Protein-nucleotide interactions detected by solid-state NMR

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1 190 Å Protein-nucleotide interactions detected by solid-state NMR Dr. Thomas Wiegand CCPN Meeting 2017, Stirling 15/07/ Å HpDnaB ATP or ATP-analogues + ssdna? Laboratory of Physical Chemistry Group Prof. Beat Meier ETH Zürich

2 DnaB helicases unwind double-stranded DNA 2 Topoisomerase DnaG primase 3 5 Parental strand DnaB helicase Taken from: -of-dna.html Adapted from: o Structural consequences of nucleotide binding (ATP & DNA)? o Structure-function relationships in protein engines? o How does DNA replication work on a molecular level? Laboratory of Physical Chemistry Group Prof. Beat Meier ETH Zürich

3 ATP-hydrolysis is coupled to molecular functioning 3 Walker,..., Gay, The EMBO Journal, 1982, 1, ; Spies, DNA Helicases and DNA Motor Proteins, Springer New York, DNA translocation

4 190 Å The helicase from Helicobacter pylori forms dodecameric assemblies 4 SF4 helicase 6 x CTD 6 x NTD 6 x NTD 6 x CTD 120 Å Molecular mass of 672 kda, 488 aa/ monomer Homology model for the HpDnaB:ADP complex (based on the AaDnaB:ADP crystal structure) Strycharska,..., Berger, Molecular Cell, 2013, 52, , Bazin,, Terradot, Nucleic Acids Res., 2015, 43,

5 Questions to be addressed by solid-state NMR 5 No single-crystals available Solid-state NMR on sedimented samples Do the nucleotides bind to the protein? Where do they bind? What are the structural consequences of nucleotide binding?

6 Approaches to probe protein-nucleotide interactions 6 Diamagnetic NMR (e.g. 31 P MAS; 13 C/ 15 N CSPs; 15 N, 13 C NCX; 13 C, 31 P and 15 N, 31 P correlations) Dynamic nuclear polarization (DNP) DNA binding to HpDnaB ATP/DNA binding Paramagnetic NMR (e.g. substitution of the Mg 2+ cofactor by Mn 2+ or Co 2+ ) EPR (e.g. Mn 2+ -Mn 2+ DEER)

7 7 Part 1 Diamagnetic solid-state NMR How to monitor nucleotide binding? How to distinguish between bound und unbound nucleotides?

8 31 P NMR to distinguish between bound and unbound nucleotides 8 5mM AMP-PNP + 5mM MgCl 2 31 P solution-state experiments allow to assign the resonances of AMP- PNP/AMP-PN HpDnaB + 5mM AMP-PNP + 5mM MgCl 2 31 P direct pulsed experiments detect AMP-PNP and AMP-PN in the solution phase of the NMR rotor HpDnaB + 5mM AMP-PNP + 5mM MgCl 2 31 P, 1 H cross-polarization (CP) experiments detect bound AMP-PNP in slightly different conformations Wiegand,...,Terradot, Böckmann, Meier, Angew. Chem. Int. Ed., 2016, 55, Laboratory of Physical Chemistry Group Prof. Beat Meier ETH Zürich Solution-phase 7.05 T Solid-phase T

9 31 P NMR: Does ssdna bind to the helicase? 9 5mM MgCl mM ssdna 31 P solution-state experiments allow to detect unbound ssdna HpDnaB + 5mM AMP-PNP + 5mM MgCl mM ssdna 31 P direct pulsed experiments detect hydrolyzed AMP-PNP (AMP-PN) in the solution-phase of the NMR rotor HpDnaB + 5mM AMP-PNP + 5mM MgCl mM ssdna 31 P, 1 H cross-polarization (CP) experiments detect bound ssdna to the helicase Wiegand,...,Terradot, Böckmann, Meier, Angew. Chem. Int. Ed., 2016, 55, Laboratory of Physical Chemistry Group Prof. Beat Meier ETH Zürich Solution-phase 7.05 T Solid-phase T

10 post-hydrolytic transition state pre-hydrolytic P chemical shifts are very sensitive to the choice of the ATP-analogue o 31 P, 1 H cross-polarization experiments allow to detect the bound ATP-analogues. o Structural inhomogeneities observed upon AMP-PNP binding. o ATP gets hydrolyzed during rotor filling. 31 P, 1 H CP-MAS T

11 post-hydrolytic transition state pre-hydrolytic DNA-binding monitored by 31 P, 1 H cross-polarization experiments 11 DNA binding ( ) DnaB + ATP-analogue + ssdna 31 P, 1 H CP-MAS T

12 Arginine sidechains of the protein bind to ssdna N- 13 C 850 MHz arginine sidechain phosphate backbone of DNA

13 CSPs indicate nucleotide binding & conformational switch of the CTD D NMR experiments HpDnaB HpDnaB + AMP-PNP + MgCl 2 Chemical-shift perturbations (CSPs) o Consequence of nucleotide-binding o Related to allosteric effects 13 C- 13 C 20 ms 850 MHz Wiegand,...,Terradot, Böckmann, Meier, Angew. Chem. Int. Ed., 2016, 55, CSP > 0.3 ppm

14 NTD δ1(13c)/ ppm How to assign such a large system? Building block approach 13C-13C ms 850 MHz + CTD =? FL Are the domains conserved? Transfer of assignments between NTD (assignment δ2(13c)/ ppm available) and FL HpDnaB Wiegand, Gardiennet,.., Terradot, Böckmann, Meier, Biomol. NMR Assign., 2016, 10, 13-23; Tuesday, 08/09/15 possible? Laboratory of PhysicalGardiennet, Chemistry Group Prof. Beat MeierBöckmann, ETH Zürich Meier, J. Biomol. NMR, 2016, 65,14 Wiegand,...,Terradot,

15 Building block approach: Assignment of the full-length protein 15 Averaged absolute 13 C and 15 N chemical shift differences between NTD and FL HpDnaB Structural differences between isolated NTD and NTD in FL DnaB And the CTD? Sequential assignment of the full-length protein Laboratory Wiegand, of Physical Gardiennet, Chemistry Group...,Terradot, Prof. Beat Meier Böckmann, ETH Zürich Meier, J. Biomol. NMR, 2016, 65,

16 16 Part 2 Paramagnetic solid-state NMR to determine protein-atp interactions Where does the metal ion bind?

17 Paramagnetic solid-state NMR allows to identify residues in NBD 17 Mn 2+ : Paramagnetic relaxation enhancements (PREs) Co 2+ : Pseudo-contact shifts (PCSs) o o o o o o I para /I dia ratio for 2D 13 C- 13 C DARR B 0 =20.0 T T 1e (Mn 2+ )= 30 ns T 1e (Co 2+ )= 0.1 ns I CP, I t1, I t2, I DARR experimental conditions Tamaki,..., Demura, J. Biomol. NMR 2016, 64,

18 NMR: Diamagnetic Mg 2+ can be substituted by paramagnetic Mn 2+ ions 18 3 types of resonances: 13 C- 13 C 20 ms 850 MHz a) Not-influenced by AMP-PNP:Mn 2+ binding (e.g. 24A) b) Attenuated upon AMP-PNP:Mn 2+ binding (e.g. 228A) c) Broadened beyond detection upon AMP-PNP:Mn 2+ binding (e.g. 203A, 351A) Wiegand, Lacabanne, Keller...,Terradot, Jeschke, Böckmann, Meier, Angew. Chem. Int. Ed., 2017, 56, Laboratory of Physical Chemistry Group Prof. Beat Meier ETH Zürich 18

19 How to determine PREs with CCPN? 19 o Assign 3D spectra of diamagnetic protein sample o Scale the spectra (use a resonance not affected by PREs, here 34V) o Assign 3D spectra of paramagnetic protein sample o Extract intensities from peak assignment lists Wiegand, Lacabanne, Keller...,Terradot, Jeschke, Böckmann, Meier, Angew. Chem. Int. Ed., 2017, 56, Laboratory of Physical Chemistry Group Prof. Beat Meier ETH Zürich 19

20 Site-specific determination of PREs from 3D experiments 20 Residues with effective distances < 15 Å (distance between Cα and the two nearest metal centers) Wiegand, Lacabanne, Keller...,Terradot, Jeschke, Böckmann, Meier, Angew. Chem. Int. Ed., 2017, 56, Laboratory of Physical Chemistry Group Prof. Beat Meier ETH Zürich 20

21 Paramagnetic solid-state NMR allows to identify residues in NBD 21 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,

22 22 Part 3 31 P, 13 C correlation experiments to probe protein-nucleotide interactions Where do the ATP-analogues and DNA bind?

23 Collaboration with Prof. C. Copéret (ETH Zürich) MAS-DNP for studying protein-dna interactions P- 13 C/ 15 N correlations suffer from weak NMR signal Taken from: ic-nuclear-polarization--dnp--.html Dynamic nuclear polarization (DNP)- enhanced MAS

24 Ravera,..., Griffin, Luchinat, Bertini, J. Am. Chem. Soc., 2013, 135, ; Wiegand,..., Copéret, Böckmann, Meier, J. Biomol. NMR, submitted. Collaboration with Prof. C. Copéret (ETH Zürich) MAS-DNP for studying protein-dna interactions 24 HpDnaB:ADP:ssDNA ρ= (SN DNP /SN NMR ) Highest sensitivity without d8-glycerol. 2 mm AMUpol radical concentration (not optimized).

25 MAS-DNP for studying protein-dna interactions T 395 GHz gyrotron Measurement time 21 h. Morag,..., Goldbourt, JACS, 2014, 136, ; Wiegand,..., Copéret, Böckmann, Meier, J. Biomol. NMR, submitted. HpDnaB:ADP:ssDNA Lowest contour level: 2.1 times noise RMSD Collaboration with Prof. C. Copéret (ETH Zürich)

26 X CHHP & NHHP Morag,..., Goldbourt, JACS, 2014, 136, Any chance for probing DNA interactions under conventional NMR conditions (in reasonable measurement time)?

27 CHHP 2D experiments for studying protein-dna interactions 27 HpDnaB:ADP:AlF 4- :ssdna Measurement time 13 d. 200 μs H-H spin diffusion time khz

28 Conclusions 28 o 31 P NMR allows to monitor nucleotide binding o 13 C/ 15 N CSPs highlight conformational changes o Paramagnetic NMR allows to probe protein- ATP interactions o DNA binding can be detected in 31 P/ 13 C correlation experiments

29 Prof. Beat H. Meier Riccardo Cadalbert Prof. Gunnar Jeschke Katharina Keller Dr. Maxim Yulikov Lyon Dr. Anja Böckmann Denis Lacabanne Dr. Laurent Terradot Dr. Joanna Timmins (Grenoble) Prof. Christophe Copéret