo

ud

y, p

ha

se

se

pa

rate

dtime spin

Low

temp

More

spin

Two phasesP

ha

se

se

pa

rate

d D

isp

ers

ed

~7mM

<100 μM

What is the structure of FUS LC


277K

Ph

as

e s

ep

ara

ted

Dis

pe

rse

d

Atomic details of FUS LC droplets

~7mM FUS LC at 25 °C derived from a original sample:

1 mM FUS LC in 50 mM MES/BisTris 150mM NaCl pH 5.5

NMR Observation volume

FUS LC remains disordered in

liquid phase separated state

• Phase-separated state

spectrum overlays with

dispersed

Translation

In liquid droplets, the majority of

FUS LC retains a disordered

structure very similar to the

dispersed state

277K

Ph

as

e s

ep

ara

ted

Dis

pe

rse

d

Atomic details of FUS LC droplets FUS LC remains disordered in

liquid phase separated state

• Phase-separated state slowed

motions compared to dispersed

277K

Seeing inside granulesFUS LC domain

• Disordered domain as a

monomer (dispersed)

• Liquid-liquid phase-separation

at physiological concentration

• Recruits RNA Pol II CTD

• FUS LC remains disordered

inside the phase-separated

state

Next steps

• Mechanism of cell regulation

of droplet assembly?

• Disease mutations (G156E)

• Atomic contacts in droplets?

• Other droplet proteins?

Burke KA, Janke AM,

Rhine CL, Fawzi NL, Molecular Cell. (2015)

Fawzi group:

Dr. Kathleen Burke

Alex Conicella

Veronica Ryan

Charlene Chabata

Abigail Janke

Daniel Ramirez Montero

Dahee Seo

Joshua Amaya

Collaborators:

Frank Shewmaker - USUHS

Yuh Min Chook and Mike Rosen - UT Southwestern

Steve McKnight - UT Southwestern

Jeetain Mittal – Lehigh

Tony Hyman – MPI Dresden

Acknowledgements:

Michael “Sparky” Clarkson and Geoff Williams

Structural biology at Brown: Wolfgang Peti, Rebecca Page,

Alexandra Deaconescu, Gerwald Jogl and their groups

Funding:

Acknowledgements

P20GM104937

P20GM103430

Nicolas Fawzi - Residue-by-Residue View of the In Vitro FUS Granules

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