Solid-State NMR Distance & Dynamics Techniques for Structure Determination of the Influenza M2 Protein

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1 Solid-State NMR Distance & Dynamics Techniques for Structure Determination of the Influenza M2 Protein Mei Hong Department of Chemistry and Ames Laboratory, Iowa State University 2nd CCPN / Extend-NMR Workshop, Hubertusstock, Germany, Oct 21, !

2 The Influenza M2 Protein Channel opens at low ph out. A tetrameric channel. TM helix (22-46): functional core. H + selectivity: His 37. Inhibited by adamantyl drugs. 2!

3 The Drug Binding Question X-ray structure Soln NMR structure D44 Stouffer, DeGrado et al, Nature, Schnell and Chou, Nature, M2 (22-46) OG ph 5.3 P : Amt : OG = 1.3 : 1 : 52. M2 (18-60) DHPC micelles ph 7.5 P : Rmt : DHPC = 1 : 53 : 40. (200 fold excess).! Structures both solved in detergents, potential for distortion.! Drug in large excess in the solution structure. 3!

4 Traditional SSNMR Strategies for Studying Ligand Binding Sites Site-specific 13 C, 15 N or 19 F labeling of the ligand and the protein: REDOR, R 2 examples: retinal in br (Griffin et al) and rhodopsin (Smith et al), Taxol with tubulin (Schaefer et al), Limitations: No general strategy for site-specific labeling of ligands. Distance reach < 5 Å, unless 19 F or 1 H is used. Small ligands likely dynamic, require very low T to freeze. At mild low T, exchange broadening may occur to destroy signals. Low concentration of drug. What did not work: 15 N-labeled Amt with 13 C labeled M2. 13 C- 15 N REDOR: short distance reach (50 Hz for 4 Å), no observable effects. 13 C-detected 1 H- 15 N REDOR: complex sequence, short 1 H T 2 (190 Hz for 4 Å, ~ 1 H T ms). 1 H spin diffusion (CHHN) very low sensitivity, drug dynamics attenuates spin diffusion. 4!

5 D15-Amt Amt Binding Site from 13 C- 2 H REDOR Jun Wang Sarah Cady DMPC bilayers, ph 7.5, 243 K Lipid filtered spectra 5!

6 Quantitative 13 C- 2 H REDOR: the Universal Curve Single 13 C-pulse REDOR Single 2 H-pulse REDOR Qualitative dephasing but long 13 C T 2. Quantitative dephasing following the universal curve. 70% 2 H inversion. 6!

7 13 C- 2 H REDOR! The orientation-dependent 13 C- 2 H REDOR frequency under MAS is 2 " CD (#,$,r) = % CD ( r)& & sin 2# sin$ ' The coupling constant! CD depends on the distance as: " CD ( r) = #2 $ µ 0 4%!& C& D r 3 = #2% $ 9.3 khz 1 ( r 1 Å) 3 " CD were calculated for #, $ and r values that correspond to various locations of the deuterons on each ring, which are sampled at 10 steps. Since each ring rotates uniaxially, " CD were averaged to give " CD. For 13 C coupled to each I = 1 spin, the 2I + 1 = 3 allowed values of the 2 H S z result in three equally spaced spectral lines of equal intensity, at 0 and ± " CD, for each orientation of the C-D vector. [ ( )] ( S S 0 ) 1 spin pair = cos " CD Nt r 7!

8 Multi-Spin REDOR to a Mobile Molecule Multiple 13 C- 2 H pairs: ( S S 0 ) M spin pairs = 1 3 M " M m=1 [ 1+ 2cos ( # CD,m Nt r )] Due to fast rotation, all 6 deuterons on each Amt ring have the same averaged coupling: ( S S 0 ) 12 deuterons = [ 1+ 2cos (" A CD Ntr )] 6 B # 1+ 2cos " CDNtr [ ( )] 6 8!

9 Example: 13 C REDOR to a CD 3 Group Each 13 C is coupled to 3 equivalent I=1 spins. ( S S 0 ) C-CD3 = 1 [ ( )] cos " CD,m Nt r = cos# + 6cos2# + 2cos 3# 27 ( ), where # = " CD,m Nt r Eq. REDOR S/S 0 value:7/27. For d15-amt, each 13 C is coupled to 6 equivalent I=1 spins in each equatorial plane. 9!

10 Distance Analysis for a 4-Fold Symmetric Bundle Coupling to 15 deuterons speeds up REDOR dephasing by (15) 1/2 ~ 3.9. Due to the 4-fold symmetry of the helical bundle, distances were parameterized in terms of pore radius R and height Z from the center of Amt. r CD = ( R 2 + Z 2 ) 1 2 Joint fitting of distances to multiple residues strongly constrained the protein structure. Klaus Schmidt-Rohr 10!

11 Ligand as a Probe of Protein Structure! SSNMR X-ray Soln NMR 11! 11!

12 Drug-Complexed M2 Structure in Bilayers! 6 x 4 protein-drug distances! 1 x 4 Trp 41 distances! 15 x 4 N-H dipolar couplings! 2 x 4 rotamer constraints! Chemical shifts Hu, Cross et al, Biophys. J., 92, 4335 (2007). 0.3 Å heavy-atom RMSD PDB: 2KQT Cinque Soto Bill DeGrado Cady, Schmidt-Rohr, Wang, Soto, DeGrado and Hong, Nature, 463, 689 (2010). 12!

13 Structures Without & With Protein-Drug Distances! DMPC bilayers, with M2-drug distances PDB: 2KQT DLPC bilayers, no M2-drug distances PDB: 2KAD 13!

14 Motional Rate Regimes Detected by NMR Fast motion: k >>!, (!: interaction strength), typically % c < 1 µs Amplitude: spectral line narrowing; Expts: WISE, DIPSHIFT, LG-CP, recoupled CSA, etc. Lower limit of k: at least 10-fold faster than!;& Exact rate: relaxation times; Slow motion: k <<!, typically % c > 10 ms Measured by 2D and 1D exchange NMR (both static & MAS); Amplitude: Nt r dependent CODEX curves, cross peak distance to diagonal in 2D spectra; Timescale: decay constant in t m -dependent exchange intensities; Number of sites: final value of CODEX curve (1/n R ). Intermediate motion: k ~!. Manifested as intensity loss due to interference with 1 H decoupling; Intensity reduction in Hahn echo or quad echo experiments; Exponential decay in undoubled-dipshift curves; Rate: T 2,1' minima in log(t 2,1' ) versus 1/T plots. 14!

15 Effects of Motions on NMR Lineshapes Example: 2-site exchange with equal-population. slow k << " A # " B k " # A $ # B intermediate fast k >> " A # " B 15!

16 S bond (" PD ) # $ Fast Motion: Order Parameter S $ = 1 ( 2 3cos2 " PM %1)& 1 2 3cos2 " MD %1 = S (" PM )& S mol S tensor: 3 x 3 matrix, 5 unknowns. If the molecule is rigid, then there is a single S tensor for the whole molecule. If the rigid molecule rotates only about a single molecular axis, then the S tensor is uniaxial with its unique axis along the director. S bond (" PD ) # $ $ = 1 2 3cos2 " PD %1 If the rigid molecule rotates about its own molecular axis & an external axis, then S is uniaxial along the molecular axis, ( ) ( ) 16!

17 Amantadine Motion: Symmetry Consideration! Due to the tetrahedral geometry, all C-D bonds lie on a diamond lattice. 3-fold axis Relative to the molecular axis: 12 CD bonds : " PM = 70, CD bonds : " PM = 0 If Amt rotates only around its own molecular axis: " = " # 1 ( 2 3cos2 $ PM %1) = " #S ( $ PM ) 12 CD bonds : 0.33"# = 40 khz 3 CD bonds : 1.0"# = 125 khz If Amt also rotates around an external axis, the bilayer normal n: ( ) # 1 ( 2 3cos2 $ Mn %1) " = " # 1 2 3cos2 $ PM %1 = " #S( $ PM )#S mol 17!

18 Drug Dynamics & Orientation in Lipid Bilayers! 12 CD bonds : 0.33"# = 40 khz 3 CD bonds : 1.0"# = 125 khz Gel phase: S mol " 1 # $ Mn " 0 Fluid phase: ( ) S mol = ±0.46 = 1 2 3cos2 " Mn #1 $ " Mn = 37, 80 2 H quadrupolar coupling (khz) Cady, Schmidt-Rohr, Wang, Soto, DeGrado and Hong, Nature, 463, 689 (2010). 18!

19 Drug Orientation When Bound to M2! S mol = " Mn = 13 ( ) ( ) = !

20 Rotational Diffusion of TM Proteins a Saffman & Delbrück, PNAS, 75, 3111 (1975). b Aisenbrey & Bechinger, JACS, 126, (2004). 20!

21 VT 13 C Spectra: Membrane-Dependent Dynamics 21!

22 Site-Specific Dynamics: the DIPSHIFT Experiment SLF technique: X magn evolution (! PDLF, PISEMA, LG-CP). Constant time: one rotor period. Original version 1 H homonuclear decoupling ( r < 5 khz: e.g. MREV-8 " exp = 2 # $ XH # k homo ( r > 5 khz: e.g. FSLG, DUBMO " 1# t 1 ( ) = % t 1 0 $ t ( )dt " obs = # XH $S XH $k hom o Doubled (4x, etc) versions " obs = 2# XH $S XH $k hom o Allows higher ( r to be used, or small couplings to be measured more accurately. Constant homonuclear decoupling time, no T 2 effects in t 1, no exponential decay. Munowitz, Griffin, Bodenhausen, and Huang, J. Am. Chem. Soc., 103, 2529 (1981). Hong, Gross, Rienstra, Griffin, Kumashiro and Schmidt-Rohr, J. Magn. Reson. 129,85 (1997). 22!

23 DIPSHIFT Time Signals 7 khz MAS, FSLG dec (k homo = 0.577), C-H rigid limit 22.7 khz, N-H rigid limit 10.5 khz Insensitive. 23!

24 Membrane-Dependent Protein Motion! All 303 K A29 CD 3 Backbone immobilization at RT allows sidechain motion to be studied. Luo, Cady, and Hong, Biochemistry, 48, 6361 (2009). 24!

25 ph-dependent Motion of the H + -Selective Imidazole! 308 K, with immobilized backbone in viral membranes. Hu, Luo and Hong, Science, in press. 25!

26 Motional Averaging by Equal-Population Two-Site Jumps Average (or sum) tensor: " = # A + # B invariant under the switching of A and B. The ) principal axis directions are: ( ) 2 = (# B + # A ) 2 ) 3 : Normal of the AOB plane ) 1 : Bisector of the AOB angle ) 2 : Normal of the bisector in AOB plane " n : direction angles between z PAS & # 1,# 2,# 3 axes * A * B ) 3 axis: 90, 90 ) 1 axis: #/2, #/2 ) 2 axis: 90 +#/2, 90 -#/2 1,2,3 convention : left to right, or large to small frequencies : " 1 > " 2 > " 3 # < 90 and # > 90 have switched 1, 2 labels. The principal values of the averaged tensor: " n = 1 2 # ( 3cos2 $ n %1) 26!

27 Two-Site Jump: Phenylene Ring Flip Consider 2 H or C-H dipolar spectra (+=0): Reorientation angle # = 120. " n = 1 2 # ( 3cos2 $ n %1) " 1 = 30 " 2 = 60 " 3 = 90 # ' $ 1 = 5 ) 8 % ( $ 2 = & 1 8 % ) * $ 3 = & 1 2 % ' # ( % = 5 8 % * + = 0.6 +! 0 for the averaged tensor. 27!

28 Possible Geometries of Imidazole Motion 180 ring flip around the C!-C" bond: #=114 for the C$-N!1 bond Uniaxial rotation: S bond = 1 ( 2 3cos2 " PD #1) C$-N!1:,=57, S= C!2-H!2:,=78,S= * A * B ) 3 axis: 90, 90 ) 1 axis: 33, 147 ) 2 axis: 57, 57 " 1 = 33 " 2 = 57 " 3 = 90 " n = 1 2 # ( 3cos2 $ n %1) # ' $ 1 = 0.56% ) ( $ 2 = &0.06% ) * $ 3 = &0.5% S " # # = 0.56 ' # ( % = 0.56% * + = 0.79 Neither motion agrees with the experimental order parameters. 28!

29 Imidazole Motional Geometry from S CH & S CN! S " # " $% 29!

30 Ring-Flip Energy Accounts for Functional Data C!2-H dipolar coupling Ring reorientation rate: ~ 5x10 4 /s Proton conduction rate: 100 2x10 4 /s k 293 K " 52 khz, k 263 K # 3.3 khz. " k #G a 293 K = e R k 263 K $ " 1 ' & ) % 263( *16 "G flip > 59 kj/mol Single-channel H + current: 18 C: 2.7 x A 37 C: 40 x A "G H+ cond # 104 kj/mol Lin and Schroder, J. Virol, Shutter (water wire) model: Hu, Luo and Hong, Science, in press. "G water # 29 $ 42 kj/mol 30!

31 Acknowledgement Iowa State University Dr. Sarah Cady, Fanghao Hu Dr. Shenhui Li Yongchao Su, Yuan Zhang Marilu Dick-Perez, Jennifer Hayes Aaron Liao, Theint Theint Tuo Wang, Yi Ding Collaborators: Prof. Wuyuan Lu (Maryland) Prof. Jacek Lubkowski (NIH) Prof. Alan Waring, Piotr Ruchala (UCLA) Prof. Robert Lehrer (UCLA) Prof. Bill DeGrado group (UPenn) Prof. Klaus Schmidt-Rohr (ISU) Prof. Olga Zabotina (ISU) Funding NIH, NSF, DOE Iowa State University 31!