[PPT] - ? 190 ATP or ATP-analogues 120 + Hp DnaB ssDNA Laboratory of PowerPoint Presentation

SLIDE 1

Protein-nucleotide interactions detected by solid-state NMR

Dr. Thomas Wiegand

CCPN Meeting 2017, Stirling 15/07/2017

Laboratory of Physical Chemistry – Group Prof. Beat Meier – ETH Zürich

120 Å 190 Å

HpDnaB

ATP

r

ATP-analogues + ssDNA

?

SLIDE 2

2 Laboratory of Physical Chemistry – Group Prof. Beat Meier – ETH Zürich

DnaB helicases unwind double-stranded DNA

Structural consequences of nucleotide binding (ATP & DNA)?
Structure-function relationships in protein engines?
How does DNA replication work on a molecular level?

Adapted from: http://biochem.pepperdine.edu/dokuwiki/doku.php?id=chem331:dnab_helicase

DnaB helicase Topoisomerase Parental strand 3’ 5’ DnaG primase

Taken from: http://love-life- science.blogspot.ch/2014/09/unzipping

of-dna.html

SLIDE 3

3

ATP-hydrolysis is coupled to molecular functioning

Walker, ..., Gay, The EMBO Journal, 1982, 1, 945-951; Spies, DNA Helicases and DNA Motor Proteins, Springer New York, 2012.

DNA translocation

SLIDE 4

4

The helicase from Helicobacter pylori forms dodecameric assemblies

Strycharska, ..., Berger, Molecular Cell, 2013, 52, 844-854, Bazin, …, Terradot, Nucleic Acids Res., 2015, 43, 8564-8576.

6 x CTD 6 x NTD 6 x CTD 6 x NTD Molecular mass of 672 kDa, 488 aa/ monomer

120 Å 190 Å

SF4 helicase

Homology model for the HpDnaB:ADP complex (based on the AaDnaB:ADP crystal structure)

SLIDE 5

5

Do the nucleotides bind to the protein? Where do they bind? What are the structural consequences of nucleotide binding?

No single-crystals available Solid-state NMR on sedimented samples

Questions to be addressed by solid-state NMR

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6

Approaches to probe protein-nucleotide interactions

Diamagnetic NMR (e.g. 31P MAS;13C/15N CSPs;

15N,13C NCX; 13C,31P and 15N,31P correlations)

Paramagnetic NMR (e.g. substitution of the Mg2+ cofactor by Mn2+ or Co2+) Dynamic nuclear polarization (DNP) EPR (e.g. Mn2+-Mn2+ DEER)

DNA binding to HpDnaB

ATP/DNA binding

SLIDE 7

7

Part 1 Diamagnetic solid-state NMR

How to monitor nucleotide binding? How to distinguish between bound und unbound nucleotides?

SLIDE 8

8 8 Laboratory of Physical Chemistry – Group Prof. Beat Meier – ETH Zürich

31P NMR to distinguish between bound and unbound nucleotides

31P,1H cross-polarization (CP)

experiments detect bound AMP-PNP in slightly different conformations

Solution-phase spectra @ 7.05 T Solid-phase spectra @ 11.74 T

31P direct pulsed experiments detect

AMP-PNP and AMP-PN in the solution phase of the NMR rotor

31P solution-state experiments allow

to assign the resonances of AMP- PNP/AMP-PN

HpDnaB + 5mM AMP-PNP + 5mM MgCl2 HpDnaB + 5mM AMP-PNP + 5mM MgCl2 5mM AMP-PNP + 5mM MgCl2

Wiegand,...,Terradot, Böckmann, Meier, Angew. Chem. Int. Ed., 2016, 55, 14164-14168.

SLIDE 9

9 9 Laboratory of Physical Chemistry – Group Prof. Beat Meier – ETH Zürich

31P NMR: Does ssDNA bind to the helicase?

31P,1H cross-polarization (CP)

experiments detect bound ssDNA to the helicase

Solution-phase spectra @ 7.05 T Solid-phase spectra @ 11.74 T

31P direct pulsed experiments detect

hydrolyzed AMP-PNP (AMP-PN) in the solution-phase of the NMR rotor

31P solution-state experiments allow

to detect unbound ssDNA

HpDnaB + 5mM AMP-PNP + 5mM MgCl2 + 0.5mM ssDNA HpDnaB + 5mM AMP-PNP + 5mM MgCl2 + 0.5mM ssDNA 5mM MgCl2 + 0.5mM ssDNA

Wiegand,...,Terradot, Böckmann, Meier, Angew. Chem. Int. Ed., 2016, 55, 14164-14168.

SLIDE 10

10

31P chemical shifts are very sensitive to the choice of the ATP-analogue

31P,1H CP-MAS NMR @ 11.74 T

31P,1H cross-polarization

experiments allow to detect the bound ATP-analogues.

Structural inhomogeneities
bserved upon AMP-PNP

binding.

ATP gets hydrolyzed during rotor

filling.

pre-hydrolytic “transition state” post-hydrolytic

SLIDE 11

11

DNA-binding monitored by 31P,1H cross-polarization experiments

31P,1H CP-MAS NMR

@ 11.74 T

DnaB + ATP-analogue + ssDNA

pre-hydrolytic “transition state” post-hydrolytic

( )

DNA binding

SLIDE 12

12

Arginine sidechains of the protein bind to ssDNA

15N-13C NCX @

850 MHz

arginine sidechain phosphate backbone of DNA

SLIDE 13

13

CSPs indicate nucleotide binding & conformational switch of the CTD

13C-13C 20 ms DARR @ 850 MHz

CSP > 0.3 ppm HpDnaB HpDnaB + AMP-PNP + MgCl2

Chemical-shift perturbations (CSPs)

Consequence of nucleotide-binding
Related to allosteric effects

+ 3D NMR experiments

Wiegand,...,Terradot, Böckmann, Meier, Angew. Chem. Int. Ed., 2016, 55, 14164-14168.

SLIDE 14

14 14 Laboratory of Physical Chemistry – Group Prof. Beat Meier – ETH Zürich

How to assign such a large system? Building block approach

NTD

1-153

CTD

152-488

FL

+ =

δ2(13C)/ ppm δ1(13C)/ ppm

?

Tuesday, 08/09/15

Are the domains conserved? Transfer of assignments between NTD (assignment available) and FL HpDnaB possible?

Wiegand, Gardiennet, .., Terradot, Böckmann, Meier, Biomol. NMR Assign., 2016, 10, 13-23; Wiegand, Gardiennet, ...,Terradot, Böckmann, Meier, J. Biomol. NMR, 2016, 65, 79-86.

13C-13C 20 ms DARR @ 850 MHz

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15

Averaged absolute 13C and 15N chemical shift differences between NTD and FL HpDnaB

15 Laboratory of Physical Chemistry – Group Prof. Beat Meier – ETH Zürich

Building block approach: Assignment of the full-length protein

Wiegand, Gardiennet, ...,Terradot, Böckmann, Meier, J. Biomol. NMR, 2016, 65, 79-86.

And the CTD? Sequential assignment of the full-length protein

Structural differences between isolated NTD and NTD in FL DnaB

SLIDE 16

16

Part 2 Paramagnetic solid-state NMR to determine protein-ATP interactions

Where does the metal ion bind?

SLIDE 17

17

Paramagnetic solid-state NMR allows to identify residues in NBD

Mn2+: Paramagnetic relaxation enhancements (PREs) Co2+: Pseudo-contact shifts (PCSs)

Tamaki, ..., Demura, J. Biomol. NMR 2016, 64, 87-101.

Ipara/Idia ratio for 2D 13C-13C

DARR

B0=20.0 T
T1e(Mn2+)= 30 ns
T1e(Co2+)= 0.1 ns
ICP, It1, It2, IDARR
experimental conditions

SLIDE 18

18 18 Laboratory of Physical Chemistry – Group Prof. Beat Meier – ETH Zürich

NMR: Diamagnetic Mg2+ can be substituted by paramagnetic Mn2+ ions

13C-13C 20 ms DARR @ 850 MHz

3 types of resonances: a) Not-influenced by AMP-PNP:Mn2+ binding (e.g. 24A) b) Attenuated upon AMP-PNP:Mn2+ binding (e.g. 228A) c) Broadened beyond detection upon AMP-PNP:Mn2+ binding (e.g. 203A, 351A)

Wiegand, Lacabanne, Keller...,Terradot, Jeschke, Böckmann, Meier, Angew. Chem. Int. Ed., 2017, 56, 3369-3373.

SLIDE 19

19 19 Laboratory of Physical Chemistry – Group Prof. Beat Meier – ETH Zürich

How to determine PREs with CCPN?

Assign 3D spectra of diamagnetic protein sample
Scale the spectra (use a resonance not affected by PREs, here 34V)
Assign 3D spectra of paramagnetic protein sample
Extract intensities from peak assignment lists

Wiegand, Lacabanne, Keller...,Terradot, Jeschke, Böckmann, Meier, Angew. Chem. Int. Ed., 2017, 56, 3369-3373.

SLIDE 20

20 20 Laboratory of Physical Chemistry – Group Prof. Beat Meier – ETH Zürich

Site-specific determination of PREs from 3D experiments

Wiegand, Lacabanne, Keller...,Terradot, Jeschke, Böckmann, Meier, Angew. Chem. Int. Ed., 2017, 56, 3369-3373.

Residues with effective distances < 15 Å (distance between Cα and the two nearest metal centers)

SLIDE 21

21

Paramagnetic solid-state NMR allows to identify residues in NBD

Long-range distance information (> 20 Å) becomes accessible, nucleotide binding domains are identified.

Wiegand, Lacabanne, Keller...,Terradot, Jeschke, Böckmann, Meier, Angew. Chem. Int. Ed., 2017, 56, 3369-3373.

SLIDE 22

22

Part 3

31P,13C correlation experiments to probe

protein-nucleotide interactions

Where do the ATP-analogues and DNA bind?

SLIDE 23

23

MAS-DNP for studying protein-DNA interactions

Dynamic nuclear polarization (DNP)- enhanced MAS

31P-13C/15N correlations suffer from weak NMR

signal

Collaboration with Prof. C. Copéret (ETH Zürich) Taken from: http://www.coperetgroup.ethz.ch/research/dynam ic-nuclear-polarization--dnp--.html

SLIDE 24

24

MAS-DNP for studying protein-DNA interactions

Collaboration with Prof. C. Copéret (ETH Zürich)

HpDnaB:ADP:ssDNA

ρ= (SNDNP/SNNMR)

Ravera, ..., Griffin, Luchinat, Bertini, J. Am. Chem. Soc., 2013, 135, 1641-1644; Wiegand, ..., Copéret, Böckmann, Meier, J. Biomol. NMR, submitted.

Highest sensitivity without d8-glycerol. 2 mM AMUpol radical concentration (not optimized).

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25

MAS-DNP for studying protein-DNA interactions

Collaboration with Prof. C. Copéret (ETH Zürich)

10.0 kHz @ 14.1 T 395 GHz gyrotron

Measurement time 21 h.

Morag, ..., Goldbourt, JACS, 2014, 136, 2292-2301; Wiegand, ..., Copéret, Böckmann, Meier, J. Biomol. NMR, submitted.

Lowest contour level: 2.1 times noise RMSD

HpDnaB:ADP:ssDNA

SLIDE 26

26

Any chance for probing DNA interactions under conventional NMR conditions (in reasonable measurement time)?

X

CHHP & NHHP

Morag, ..., Goldbourt, JACS, 2014, 136, 2292-2301.

SLIDE 27

27

CHHP 2D experiments for studying protein-DNA interactions

Measurement time 13 d.

200 μs H-H spin diffusion time 11.74 T @ 17.0 kHz

HpDnaB:ADP:AlF4

:ssDNA

SLIDE 28

28

Conclusions

31P NMR allows to monitor nucleotide binding
13C/15N CSPs highlight conformational

changes

Paramagnetic NMR allows to probe protein-

ATP interactions

DNA binding can be detected in 31P/13C

correlation experiments

SLIDE 29

Prof. Beat H. Meier

Riccardo Cadalbert

Prof. Gunnar Jeschke

Katharina Keller

Dr. Maxim Yulikov
Prof. Christophe Copéret
Dr. Anja Böckmann

Denis Lacabanne

Dr. Laurent Terradot
Dr. Joanna Timmins