Georgia State University

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1 Chemical Exchange and Calcium Signaling Jenny J. Yang Chemistry Department Center for Diagnostics & Therapeutics Georgia State University

2 Ligand Protein interactions by NMR Molecular Recognition: Calcium signaling CaM regulation of gap junctions Calcium kinetics Chemical exchange NMR time scale Fast exchange for binding constants Slow exchange for tight binding Single vs. multiple binding mode Applications for monitoring calcium signaling CaM regulation of gap junctions

3 Ca2+ Homeostasis and Signaling ([Ca2+]e 10 3 M) Ca2+ channels GPCR E NCX STIM1 RTK IP3R RyR SERCA SERCA ([Ca2+]i M) MCU/MiCa Letm1 Ca2+ IP3R Uniporter RyR CaM NCX/HCX Genes transcription (NFAT, CREB, DREAM) [Ca2+]I activated Buffers Buffers Effectors Ca2+ Ca2+iOscillation frequency 10-6 μs 10-3 ms 1 s 103 min 106 h

4 Binding Modes between CaM and Its Targets Collapsed NMDA receptor NR1 C1 Ca 2+ pump Extended CaMKII 1-7 Cav IQ motif of myosin V CNG channel enos CaMKI skmlck mglur7a 1-14 Ca 2+ Anthrax exotoxin CaMKK 1-16 SK channel RyR MARCKS Glutamate decarboxylase Dr. Hing-cheung Wong

5 Questions How do we know the protein is conformational ensemble? Calcium binding process? Affinity? Multiple binding sites? Calcium induced conformational changes? Other metal ions such as Pb 2+ have the same effect on affinity and conformational change? protein/peptide interaction?

6 Typical Motion Time Scale for Physical Processes

7 NMR Dynamic Experiments Initial NMR dynamics experiments in 1970s. Rapid advancements due to ability to label specific positions in bio-molecules and methodologies development Magnetization exchange spectroscopy (EXSY)-slow exchange 0.5 s -1 to over 50 s -1 CPMG relaxation dispersion: chemical shifts100~2000s -1, R 1 rho can extend to more rapid exchange (dot) Residual dipolar coupling (RDC) and PRE Spin relaxation for ns-ps CPMG and PRE are sensitive to low-lying excited states with populations > 0.5% H/D exchange can detect high energy excited states with much lower population A.Mittermaier, L. Kay (2009) Trends Biochem. Sci. 34, 601. A. Mittermaier, L. Kay (2006) Science. 312, 224 K. Wuthrich, G Wagner,(1978) Trends Biochem. Sci. 3, 227

8 Effects of Chemical Exchange on NMR Spectra Chemical exchange refers to any process in which a nucleus exchanges between two or more environments in which its NMR parameters (e.g. chemical shift, scalar coupling, or relaxation) differ. DNMR deals with the effects in a broad sense of chemical exchange processes on NMR spectra; and conversely with the information about the changes in the environment of magnetic nuclei that can be derived from observation of NMR spectra. K ex Conformational equilibrium K B Chemical equilibrium

9 Types of Chemical Exchange Intramolecular exchange Motions of sidechains in proteins Helix-coil transitions of nucleic acids Unfolding of proteins Conformational equilibria Intermolecular exchange Binding of small molecules to macromolecules Protonation/deprotonation equilibria Isotope exchange processes Enzyme catalyzed reactions A M+L B ML Because NMR detects the molecular motion itself, rather the numbers of molecules in different states, NMR is able to detect chemical exchange even when the system is in equilibrium

10 NMR Time Scale Time Scale Chem. Shift, d Coupling Const., J T2 relaxation Slow k << d A - d B k << J A - J B k << 1/ T 2, A - 1/ T 2, B Intermediate k = d A - d B k = J A - J B k = 1/ T 2, A - 1/ T 2, B Fast k >> d A - d B k >> J A - J B k >> 1/ T 2, A - 1/ T 2, B Sec NMR time-scale refers to the chemical shift timescale. The range of the rate can be studied s -1 for H can be extended to faster rate using 19 F, 13 C and etc.

11 2-state First Order Exchange A k1 B k -1 Lifetime of state A: t A = 1/k +1 Lifetime of state B: t B = 1/k -1 Use a single lifetime 1/ t =1/ t A + 1/t B = k +1 + k -1

12 Rationale for Chemical Exchange For slow exchange For fast exchange FT FT Bloch equation approach: dm AX /dt = -(Dω A )M AY - M AX /t A + M BX /t B dm BX /dt = -(Dω B )M BY - M BX /t B + M AX /t A

13 2-state 2nd Order Exchange M+L k +1 ML k -1 K d = [M] [L]/[ML] = k -1 /k +1 K d = M k on = k +1 ~ 10 8 M -1 s -1 (diffusion-limited) k -1 ~ s -1 Lifetime 1/ t =1/ t ML + 1/t l = k -1 (1+f ML /f L ) f ML and f L are the mole fractions of bound and free ligand, respectively

14 Slow Exchange k << δ A -δ B A k1 B k -1 Separate lines are observed for each state. The exchange rate can be readily measured from the line widths of the resonances Like the apparent spin-spin relaxation rates, 1/T 2i,obs 1/T 2A,obs = 1/T 2A + 1/t A = 1/T 2A + 1/k 1 1/T 2B,obs = 1/T 2B + 1/t B = 1/T 2B + 1/k -1 line width Lw = 1/pT 2 = 1/pT 2 + k 1 /p Each resonance is broadened by D Lw = k/p Increasing temperature increases k, line width increases

15 Slow Exchange for M+L k 1 ML k- 1 Separate resonances potentially are observable for both the free and bound states M F, M B, L F, and L B The addition of a ligand to a solution of a protein can be used to determine the stoichiometry of the complex. Once a stoichiometric mole ratio is achieved, peaks from free ligand appear with increasing intensity as the excess of free ligand increases. Obtain spectra over a range of [L]/[M] ratios from 1 to 10

16 Slow Exchange for M+L k 1 ML For free form 1/T 2L,obs = 1/T 2L + 1/t L = 1/T 2L + k -1 f ML /f L 1/T 2M,obs = 1/T 2M + 1/t M = 1/T 2M + k -1 f ML /f M For complex form 1/T 2ML,obs = 1/T 2ML + 1/t ML = 1/T 2ML + k -1 k- 1 Measurements of line width during a titration can be used to derive k -1 (k off ).

17 19 F spectra of the enzyme-inhibitor complex at various mole ratio of carbonic anhydrase:inhibitor Free inhibitor 1:4 1:3 1:2 1:1 1:0.5 Bound ligand At -6 ppm the broadened peak for the bound ligand is in slow exchange with the peak from free ligand at 0 ppm. The stoichiometry of the complex is 2:1. No signal from the free ligand is visible until more than 2 moles of inhibitor are present. Roberts, GCK NMR of Macromolecules

18 Coalescence Rate For A B equal concentrations, there will be a rate of interchange where the separate lines for two species are no longer discernible The coalescence rate k c = p Dd / 2 = 2.22 Dd Dd is the chemical shift difference between the two signals in the unit of Hz. Dd depends on the magnetic field

19 Coalescence Temperature Since the rate depends on the DG of the inversion, and the DG is affected by T, higher temperature will make things go faster. Tc is the temperature at which fast and slow exchange meet. T>Tc, fast exchange T<Tc, slow exchange T we can calculate the DG of the process DG = R * T C * [ ln ( T C / Dd ) ] T C

20 Fast Exchange k >> δ A -δ B A k1 k -1 B A single resonance is observed, whose chemical shift is the weight average of the chemical shifts of the two individual states δ obs = f A δ A +f B δ B, f A + f B = 1 For very fast limit 1/T 2,obs = f A /T 2A + f B /T 2B For moderately fast 1/T 2,obs = f A /T 2A + f B /T 2B + f A f B2 4p (Dd AB ) 2 / k -1 Maximal line broadening is observed when f A = f B = 0.5

21 Line Shape Simulation

22 Fast Exchange k >> δ A -δ B M+L k+1 k-1 ML For M δ M,obs = f M δ M +f ML δ ML For L δ L.obs = f L δ L +f ML δ ML 1/T 2,obs = f ML /T 2,ML + f L /T 2,L + f ML f L2 4p (d ML -d L ) 2 / k -1 A maximum in the line broadening of ligand or protein resonances occurs during the titration at a mole ratio of approx. ligand:protein 1:3 The dissociation constant for the complex can be obtained by measuring the chemical shift of the ligand resonance at a series of [L].

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25 NMR Titration Both T6(CH3) at 1.1 ppm from DNA (A) and L(CNH 2 ) methyl from ligand (B) are in fast exchange. Expanded regions from 300 MHz 1 HNMR spectra from complexes between L(NH 2 ) and d(ggtaatacc) 2 recorded at 10 ºC (Craik)

26 Binding of Inhibitor to Enzyme H I * * H I * H k off k unf * H S * H * k off K d = = k on [protein][ligand] [protein- ligand] H S NMR studies are done at mm, making it difficult to determine K d with any accuracy if the binding is very tight (nm K d ).

27 Measuring Binding Constant M+L k+1 k-1 ML δ M,obs = f M δ M +f ML δ ML, The total change in δ of M Dd Mo = δ ML - δ M At [L], Dd M = δ M,obs - δ M If [M] is fixed, Dd M = Dd Mo [L]/([L]+K d ) At 0.5 Dd Mo, K d = [L] Similar for H=L, K d = K a Dd H = Dd Ho [H]/([H]+K a ) A - +H + k+1 k-1 AH Inhibitor G-3-P binds to Triosephapte Isomerase -JMB, :319

28 NMR studies of Ca(II) Binding to cntnc [Ca]t/cTnC cntnc is the N-terminal domain of TnC from cardiac muscle with a single calcium binding site II. Peak at 10.3 ppm is G70 at the calcium binding site. Monica et al., 1997

29 fractional change of chemical shifts Monitoring Calcium-binding by NMR CaM-CD2-III-5G Ca mm mm mm 0 mm ppm K d = 144 ± 27 um [Ca(II)], (mm)

30 The Ionization of His in Oxy & DeOxyhemglobin C2H 7.7 ppm 8.7 ppm C4H 7.0 ppm 7.4 ppm Bohr effect: Hb takes up H + on releasing O2 The pka of His-b-146 changes by more than 1pH unit upon ionization due to the stabilization of the charged form by Asp94 in the deoxy structure.

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32 NH Calcium Induced Chemical Shift Changes NH 0.4 D15 F21 M23 N60 Chemical shift change (ppm) L58 G61 N D17 Q22 I Residues D D62 L63 N Chemical shift change (ppm) I18 Chemical shift change (ppm) Q w.t. CD2-D Residues Residues

33 Manganese Relaxation of CD2-6D15 D26 D71 T86 T5 H A D62 log (Di -1 ) D N90 I18 A54 Cyan: without Mn 2+ Purple: with Mn log (r) Yang et al. (2005) JACS, 127, Mildvan, AS, and Cohn M, 1970, Adv. Enzymol. Relat. Areas Mol. Biol., 1970, 33, 1-70

34 Developing Calcium Sensors by Design 1. Highly targeting specificity 2. Simple stoichiometric interaction mode to ease calibration 3. Tunable affinities, selectivity & kinetics 4. Minimal perturbation on signaling without using natural calcium binding proteins JACS, 2002, 2005, 2007 Biochem, 2005, 2006, PEDS, 2007, protein science 2008 J. Zhou, A. Hofer, J.J. Yang, Biochem, 2007 A.Holder, J.J. Yang, Biotech 2009 S. Tang,.O. Delbano J.J. Yang, PNAS, 2011

35 Catcher: Ca 2+ Sensor for Detecting High Concentration fluorescence Normalized fluorescence Relative amount of Ca-bound CatchER F 488 F 395 A Wavelength (nm) [CatchER] M Equilibrium dialysis coupled with ICP-OES x - x - 1 H, 15 N-HSQC x - Ca 2+ x - x - S. Tang,.O. Delbano J.J. Yang, PNAS, 2011

36 Fast Kinetics of CatchER normalized fluorescence (%) [Ca 2+ ] µm time (s) normalized fluorescence (%) [EGTA] µm time (s) k on = k off /K d = 3.9 x 10 6 M -1 s -1 Fast Kinetics: k off = 700 s -1 S. Tang,.O. Delbano J.J. Yang, PNAS, 2011

37 CatchER s Ca 2+ Induced Chemical Shift Changes Y182 G228 S. Tang,.O. Delbano J.J. Yang, PNAS, 2011

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41 Integration of Calcium Signaling Via CaSR LB1 LB2 Site 2 Site 1 Site 3 Site 4 site5 Identification of Ca 2+ binding sites in ECD of CaSR How can CaSR sense the change of Ca 2+ o within a narrow range? (multiple sites? cooperativity?) Identification of CaM binding region in c-tail of CaSR Y. Huang, JJ Yang, J Biol Chem. 2007; Yun Huang.. JJ Yang Biochemistry 2009; Y Hang, JJ Yang, JBC 2010

42 Subdomain Approach LB1 LB2 site4 site3 site1 site2 site3 site2 site5 Yun Huang.. JJ Yang Biochemistry 2009

43 Two Distinct Ca 2+ -Binding Processes Revealed by NMR Relative change of chemical shift K d : 1.6 ± 0.1 mm n Hill : 2.3 ± [Ca 2+ ] (mm) ANS + Protein + Ca site3 site1 site2 Fluorescence Intensity ANS + Protein ANS Relative change K d1 = 0.7 ± 0.1 mm K d2 = 6.4 ± 0.8 mm n hill = 2.9 ± 1.2 Yun Huang.. JJ Yang Biochemistry 2009; wavelength (nm) [Ca 2+ ] mm

44 2-15 N (ppm) 2D-NMR Revealed CaSR ECD Binds to Ca H (ppm) 44 By You Zhuo

45 The Principle of Saturation Transfer Difference (STD) 45

46 [L-Phe] Monitoring the Ligand-Protein Interaction via STD NMR 15 STD-AF 10 5 Kd = 30 ± 5 mm L-Phe (mm) Phe only Phe+Ca 2+ STD-AF Kd = 15 ± 5 mm L-Phe (mm) 46 Chen Zhang, You Zhuo.. Jenny Yang, JBC 2014

47 Monitoring the Ligand-Protein Interaction via STD NMR 47

48 Residues in the linker of Calmodulin underwent sequential phases of conformational change

49 2:1, Ca:CaM 1:1, Ca:CaM 0:1, Ca:CaM a G113 G113 Chemical Exchange in Rat CaM G96 Fast Exchange b G59 I II G23 Slow Exchange Indicates: Initial occupancy of C- terminal domain Suggests: Temporal occupancy of N- terminal domain at low [Ca] and/or domain coupling Kirberger M, Yang JJ, JIBC, Fast Intermediate Slow II I Rat CaM I V

50 Similarities in Chemical Exchange with Ca 2+ Titration No exchange Slow exchange Fast exchange Paramecium CaM Slow then fast exchange Jaren, O.R., et al., Calcium-induced conformational switching of Paramecium calmodulin provides evidence for domain coupling. Biochemistry, (48): p Sun, H., et al., Mutation of Tyr138 disrupts the structural coupling between the opposing domains in vertebrate calmodulin. Biochemistry, (32): p I intermediate exchange II Slow exchange II I Rat CaM Fast exchange I V Kirberger M, Yang JJ, JIBC, 2013.

51 Identifying CaM Binding Region in Gap Junction Connexins Score Cx50_m Cx46_h Cx44_s Cx43_h CaMKI_h CaMKII_h NH 2 COOH TKKFRLEGTLLRTYVCHI RGRVRMAGALLRTYVFNI RGKVRIAGALLRTYVFNI HGKVKMRGGLLRTYIISI FAKSKWKQAFNATAVVRH NARRKLKGAILTTMLATR 310 xxbxb#xxx#xxxx#xxx Y. Zhou. JJ Yang JBC. 2007; Zhou Y, Yang JJ. Biophys J ; Y. Chen,.. JJ Yang Biochem J. 2011

52 CaM Binding Peptide Fragments of Cx43 Competition Assay Red : Ca-CaM + Cx43-3p Blue : Ca-CaM-Cx43-3p + Cx43-2p

53 [Cx43]/[CaM] 0:1 0.4:1 0.8:1 1.2:1 Monitoring Cx Peptide and Calmodulin [Cx44]/[CaM] 0: T29 T A : : : Dppm-H K94 D64 G33 G61 G25 K148 A57 1.2: [Cx43]/[CaM] 2.0:1 Y. Zhou. JJ Yang JBC T29 T H (ppm) A H (ppm)