Solid-state NMR studies of membrane protein structure and dynamics in synthetic lipids and cell membranes

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1 Solid-state NMR studies of membrane protein structure and dynamics in synthetic lipids and cell membranes Vlad Ladizhansky University of Guelph, Ontario Canada Winter School on Biomolecular NMR January 15, 2016

2 Bacteriorhodopsin (haloarchaea) Halorhodopsin (haloarchaea) Sensory Rhodopsin I- Transducer Complex (haloarchaea) Proteorhodopsin (proteobacteria) Anabaena Sensory Rhodopsin (cyanobacteria) Chlamydomonas CSRA Chlamydomonas CSRB Cytoplasm Original Microbial Rhodopsins (Haloarchaeal) Retinal Schiff Base Bacteriorhodopsin (BR) - H + pump Halorhodopsin (HR) Cl - pump Sensory rhodopsins I and II (SRI and SRII) - photosensors Bind all-trans retinal via lysine Schiff base Retinal photoisomerizes to 13-cis and causes conformational changes Spudich and Jung (2005) Handbook of Photosensory Receptors; Wiley-VCH

3 Signal Transduction Cascade of Anabaena Sensory Rhodopsin Model of photoactivation ASRT: DNA complex: Wang et al., J. Mol. Biol Anabaena sensory rhodopsin (ASR) found in fresh water cyanobacterium Anabaena sp. PCC 7120 ASR interacts with soluble tetrameric transducer (ASRT), which dissociates upon photoactivation and regulates expression of photosynthesis- and circadianrelated genes (Jung et al., 2003, Mol. Microbiol.; Vogeley et al, 2004, Science; Vogeley et al, 2007, JMB; Jung, 2007, Photochem. Photobiol.; Kondoh et al, 2011, JACS)

4 Solid-state NMR of ASR 15 N 1D NMR 2D MHz 600 MHz Single all-trans retinal conformation X-ray data consistent with two simultaneous conformers, all-trans and 13-cis,15-syn Excellent resolution Most of the protein is observed L. Shi et al., Angew. Chem. Int. Ed., 2011

5 Structure Determination Samples with alternate 13 C labeling pattern using [1,3-13 C] glycerol or [2-13 C] glycerol as carbon sources Experiments to measure interatomic distances 2D PDSD carbon-carbon correlation 2D CHHC 3D HBR 2 LeMaster, Hong, Oschkinat

6 Solid state NMR structure of ASR Unambiguous distance constraints Intraresidue contacts: 1293 Sequential: 663 Short (2 i-j 4): 532 Long ( i-j >4): 211 H-bonds constraints 100 Dihedral angle constraints 372 E D E D Lowest energy structure 10 (out of 100) lowest energy structures RMSD ~ 2.1 Å for all backbone atoms

7 Oligomerization and supramolecular organization Bacteriorhodopsin lattice of trimers, but not in detergents Halorhodopsin trimers in membranes and detergents Proteorhodopsin hexamers Sensory rhodopsins heterotetramers (rhodopsins are dimers) SDS-PAGE suggests that ASR is trimeric in detergent Oligomerization state may be different in lipids (e.g. BR)

8 Visible range CD of retinal proteins is sensitive to their oligomerization state Bacteriorhodopsin (BR) in DMPC Trimer monomer Halorhodopsin (HR) in DDM Gloebacter rhodopsin (GR) in DDM Wavelength Wavelength Wavelength (Heyn et al. (1981) Biochemistry 20, 840. PDB 2NTU Sasaki et al. (2009) Photochem & Photobiol. 85,130. PDB 1E12 Tsukamoto et al. (2013) FEBS Lett. 587, 322. BR HR No structure available; Proposed trimers & pentamers

9 Confirming ASR trimerization by CD Bacteriorhodopsin (BR) in DMPC ASR in DDM ASR in DMPC/DMPA Trimer monomer Wavelength Wavelength Wavelength PDB 2NTU Similar CD shapes indicate similar oligomerization In lipid and detergent BR S. Wang et al, J. Am. Chem. Soc., 2012

10 Paramagnetic Relaxation Enhancements in ASR Nitroxide labels (MTSL) attached to Cys residue introduced into the putative interfacial region MTSL Red: MTSL-labeled S26C ASR (paramagnetic) Blue: MMTS-labeled sample (diamagnetic S-methyl) NCA correlation 13 C- 13 C correlation

11 Paramagnetic relaxation enhancements MTSL NCA correlation 13 C- 13 C correlation MTSL Carbon-Carbon correlation H N HA HB HG HD

12 Paramagnetic Relaxation Enhancements MTSL site B C D Cytoplasmic B A E E A D Top view from the cytoplasmic side G Strong PREs (green) from the C26-MTSL observed in the helices D and E, but not C, F, and G are against the expectations from the structure F Extracellular

13 Oligomeric organization: X-ray vs SSNMR NMR C26-C26 DEER measurement B D X-ray C G DEER Signal d e-e =3.38±0.16 nm Time [µs] DEER measurements: S. Milikisiyants, S. Wang, R. A. Munro, M. Donohue, M. Ward, L. Brown, T.I. Smirnova, V. Ladizhansky, A.I. Smirnov, in preparation. NMR structure: S. Wang et al, Nature Methods 2013, 10, X-ray structure: L. Vogeley et al, Science 2004, 306,

14 View View from from Cytoplasm Cytoplasm F E Monomer structure: X-ray vs SSNMR D B C G E A E Cytoplasm Periplasm F D C A D C G B F Cytoplasm G A Differences are on the cytoplasmic side Cytoplasmic halves of helices A, E, F B Grey: Xray Red: SSNMR Periplasm X-ray structure: 1XIO NMR structure: PDB: 2M3G

15 Supramolecular organization of ASR SAXS 2D hexagonal lattice, lattice constant ~66 Å M. Ward et al, Biophys. J., 2015

16 Confirming ASR trimerization in E. coli membranes by CD in the visible range ASR in DDM ASR in DMPC/DMPA ASR in E. coli membrane Wavelength Wavelength Similar bilobe shapes in detergents, lipids, E. coli indicate similar oligomerization S. Wang et al, J. Am. Chem. Soc., 2012; M. Ward et al, Biophys. J., 2015.

17 ASR in E.coli membrane: sample preparation Targeted labeling of the protein of interest T. Jacso et al, Angew. Chem (2011), 51, 432; M. Renault et al, PNAS (2012), 109, Sucrose gradient to separate inner membrane (IM) and outer membrane (OM) 2-phase system to remove IM vesicles not containing ASR FTIR α β Sucrose gradient: S. Wagner et al, Mol Cell Proteomics (2007), 6, phase separation: H. Everberg et al, J. Chromatogr A, (2006), 1118, 244.

18 ASR in E.coli membrane ASR is ~10% of the protein content, crowded spectra RED: proteoliposomes 900 Black: ASR in E.coli Use of the 900 MHz spectrometer at the National High Magnetic Field Laboratory ~ 2x sensitivity Enables 3D spectroscopy

19 3D spectroscopy of ASR in E.coli In total ~100 residues have been assigned in the ASR in the E.coli membrane through chemical shift mapping M. Ward et al, Biophys. J., Black E. coli membrane Red - Proteoliposomes Blue control (no ASR)

20 Anabaena Sensory Rhodopsin in the E. coli inner membrane 15 N chemical shift perturbations 13 C α chemical shift perturbations Green-conserved Yellow- perturbed All-trans retinal Conserved retinal pocket High degree of structural conservation Changes in residues facing the interior of the protein & the bilayer

21 Topology and Dynamics of ASR by H/D Exchange H 2 O/D 2 O H/D exchange data recorded using 2D NCA and 3D NCACX Cytoplasm SNR Periplasm Shi et al, 2011, Angew. Chem; Wang et al, 2013, Nature Methods Cytoplasmic side more exposed Protein assymetrically positioned in membrane

22 H/D exchange to probe intermediate states Model of photoactivation Experimental setup Illuminate with sample in D 2 O C-terminus (helix G) is a putative binding motif for ASRT Measure changes in the H/D pattern Protein goes through multiple intermediate states in the photocycle Conformational rearrangements detected through changes in solvent-accessible surface using H/D exchange S. Wang et al., Biophys J. 2011, 101, L23-25.

23 H/D exchange to probe intermediate states Helix G SNR Complete exchange of the cytoplasmic sides of helices F,G H/D data are consistent with possible movement of helix G In part in: S. Wang et al., Biophys J. 2011, 101, L23-25; S. Wang et al, Nature Methods, 2013, 10,

24 Hierarchy of Motions Local motions, ps-ns Collective motions, ns-us Slow exchange or static disorder H E HN C α COOH R D! Fast motions on the time scale of 1/D IS Order parameter S = D expt / D rigid report on the overall amplitude of motions Relaxation rates characterize the time scales Slow motions > 100 s of ms

25 1 H- 15 N/ 13 C DIPSHIFT Measurements 13 C- 1 H TMREV 13 C- 1 H DIPSHIFT 15 N- 1 H TMREV 15 N- 1 H DIPSHIFT A64 is in the BC loop S115 is in helix D C134 is in helix E Uses afterglow magnetization to record complimentary NCA and NCO planes TMREV: Hohwy et al, JACS, 2000,122, 3218 Afterglow: Banigan et al JPC B, 2012, 116, 7138.

26 15 N- 13 C and 15 N- 13 Cα Order Parameters 3D ZF TEDOR pulse sequence 15 N- 13 C α S47: helix B G145: flank of helix E Y51: flank of helix B R72: flank of helix C 15 N- 13 C Michal & Jelinski, JACS, 1997, 119, 9059; Jaroniec et al, JACS 2002, 124, 10728

27 Order Parameters N- 1 H couplings and C- 1 H couplings using TMREV N- 13 C α couplings and N- 13 C couplings using 3D ZF TEDOR Rigid backbone, order parameter is close to unity

28 Transverse relaxation in solid-state NMR 1 H 1 H 1 H 1 H 1 H T 1ρ at moderate spinning frequencies Decoupling 15 N 1 H 1 H T 1ρ at fast spinning frequencies, >45 KHz 1 H 1 H 1 H 15 N Lewandowski, Sass, Grzesiek, Blackledge, Emsley, J Am Chem Soc ,

29 Transverse relaxation measurements Relaxation measurements done at 8 C, 50 khz MAS, spin lock of 12 khz K60 T 1ρ ~189ms Q66, T 1ρ ~77ms Q195, T 1ρ ~91ms A71, T 1ρ ~435ms T170, T 1ρ ~286ms Y171, T 1ρ ~417ms

30 Transverse relaxation measurements Slow motions in the BC, FG loops ω R 2 τ c 2 << 1 R 1ρ ~ 2 15 (b IS / 2)2 (1 S 2 )τ c Kurbanov, Zinkevich, Krushelnitsky, J. Chem. Phys. 2011, 135,

31 Time scales of motions using Model-Free approach Time scales of motions Helices: low 10 s of nanoseconds B-C and F-G loops: up to 100 nanoseconds Good et al, J. Am. Chem. Soc. 2014, 136, 2833

32 Possibility of collective motions? 3D Gaussian Axial Fluctuation Model (GAF) From Lewandowski, Acc Chem Res 2013, 46, σ α, σ β, σ γ define axes of diffusive motions in GAF J. Lewandowski, U. of Warwick Anisotropic motion of a helix averages differently oriented dipolar tensors (e.g., 13 C α - 1 H, 15 N- 1 H) in a different manner

33 3D GAF fitting 7 Helices, B-C and F-G loops fit independently For each secondary structure element R 1ρ, S CαHα, and S NH are fit simultaneously. Time scales approximately the same as in the model free approach Local motions are neglected in the fit Amplitudes are overestimated

34 Solid-state NMR structure determination Protein dynamics Summary Excellent resolution demonstrated for many classes of MPs Already sufficient sensitivity, likely to improve in the years to come First structures available, many more studies under way New technologies & methodologies to unmask stochastic contributions to the relaxation pathways Both fast and slow motions, chemical exchange processes can be probed In-membrane and in-cell NMR Effects of environment Prospects of de novo structural characterization in cell membranes

35 Acknowledgements NSERC Canada Research Chair program Canada FoundaNon for InnovaNon Ontario Ministry of Research and InnovaNon Students Daryl Good Meaghan Ward Rachel Munro Former members Lichi Shi Izuru Kawamura (Yokohama Nat Univ) Shenlin Wang (Peking University) Collaborators Leonid Brown (Guelph) Alex Smirnov (NCSU) Hongjun Liang (Colorado Sch of Mines) Kevin Jung (Sogang U) Takashi Okitsu (Kobe Pharm. Univ.) Akimori Wada (Kobe Pharm. Univ.) Jozef Lewandowski (U. of Warwick) Ivan Hung (NHMFL) Peter Gor kov (NHMFL) Henry Stronks (Bruker Canada) Michael Fey (Bruker USA) Howard Hutchins (Bruker USA) Wurong Zhang (Bruker USA) Jochem Struppe (Bruker USA) Werner Maas (Bruker USA)