Probing Ca -dependent Conformational Changes in Calmodulin by H/D Exchange: Effect of Ionic Strength and Peptide Binding

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1 Probing Ca -dependent Conformational Changes in Calmodulin by H/D Exchange: Effect of Ionic Strength and Peptide Binding Mei Zhu, Madeline A. Shea, Michael L. Gross 1 Washington University Center for Biomedical and Bioorganic Mass Spectrometry:An NIH supported Resource Center; Department of Chemistry;Washington University; St. Louis, MO ( 2 Department of Biochemistry; College of Medicine; University of Iowa; Iowa City; IA52242.

2 1. Purpose: Overview * A novel H/D exchange protocol is applied to examine the degree of conformational changes in calmodulin induced by Ca binding and peptide binding. 2. Methods: * An immobilization procedure for protein in H/D exchange is coupled online with ESI mass spectrometry 3. Novel Aspects: * Influences of metal-ion and peptide binding on protein conformation are determined by ESI MS. * Effects of ionic strength and changes in calmodulin as isolated from various organisms are explored.

3 Introduction: What is Calmodulin? Fig. 1: Calmodulin. * A small, acidic protein of 148 AA, ~ 17 kda * Contains one central helix, two globular domains four Ca -binding sites * Binds other proteins/peptides, serves as Ca -dependent regulator in many metabolic pathways

4 1. Materials: Methods * "Calcium-free" pocine calmodulin ("pocam": MW Da) was obtained from Ocean Biologics Co., Edmonds, WA. * Wild-type paramecium calmodulin ("PCaM": MW Da), rat calmodulin ("rcam": MW 16706) and their half domains were expressed by Professor Madeline A.Shea'a group at Univ. of Iowa. * Melittin (from bee venom) and mastoparan (from Polistes Jadwagae) were purchased from Sigma. 2. Instrumentation: * HPLC was carried out using a Waters 600MS pump with an isocratic flow of 14.9% H2O, 5.1% acetic acid and 80% CH3CN. * The flow, split by a LC-packing splitter, was introduced into a Microtech Zorbax C-18 SBW column (10 mm x 0.32 mm) with a flow rate of 33 µl/min. * ESI MS was done with a Finnigan LCQ operating in the full-scan mode. 3. Sample Preparation: * The PCaM, rcam and their half protein stock solutions (200 µm) were prepared by diluting with appropriate amounts of 50 mm HEPES/0.1 M KCl buffer (ph 7.4). * The pocam stock solution (e.g., 200 µm) was prepared by dissolving 1 mg protein in 298 µl 50 mm HEPES/0.1 M KCl buffer (ph 7.4).

5 3. Sample Preparation (continue): * The H/D amide exchange experiment was carried out in 99% D2O and different ionic strengths. The pocam stock solutions (e.g., 3mM) were more concentrated and were prepared in 50 mm HEPES aqueous buffer alone (ph 7.4), in HEPES buffer with 100 mm XCl (X = Li, Na, K, Cs or + NH 4 ) or KNO 3(pH 7.4), or in NH4OAc buffer (ph 7.0, adjusted with NH OH) with different concentrations (e.g., 20 mm or 400 mm). 4 * For the Ca titration in 99% D2O, 2 µl aliquots of protein were taken and incubated with 2 µl of CaA 2 ( A = Cl, NO3 or CH3COO) solution at various calcium concentrations at room temperature for 30 min. For the Ca titration in 90% D O, 7.5 ul of protein and 2.5 ul of CaA were incubated 2 2 instead. * For titration of the CaM-peptide complex with Ca, 200 µm of CaM and 200 µm of peptide were incubated in HEPES/KCl aqueous buffer at room temperature for at least 1 h. 7.5 µl aliquots of CaM-peptide stock solution were taken and incubated with 2.5 µl CaCl 2 at various concentrations in HEPES/KCl buffer for 30 min or 1 h.

6 4. H/D Exchange Protocol: (Fig. 2) * Following incubation, D2O (with the same buffer and salt as in aqueous buffer) was added to initiate exchange at ph 7.4 for HEPES buffer system and 7.0 for NH4OAc system, D2O/H2O = 99/1 or 90/1. After 1 h, the exchange was quenched with 1 M HCl (bringing the ph to 2.5). The exchange solution was then injected onto the C-18 SBW column, which was cooled to 0 ºC with ice. The extent of H/D exchange was measured after 1 h for all experiments because the exchange was nearly constant at that time. * The protein, concentrated and immobilized on the column, was washed with chilled formic acid solution (ph 2.5) to allow fast-exchanging active H's on side chains to be converted back to an H form, and to remove salts (H2O/D2O > 100/1). * An isocratic flow of solvent with high organic composition (ph 2.5) was used to elute rapidly the protein for ESI-MS determination of its molecular weight.

7 Fig.2: H/D Exchange Protocol CaM CaCl2 Peptide KCl HEPES H2O xµl KCl HEPES D2O 90~99xµL CaM CaCl2 Peptide KCl HEPES D2O 90~99xµL H2O 1xµL H/D 1 Hr Take out yµl Quench Waste Mass analyzer Waste Mass analyzer Loading 1~200 µl Injector Solvent pump Injector Solvent pump Waste Mass analyzer Washing Back exchange 0 ºC ph 2.5 Waste Mass analyzer Eluting ph 2.5 Injector Solvent pump MS Analysis Injector Solvent pump

8 Results and Discussion 1. Porcine Calmodulin (pocam)-calcium Interactions A. Ca-Titation: ( Fig.3) * With the addition of Ca, pocam undergoes less H/D exchange, indicating the formation of Ca -bounded calmodulin species that have tighter, less solvent-accessible structures than apo-calmodulin. * The resulting titration curve (Fig. 3) shows two breaks: [Ca ]total = 0.04 mm and 0.25 mm, respectively, which may correspond to CaM complexed Ref.1 with two Ca and four Ca, according to a fractional species calculation (Fig. 3 lower panel). Major conformational changes occurred after CaM became bound to two Ca. After four Ca were incorporated, no further changes in H/D exchange were observed. {Ref. 1: Nemirovskiy, O; Giblin, D; Gross, M. JASMS, (1999), 10, }

9 Fig. 3: H/D Exchange Data for CaM Titration With Ca ( 99%D2O; [CaM] = 15 µm; [NH OAc] = 20 mm; ph = pd = 7.0; T = 25 ºC ) Number of Hydrogens Exchanged in 1 Hr Calmodulin Binding Fractions vs Total Calcium 1.0 Fractional Abundance Tota l Concentration of Ca ( mm ) Fraction 0 Ca Fraction 1 Ca Fraction 2 Ca Fraction 3 Ca Fraction 4 Ca

10 B. Effect of Ionic Strength (Fig. 4 A,B,C; Table 1) * Owing to on-column desalting and concentrating the protein, we now can use normal buffers and high concentrations of salts to mimic a biological system. * The largest extent of H/D exchange was observed when using low ionic strength conditions such as 2 mm NH4OAc buffer, ph 7.0 (Fig. 4A). At higher ionic strength (e.g., 100 mm KCl, 50 mm HEPES buffer, ph 7.4), pocam shows less conformational changes than it does in the absence of + K. Replacing KCl with KNO had no effect (Fig. 4B) * Changing K to other alkali ions or to NH 4 shows similar effects as compared to the Ca-titrations in HEPES buffer with no salt added. H/D exchange in solutions containing other metal salts also points to lower conformational changes upon binding to Ca (Fig. 4C and Table 1). * A possible explanation for the effect of different media is that alkali ions or ammonium ion stabilize the protein and prevent association of Ca with CaM, leading to lower conformational changes and less protection from H/D exchange.

11 Fig.4A: H/D Exchange Data for Porcine CaM Titration With Ca at Different Ionic Strength( [CaM] = 15 µm; 99% D2O; [NH4OAc] = 2 or 20 mm; ph = pd = 7.0; T = ºC ) Number of Hydrogens Exchanged in 1 Hr du(in 20 mm NH4OAc) du(in 2 mm NH4OAc) Total Concentration of Ca (mm)

12 Fig. 4B: H/D Exchange Data For Porcine CaM Titration With Ca at Different Ionic Strength ([CaM] = 15 µm; 99% D2O; + [HEPES] = 50 mm; ph = pd = 7.4; [K ] = 0 or 100 mm ) Number of Hydrogens Exchanged in 1 Hr du (no K+) du (0.1M KCl) du (0.1M KNO3) Total Concentration of Ca (mm)

13 Fig. 4C: H/D Exchange Data for Porcine CaM Titration With Ca in Different Salt Media. ([CaM]=15 µm; 99% D2O; [HEPES] = 50 mm; ph = pd = 7.4; [salt] = 0 or 100 mm ) Number of Hydrogens Exchanged in 1 Hr du (no M+) du (0.1M LiCl) du (0.1M NaCl) du (0.1M KCl) du (0.1M CsCl) du (0.1M NH4Cl) Total Concentration of Ca (mm)

14 Table 1: Comparison of the extent of H/D exchange in different media. M + Added Extent of H/D Exchange Na + 5 K + 6 Li + 6 NH 4 + Cs + 10 None 16 None 17 None 21 9 H/D Exchange Conditions #1: In H 2 O: [MCl] = 100 mm; [HEPES] = 50 mm; [Na + ] 20 mm; [CaCl 2 ] = mm; [CaM] = 1.5 mm; ph = 7.4 In D 2 O: [MCl] = 100 mm; [HEPES] = 50 mm; [Na + ] 20 mm; [CaCl 2 ] = 0-1 mm; [CaM] = 15 M; 99% D 2 O; pd = 7.4 #2: Similar to #1, but without MCl #3: Similar to #1, but without MCl; in D 2 O [HEPES] = 5 mm; [Na + ] 2 mm #4: In H 2 O: [NH 4 OAc] = 200 mm; [CaM] = 1.5 mm [Ca(OAc) 2 ]=0-100 mm; ph = 7.0 In D 2 O: [NH 4 OAc] = 20 mm; [CaM] = 15 M; [Ca(OAc) 2 ]= 0-1 mm; 99% D 2 O; pd = 7.0 None 35 #5: Similar to #4, but in H 2 O: [NH 4 OAc] = 20 mm; in D 2 O: [NH 4 OAc] = 2 mm.

15 2. Porcine Calmodulin-Peptide Binding * Because Ca /peptide/cam complexes dissociate under quench conditions, we were able to measure readily the influence of peptide binding on CaM and determine the stoichiometry of these complexes. A 1:1 ratio of CaM:peptide complex was observed in the CaM titration with melittin or mastoparan (titration curves not shown). * H/D exchange of these complexes indicates that the "ternary" complex has significantly lower solvent accessibility. The titration of the pocam:melittin or pocam:mastoparan complex with Ca shows there is higher affinity for Ca and larger conformational changes as compared to when no peptide was bound to calmodulin (Fig. 5). The titration Melittin ( ML ) = GIGAVLKVLTTGLPALISWIKRKRQQ-NH2 ( 26 AA) Mastoparan P ( MP(p) ) = VDWKKIGQHILSVL-NH2 (14 AA) curves for pocam:peptide complex suggests that calmodulin serves as a better "switch" in biological communication when it is able to bind to other target proteins. * Although the X-ray crystal structure and NMR structure of pocam:melittin or pocam: mastoparan are not reported in the literature, they are likely to be similar to that of the complex with peptide M13 (a CaM binding domain in myosin light chain kinase).the Ref.2 3D structure of the CaM:ML complex was modelled on the basis of the known Ref.3 structure of CaM:M13 complex. Both N and C-terminal domains interact with the peptide in holo-cam (Fig. 6). The Ca -titration curves (Fig. 5) showed that the structure collapse occurred after some fraction of the binding sites became occupied with Ca. After four Ca ions were bound, no further changes in comformation were observed.

16 Fig.5: H/D Exchange Data For pocam, pocam-ml and pocam-mp(p) Complexes Titrating With Ca.( [pocam] = [ML] = [MP(p)] = 15 µm; ph=pd=7.4; 90% D O; [HEPES]=50mM; [KCl] =100mM; T=22-29 ºC) 2 Number of Hydrogens Exchanged in 1 Hr du(pocam) du(pocam-ml) du(pocam-mp(p)) Total Concentration of Ca (mm)

17 Fig. 6: Comparison of the Structure of Calmodulin (c,d) with the Rev2 Model Proposed Structure for thecam-melittin Complex (a,b). (a) and (b): Two different views of the 3-D structure of the Rev2 model proposed for CaM-ML complex, showing the reverse orientation of the peptide (referred to N- Ref.2,3 terminal of CaM). {Ref.2: Scaloni,etc. J. Mol. Biol. (1998), 277, Ref.3: Ikura, etc. Science (1992), 256, }. (c) X-ray crystal structure of Ref.4 CaM. { Ref. 4: Babu,etc. J. Mol. Biol. (1988), 204, } Ref.5 (d) NMR structure of CaM. {Ref. 5: Ikura,etc. Biochemistry, (1991), 30, }

18 3. Comparison of Calmodulins from Different Organisms A. Sequence Difference: (Table 2) * We are interested in determining whether CaM from a simple organism (e.g., paramecium) has different properties than CaM from mammalian cells. * Table 2 shows the sequences of porcine, rat and paramecium calmodulins. Porcine and rat calmodulins share the same sequence, but pocam is acetylated at the N-terminus and trimethylated at Lys115. * Paramecium is a protist, a single cell organism. The sequence difference in paramecium calmodulin (PCaM) is highlighted in purple, and the Ca-binding sites are marked with boxes. The major differences occur in the C-terminal domain, and four amino acid changes are found in Cabinding sites.

19 Table 2: The Sequences of Calmodulins. Top, green: Rat Calmodulin (rcam) or Porcine Calmodulin (pocam, modification in A1, K115) Bottom, purple: Paramecium Calmodulin (PCaM) A D Q L T E E Q I A E F K E A F S L F D K D G D G T I T T K E L G T V M R S L A E Q L T E E Q I A E F K E A F A L F D K D G D G T I T T K E L G T V M R S L G Q N P T E A E L Q D M I N E V D A D G N G T I D F P E F L T M M A R K G Q N P T E A E L Q D M I N E V D A D G N G T I D F P E F L S L M A R K M K D T D S E E E I R E A F R V F D K D G N G Y I S A A E L R H V M T N L M K E Q D S E E E L I E A F K V F D R D G N G L I S A A E L R H V M T N L G E K L T D E E V D E M I R E A D I D G D G Q V N Y E E F V Q M M T A K G E K L T D D E V D E M I R E A D I D G D G H I N Y E E F V R M M V S K

20 B. Ca-Titration: (Fig. 7, 8) * As we expected, rcam showed a similar Ca-titration curve as that of pocam (Fig. 7), probably due to their sequence similarities. However, PCaM showed greater conformational changes and larger protection from H/D exchange than rcam or pocam. Differences Ref.6,7 in Stokes radii are consistent with the titration data; i.e., for both PCaM and rcam, the Stokes radii decrease upon binding Ca, but PCaM undergoes a greater change. * To understand better the difference between PCaM and rcam, we titrated half proteins representing the N and C domains with Ca, and followed the H/D exchange. The N- and C-terminal domains of rcam and PCaM were engineered and expressed in E. Coli by Dr. Shea's group in Univ. of Iowa. * The N-terminal domain (PCaM 1-75 and rcam 1-75) showed similar behavior in the Ca-titration, whereas a major difference occurred for the C-terminal domains (PCaM and rcam76-148) (Fig. 8). This is consistent with the fact that the major sequence differences are in the C-terminal domains of these two calmodulins. For both "half CaMs" containing the C-terminus, the difference in H/D exchange in going from apo to holo is greater than that for "half CaMs" containing the N-terminus. However, this difference is larger in "half PCaMs" (~ 10 amide H's) than it is in "half rcams"(~ 4 amide H's). {Ref. 6: Jaren, O.R.; Harmon, S.; Chen, A.F.; Shea, M.A. Biochem. (2000), 39, Ref. 7: Sorensen, B.R.; Shea, M.A. Biophys. J. (1996), 71, }

21 Fig. 7: H/D Exchange Data For Three Kinds of Calmodulins Titration With Ca ( 90% D2O; [CaM] =15 µm; [HEPES] = 50 mm; [KCl] = 100 mm; ph = pd = 7.4; T = ºC ) 130 Number of Hydrogens Exchanged in 1 Hr du(pocam) du(rcam) du(pcam) Total Concentration of Ca (mm)

22 Fig.8: H/D Exchange Data For Halves of rcam and PCaM Titration With Ca (90% D2O; [rcam1-75] = [rcam76-148]= [PCaM1-75] = [PCaM76-148] = 15 µm; [HEPES] = 50 mm; [KCl] = 0.1 M; ph = pd = 7.4; T = ºC) Number of Hydrogens Exchanged in 1 Hr du(rcam1-75) du(rcam76-148) du(pcam1-75) du(pcam76-148) Total Concentration of Ca (mm)

23 C. CaM-Melittin Binding Properties (Fig. 9) * In the absence of Ca, the three CaMs (pocam, rcam and PCaM) showed nearly identical extent of H/D exchange with and without melittin. Although lack of protection seems inconsistent with the fact that apo-cam is a weak Ref.8 binder for melittin (K d = 10 µm), it may occur because the equilibrium involved in CaM-melittin binding is slower than H/D exchange. * Ca -binding in the presence of melittin affords an increase in protection for the three CaMs. The increase is nearly the same for pocam and rcam, in accordance with their high homology. PCaM, however, shows a greater increase in protection. * The difference in protection of holo rcam and pocam offered by mellitin is ~19 amide H's, whereas for PCaM, the difference is ~14 amide H's. * Melittin increases the affinity of CaM for Ca as indicated by the shift in the titration curves to lower total concentration of Ca. The structure collapse occurred after some fraction of the binding sites were occupied with Ca, and no further changes were observed after CaM was saturated with Ca (Fig. 9). { Ref. 8: Maulet, Y.; Cox, J.A. Biochem. (1983), 22, }

24 Fig.9: H/D Exchange Data For rcam, pocam, PCaM and their Melittin Complexes Titrating With Ca.([rCaM]=[poCaM]=[PCaM]=[ML]= 15 µm; ph=pd=7.4; 90% D O; [HEPES] = 50 mm; [KCl] = 100 mm; T = ºC) 2 Number of Hydrogens Exchanged in 1 Hr du (rcam) du (rcam:ml) du (pocam) du (pocam-ml) du (PCaM) du (PCaM-ML) Total Concentration of Ca (mm)

25 C. CaM-Melittin Binding Properties (continued): (Fig. 10) * To understand further the Ca-dependent CaM-melittin binding, we titrated the "half PCaM":melittin complexes with Ca and followed the H/D exchange. Both N and C-domains of PCaM interacted with melittin in a Ca-dependent manner, and the ternary complexes showed greater protection against H/D exchange. Interestingly, when saturated with Ca in the presence of melittin, PCaM1-75 showed similar protection against H/D exchange as did PCaM (Fig.10). This titration data are consistent with a proposed structure for holo CaM:ML complex (Fig. 6), in which melittin binds to the central region and interacts with both domains of CaM. * A titration in which the two half PCaMs competed for melittin as a function of Ca concentration (Fig. 10: orange and green curves) was also carried out. After incubating 1:1:1 PCaM1-75:PCaM76-148:Melittin for 1 h, various con- centrations of Ca were added. In the presence of another half of PCaM, the N- domain (PCaM1-75) showed a similar Ca- titration curve (orange curve) as it did in the absence of C-domain (pink curve). However, the C-domain (PCaM ) showed less protection against H/D exchange (green curve) than it did in the absence of N-domain (light blue curve). A possible explanation is that melittin binds first to the N-domain of PCaM and causes a conformational change that increases the affinity of the C-domain.

26 Fig.10: H/D Exchange Data For Two Halves of PCaM and Their Melittin Complexes Titrating With Ca in the Presence or Without the Other Half of PCaM (90% D2O; [PCaM1-75] = [PCaM76-148] = [ML] = 15 µm; [HEPES] = 50 mm; [KCl] = 0.1 M; ph = pd = 7.4; T = ºC). Number of Hydrogens Exchanged in 1 Hr du (PCaM1-75) du (PCaM1-75:ML*) du (PCaM1-75:ML) du (PCaM76-148) du (PCaM76-148:ML*) du (PCaM76-148:ML) Total Concentration of Ca (mm)

27 Conclusions 1. Calmodulin-Calcium Interactions * Ca-bound CaMs show a lower extent of H/D exchange than apo-cams, which indicates that upon Ca binding, the proteins fold to the structures that are less solvent accessible. * As the ionic strength increases, pocam shows less conformational changes when it binds to Ca. The alkali metal ions, K, Na and Li, are more effective + + in diminishing the conformational change than Cs and NH. * rcam and pocam show similar Ca-titration curves. PCaM shows a greater difference in H/D exchange between apo and holo forms, indicating a greater protection for holo PCaM, than do mammalian CaMs. * The N-domains (PCaM1-75 & rcam1-75) show similar behavior in the Catitration. The C-domains of CaMs (PCaM & rcam76-148) show greater protection in H/D exchange and higher affinity for Ca than do the N-domains. The major difference occurred for the C-domains. This is consistent with the fact that the major sequence differences are in the C-domains of these two CaMs. 4

28 2. Ca-dependent Calmodulin-Peptide Interactions * The binding of Ca to CaM increases the binding affinity between CaM and peptides such as melittin or mastoparan. The structure of the complex "collapses" after some fraction of the Ca-binding sites become occupied with Ca. No further changes in conformation are observed after four Ca ions are bound. * The CaM:Melittin and CaM:Mastoparan complexes have higher affinity for calcium, lower solvent accessibility, and exhibit greater protection against H/D exchange than CaM themselves. The Ca-titration curves suggest that CaM serves as a better "switch" in biological communication when it is bound to other target proteins. * rcam:ml and pocam:ml complexes show similar changes in conformation when binding to Ca, whereas PCaM:ML shows greater comformational changes. The additional protection due to melittin binding with holo PCaM is less, however, than that is for holo rcam and pocam.

29 2. Ca-dependent Calmodulin-Peptide Interactions (continued) * Both N and C-domains of PCaM (PCaM1-75 and PCaM76-148) interact with melittin upon binding Ca. The half-pcam:ml complexes show more protection against H/D exchange than half -PCaMs themselves. A titration in which the two half PCaMs (N and C-domains) compete for melittin as a function of Ca concentration suggests that melittin binds first to the N- domain of PCaM and causes a conformational change that increases the affinity of the C-domain. Acknowledgement * This work is supported by the National Center for Research Resources of the NIH (grant No. P41RR00954). * We thank Dr. Brenda R. Sorensen, Wendy S. Van Scyoc and Dr. Olav R. Jaren in Univ. of Iowa for their kind help in data explanation, and for providing paramecium and rat calmodulins.

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