Settimo Et Al 2007 Biopolymers

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Conformational Changes Upon Calcium Binding and Phosphorylationin a Synthetic Fragment of Calmodulin

Luca Settimo,1 Serena Donnini,2 André H. Juffer,2 Robert W. Woody,3 Oriano Marin41 CRIBI Biotechnology Centre, University of Padova, via U.Bassi, 58/b, 35131 Padova, Italy

2 The Biocenter and the Department of Biochemistry, University of Oulu, P.O. Box 3000,

FIN-90014 University of Oulu, Finland

3 Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870

4 Department of Biological Chemistry, University of Padova, Viale G. Colombo 3, 35121 Padova, Italy

Received 5 June 2006; revised 8 October 2006; accepted 11 December 2006

Published online 15 December 2006 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.20657

This article was originally published online as an accepted preprint. The ‘‘Published Online’’ date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at [email protected]

INTRODUCTION

Calmodulin (CaM) is a ubiquitous eukaryotic Ca2þ-

binding protein that binds and activates different tar-

gets1 and has a very important physiological role. Sev-

eral structures of CaM have been reported using X-

ray crystallography and nuclear magnetic resonance

(NMR) (see e.g., reviews in Refs. 2 and 3).

ABSTRACT:

We have recently investigated by far-UV circular

dichroism (CD) the effects of Ca 2 þ binding and the

phosphorylation of Ser 81 for the synthetic peptide CaM

[54–106] encompassing the Ca 2 þ-binding loops II and III

and the central a helix of calmodulin (CaM) (Arrigoni

et al., Biochemistry 2004, 43, 12788–12798).Using

computational methods, we studied the changes in the

secondary structure implied by these spectra with the aim

to investigate the effect of Ca 2 þ binding and the functional

role of the phosphorylation of Ser 81 in the action of the

full-length CaM. Ca 2 þ binding induces the nucleation of

helical structure by inducing side chain stacking of

hydrophobic residues. We further investigated the effect of

Ca 2 þ binding by using near-UV CD spectroscopy.

Molecular dynamics simulations of different fragments

containing the central a-helix of CaM using various

experimentally determined structures of CaM with bound

Ca 2 þ disclose the structural effects provided by the

phosphorylation of Ser 81. This post-translational

modification is predicted to alter the secondary structure in

its surrounding and also to hinder the physiological

bending of the central helix of CaM through an alteration

of the hydrogen bond network established by the side chain

of residue 81. Using quantum mechanical methods to

predict the CD spectra for the frames obtained during the

MD simulations, we are able to reproduce the relative

experimental intensities in the far-UV CD spectra for our

peptides. Similar conformational changes that take place

in CaM [54–106] upon Ca 2 þ

binding and phosphorylation may occur in the full-length CaM.

# 2006 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 88:

373–385, 2007.

Keywords: calmodulin; calcium; phosphorylation;

conformational changes; circular dichroism

Correspondence to: Luca Settimo; e-mail: [email protected]

This article contains supplementary material available via the Internet at http://

www.interscience.wiley.com/jpages/0006-3525/suppmat.

Conformational Changes Upon Calcium Binding and Phosphorylationin a Synthetic Fragment of Calmodulin

VVC 2006 Wiley Periodicals, Inc.

PeptideScience Volume 88 / Number 3 373


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These studies revealed that CaM has a dumbbell-like

shape with N- and C-terminal lobes separated by a central

helix. Calmodulin-like domains have been identified also in

other proteins.4 Two EF-hand Ca2þ-binding motifs are pres-

ent in each lobe of CaM. When CaM binds Ca2þ, a confor-

mational change occurs in each lobe of CaM with exposureof hydrophobic residues that bind the target.3 All the struc-

tures reported for the full-length CaM can be classified in

one of the following three functional states: (i) Ca2þ-free

CaM, such as the structure determined using NMR 5; (ii)

Ca2þ-bound CaM, as the structure published by X-ray crys-

tallography by Chattopadhyaya et al.,6 which reported a

straight and continuous central a-helix of CaM, or by Fallon

and Quiocho,7 which, in contrast, reported the central a-

helix of CaM bent in the middle; (iii) Ca2þ-bound CaM in

complex with a ligand or target, as the structure determined

by X-ray crystallography by Meador et al.8 or by NMR by

Elshorst et al.9 The central helix of CaM has a fundamental

role since it assures the correct positioning of the N- and C-

terminal lobes in the binding and activation of the target,

and it is very flexible in solution in both apo5,10 and Ca2þ-

bound form.9,11 The conformational changes provided by

Ca2þ binding and the conformation and flexibility of the

central helix of CaM have been widely studied and investi-

gated using different methods such as: (i) NMR 5,9–16; (ii)

small-angle X-ray scattering17,18; (iii) spin-label electron para-

magnetic resonance19; (iv) optical spectroscopic techniques

such as far-UV CD,20 Fourier transform infrared spectros-

copy,21,22 fluorescence,23,24 optical rotatory dispersion mea-

surements25; (v) structural studies on protein mutants at res-

idues in the central helix 26–31; (vi) cross-linking.32

Several modeling studies and molecular dynamics (MD)

simulations have been performed on CaM and they have

been reviewed recently.3 In particular, MD simulations have

been performed on the full-length CaM (i) in the Ca2þ-

loaded form33–39; (ii) in the Ca2þ-free form40; (iii) in the

Ca2þ-loaded form in complex with a target peptide.41 Also

the central helix of CaM (fragment 65–92) has been analyzed

using MD.35 All these MD studies underscore the flexibility

of the middle part of the central helix of CaM (corre-

sponding approximately to the region encompassed by resi-

dues 74–82), important for the correct functioning of CaM.

CaM is phosphorylated by different kinases as reviewed in

Ref. 42. In particular, the casein kinase 2 (CK2) is a serine/

threonine kinase that phosphorylates Thr 79, Ser 81, Thr 110,

and Thr 117 in the full-length CaM.43–47 Thr 79 and Ser 81,

located in the middle of the central a helix of CaM, are the

most phosphorylated residues in the full-length protein.44,46,47

The interaction between CK2 and the central a helix of CaM

has been recently studied by molecular modeling.48

We have recently reported the synthesis, purification, and

characterization of the fragment encompassing residues 54–

106 of human CaM, wild-type and phosphorylated in posi-

tion 81.47 These peptides encompass the Ca2þ-binding loops

II and III and the central a-helix of the full-length CaM. We

will refer to these two peptides as CaM [54–106] and CaM[54–106]pS81, respectively. In the same study, we investi-

gated by far-UV CD the effect of Ca2þ binding and phospho-

rylation of residue 81 on CaM [54–106] (see Figure 6 in

Ref. 47). We also found that CaM [54–106]pS81 could not be

phosphorylated at Thr 7947 despite the fact a phosphorylated

side-chain at n þ 2 is a strong positive determinant for CK2

phosphorylation of short peptides.49

It is generally known that phosphorylation of CaM

decreases the binding and activation of CaM-dependent tar-

gets.42 However, there are no studies in the literature that

investigate the structural consequences of the phosphoryla-

tion of CaM. As reported recently by Doig and coworkers,

the structural effects consequent to protein phosphorylation

are very variable (e.g. phosphorylation can stabilize or desta-

bilize a helices) and highly dependent upon the position and

possible interactions that the phosphorylated residue can es-

tablish with the neighboring residues.50,51

In the present study, we (i) analyze spectroscopic data for

the fractions of secondary structure elements using several

methods; (ii) experimentally analyze the effect of Ca2þ bind-

ing using near-UV CD; (iii) investigate conformational

changes induced by Ca2þ binding and phosphorylation of

Ser 81 in our peptides[54–106] using molecular modeling

and MD simulation; (iv) rationalize the experimentally

observed change in far-UV CD spectra by calculating theoret-

ical CD spectra using structures from the MD trajectories.

Our models also explain why CK2 is not able to phosphoryl-

ate Thr 79 in CaM[54–106]pS81.

This study aims to investigate the structural–functional

role correlated to Ca2þ binding and phosphorylation in the

full-length CaM by using a fragment of CaM.

MATERIALS AND METHODS

Spectroscopic MeasurementsCircular dichroism (CD) spectra were recorded at 258C at pH 7.5

on a Jasco (Tokyo, Japan) Model J-710 spectrometer equipped with

a thermostated cell-holder and a Neslab RTE-110 water circulating

bath. The instrument was calibrated with (þ)-10-camphorsulfonic

acid.52 The far-UV CD spectra of the peptides [54–106]CaM and

[54–106]pS81CaM in the absence or presence of Ca2þ, which are

analyzed with computational methods, have been already

reported.47

374 Settimo et al.

Biopolymers (Peptide Science) DOI 10.1002/bip


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Near-UV CD spectra were taken using the same buffer and tem-

perature, in the presence and absence of 10 m M CaCl2. The results

were expressed as the mean residue ellipticity.53

Computational Analysis of the Far-UV CD SpectraThe secondary structure fractions of the peptides CaM[54–106] and

CaM[54–106]pS81 in the absence and presence of Ca2þ were deter-

mined from CD spectra47 using the CDPro software package that

includes SELCON3, CDSStr, and CONTIN/LL.54–58 The SP43

(IBasis 4) and SDP48 (IBasis 7) protein reference sets were used.

SP43 contains 43 proteins having known structures and CD spectra

ranging from 190 to 240 nm. SDP48 contains five unfolded proteins

in addition to those in SP4357 and was used because it gives better

results with protein fragments and partially unfolded proteins.

These large protein reference sets were chosen since our CD

spectra lacked short-wavelength data (180–190 nm). In addition,

K2D,59,60 implemented in DICHROWEB,61,62 and the method

described by Scholtz et al.63 were also used to predict secondary

structure fractions from the CD spectra.

Molecular Dynamics SimulationsThree different experimentally determined structures of Ca2þ-CaM

were downloaded from the Protein Data Bank (PDB)64: (i) the en-

semble of 26 structures determined by NMR in solution by Elshorst

et al.9 (PDB code: 1cff); the crystal structures determined (ii) by

Chattopadhyaya et al.6 (PDB code: 1cll) and (iii) by Fallon and

Quiocho7 (PDB code: 1prw).

Eight MD simulations were carried out for different fragments

of these structures as described in Table I (abbreviations in Table I

will be used throughout the text of this report).

The addition of the phosphate groups for the simulations of the

fragments having the phosphoserine in position 81 (pS81 in Table I)was done using SYBYL 7.0 (Tripos, St. Louis). Except for some pre-

liminary calculations using GROMOS 96,65,66 all molecular dynam-

ics (MD) simulations were performed with the Gromacs suite of

programs67,68 utilizing the OPLS force field.69 Force-field parame-

ters for the phosphoserine were adapted from the phosphotyrosine

parameters used by Price and Jorgensen.70

The protein structures were placed in the center of a dodecahe-

dral box, which was subsequently filled with TIP4P water molecules

(TIP4P ¼ transferable intermolecular potentials with four point

charges).71 The N-terminus and C-terminus were charged. The size

of the box was chosen so as to contain the protein and at least

1.2 nm of solvent on all sides. The number of water molecules was

roughly 15,000 for the fragments of CaM encompassing residues

54–106 and 10,000 for the fragments of CaM encompassing residues

64–93. Phosphoserine was considered in its completely dissociatedform (charge: 2) in agreement with the pK a reported for this

residue.72

The peptides 54–106 were simulated with two Ca2þ atoms taken

from the crystal structure (bound to the Ca2þ-binding loops II and

III), whereas the peptides encompassing residues 64–93 were simu-

lated without Ca2þ since these fragments correspond to the central

a-helix of CaM and do not contain Ca2þ-binding sites. Sodium

counterions were added in each simulation to maintain the overall

charge of the system equal to zero.

Coulomb interactions were treated with fast particle mesh Ewald

(PME),73,74 using a grid spacing of 0.12 nm and cubic interpolation.

The cut-off distances for the Coulomb and Lennard-Jones interac-

tions were 0.8 and 1.4 nm, respectively. Interactions between atoms

that were within 0.8 nm were evaluated every step, while interac-tions between atoms within the longer cutoff distance were eval-

uated every 3 steps. Constant pressure and temperature were main-

tained by a weak coupling of the system to an external bath at 1 bar

and 300 K using the Berendsen barostat and thermostat75 with cou-

pling times of 1 and 0.1 ps, respectively. The bond distances and

bond angles of water were constrained using the SETTLE algo-

rithm.76 All other bond distances were constrained with the LINCS

algorithm.77 Dummy nonpolar hydrogen atoms78 were employed.

The integration time step was 4 fs. Prior to the simulations, the

potential energy of each system was minimized using steepest

descent, followed by a 20-ps MD simulation with position restraints

on the protein to relax the water molecules. 100-ns simulations of

the peptides 1cll[54–106]and 1cll[54–106]pS81 and 20-ns simula-

tions for the remaining fragments ([64–93] of 1cll, 1prw and model

4 of 1cff) were performed (Table I).

The conformations generated in the MD simulations were ana-

lyzed using the analysis tools implemented in the Gromacs suite of

programs, such as the secondary structure predictions using DSSP.79

Theoretical Calculations of CD SpectraThe computation of CD spectra from the protein structure was per-

formed using the matrix method80 as described in Sreerama and

Table I MD Simulations Carried Out in This Study

Abbreviation CaM[fragment] Initial Coordinates (pdb code) Length of the MD

1cll[54–106] 54–106 1cll (with 2 Ca2þ) 100 ns

1cll[54–106]pS81 54–106 1cll (with 2 Ca2þ) 100 ns

1cll[64–93] 64–93 1cll 20 ns1cll[64–93]pS81 64–93 1cll 20 ns

1prw[64–93] 64–93 1prw 20 ns

1prw[64–93]pS81 64–93 1prw 20 ns

1cff_4[64–93] 64–93 1cff_model#4 20 ns

1cff_4[64–93]pS81 64–93 1cff_model#4 20 ns

The abbreviations used in the first column are used through all the text of this manuscript. Refer to the

Methods section for further details.

Conformational Changes in CaM 375



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Woody.54 In particular, the average CD spectrum over the whole tra-

jectory was calculated with the following steps: (i) generation of the

snapshots corresponding to the structures of the peptides being

simulated picked up every 100 ps for each trajectory (Table I), (ii)

calculation of the CD curve using theoretical quantum-mechanical

methods including aromatic residues54 for each of these frames, (iii)

average of the theoretical CD spectra (for all the frames of the whole

trajectory).

FiguresFigures were prepared using Molscript81 and Raster3D.82

RESULTS ANDDISCUSSIONS

Analysis of the Far-UV CD Spectra

Table II reports the percentage of secondary structure ele-

ments calculated from the far-UV CD spectra of CaM[54–

106] and CaM[54–106]pS81 in the absence and presence of

Ca2þ (see Figure 6 in Ref. 47) using five different methods

described in the Methods section. We used more than one

method to predict the secondary structure fractions from

these experimental curves to make the results more robust.

CDPro analysis using the 43-protein basis set gives significant

b-sheet contents, ranging from ca. 20–50% (Table II). These

may result from the limited wavelength range. A basis set

that includes unfolded proteins (48-protein basis) has been

shown57 to give more accurate analysis of secondary struc-

ture when studying partially unfolded proteins and peptide

fragments. With this basis set, the b-sheet content is de-

creased by about 10% for each of the fragments (Table II).

As can be seen in Table II, the predictions from the CD

curves of the peptide CaM[54–106] and CaM[54–106]pS81

show an increase of the a-helical content in the presence

of Ca2þ and a decrease in the a-helical content upon phos-

phorylation of Ser 81.

In the following sections, we investigate the structural

effects correlated to Ca2þ binding and phosphorylation of

Ser 81, using mainly molecular modeling and MD simula-tions rationalizing the results with the experimental data.

Effect of Ca2þ Binding

As can be seen in Table II, all the methods predict an increase

of the a-helical content of the peptide in the presence of

Ca2þ. For CaM[54–106], the a-helical content increases by

12–24% in the presence of Ca2þ. Surprisingly, the addition

of Mg2þ instead of Ca2þ does not change the CD spectrum

from the profile of apo-CaM[54–106] (Figure 6A in Ref. 47).

Interestingly, the CD spectra for the full-length CaM in

the absence and presence of Ca2þ and Mg2þ are similar to

the curves for our peptide CaM[54–106]. In fact, for the full-

length CaM, there is also an intensification of the dichroic

signal at 222 nm content upon Ca2þ binding,20,24,83,84 and

there is no change in the ellipticity upon Mg2þ binding.85

Ellipticities for CaM can vary with the ionic strength,24 lead-

ing to different values of ellipticities and a-helix fractions

reported in different studies (see e.g. Ref. 84). Brokx and

Vogel86 suggested that in CaM, as has been claimed with

Table II Fractions (Percentages) of Secondary Structure Elements From the Experimental CD Spectra for the Peptides CaM[54–106]

and CaM[54–106]pS81 in the Absence (apo) or Presence of Ca2þ (the Spectra are Reported in Figure 6 in Ref. 47)

CaM[54–106]apo CaM[54–106]-Ca2þ CaM[54–106]pS81apo CaM[54–106]pS81-Ca2þ

Total

a

a

Total

b

a

Remaining

b

Total

a

a

Total

b

a

Remaining

b

Total

a

a

Total

b

a

Remaining

b

Total

a

a

Total

b

a

Remaining

b

CONTIN/LL 12.1c 30.2c 57.7c 27.1c 17.9c 55.1c 10.0c 32.6c 57.5c 21.8c 24.6c 53.5c

8.7d 18.1d 73.3d 25.6d 9.0d 65.3d 6.3d 17.2d 76.6d 18.7d 12.9d 68.5d

SELCON3 9.4c 29.4c 59.5c 26.3c 18.9c 55.0c 10.1c 28.6c 55.6c 17.3c 26.7c 53.1c

7.4d 18.4b 61.6b 26.2b 9.1b 63.9b 8.6b 18.1b 67.0b 17.1 18.5 62.3b

CDSStr 6.0c 33.0c 60.0c 30.0c 19.0c 51.0c 5.0c 32.0c 62.0c 19.0c 25.0c 56.0c

5.0d 21.0d 73.0d 28.0d 10.0d 61.0d 4.0d 21.0d 74.0d 18.0d 15.0d 66.0d

K2D 8 41 50 25 15 60 7 49 44 10 34 56

Scholtz 9 — — 25 — — 7.1 — — 18 — —

Different methods were used as described in the methods section.a The total fractions of a-helix and b-structure in CONTIN/LL, SELCON3, and CDSStr implemented in CDPRO are reported, respectively, as the sum of

regular (ar) plus distorted (ad) a-helix and as the sum of regular (br) plus distorted (bd) b-structure.b For CONTIN/LL, SELCON3, and CDSStr the ‘‘remaining’’ corresponds to the sum of turns (T) and unordered (U) whereas for K2D it corresponds to

the prediction of ‘‘random structure’’.c Analysis using SP43 protein reference set.d Analysis using SP48 protein reference set.

376 Settimo et al.



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Ca2þ binding to troponin-C,87 a change in the alignment of

the a-helices with respect to one another could also be re-

sponsible for this increase in the CD signal, a mechanism

originally suggested by Manning.88 Even though NMR shows

that Ca2þ-CaM has a ‘‘high degree of mobility near the mid-

dle of the central helix’’,11 apparently similar to what is seen

in the apo form,5 some studies clearly show that Ca2þ bind-

ing rigidifies and increases the a-helical content of the central

helix of CaM: (i) a peptide corresponding to the third Ca2þ-

binding loop of CaM binds a multivalent cation (lanthanide)

and provides a-helix nucleation,89 CaM[54–106] contains

the second and third Ca2þ-binding loop of CaM at the N-ter-

minus and C-terminus, respectively so a similar effect might

be invoked for both termini of our peptide, which could help

stabilize the central a-helix; (ii) a spin-label electron para-

magnetic resonance study shows rigidification of the central

part76–81 of CaM upon Ca2þ binding19; (iii) Fourier trans-

form infrared spectroscopy studies show an increased portion

of a-helix in the presence of Ca2þ 21,22; (iv) a study in which

optical rotatory dispersion and proteolysis with trypsin were

used shows an increase of the a-helix content upon Ca2þ

binding25; (v) fluorescence studies23,24 show that the central

helix of CaM undergoes conformational changes upon Ca2þ

binding, and in particular Yao and coworkers report that the

central helix ‘‘becomes more extended and rigid.’’23

To further investigate experimentally the effect of Ca2þ

binding, we measured the near-UV CD in the absence and

presence of Ca2þ for CaM[54–106] (Figure 1).

The near-UV CD spectra in the presence and absence of

Ca2þ for CaM[54–106] are very similar to the published

near-UV CD spectra for the full-length CaM84,85,90: the

stronger intensities in the near-UV CD spectrum in the pres-

ence of Ca2þ show that the aromatic side chains in our pep-

tide (Phe 65, 68, 89, 92, and Tyr 99) are in a more rigid struc-

ture. In addition, as mentioned earlier, it is interesting to see

that CaM[54–106] can distinguish between Ca

2þ

and Mg

2þ

.Therefore, the radius of the metal is very important in this

conformational change, as already anticipated by Chao

et al.91 Ca2þ has a larger radius than Mg2þ (ionic radii are 0.99

and 0.65 Å for Ca2þ and Mg2þ, respectively 92). Therefore for

Ca2þ, the charge is distributed in a larger volume and over a

larger surface in comparison to Mg2þ. As a consequence, a

larger number of residues (at least five in each Ca2þ-binding

loop of CaM) can interact with Ca2þ, in comparison with the

smaller Mg2þ. Thus, the conformational change is expected to

take place when more residues are involved in the contact with

the metal ion.

The N-terminal half of the CaM[54–106] fragment, from

residue 54 to 76 of the structure determined by Chattopad-

hyaya et al.6 is displayed in Figure 2. The formation of a

hydrophobic cluster when Ca2þ is bound is evident. This

cluster, formed by Ile 63 and Val 55 of the Ca2þ-binding loop

II with Phe 68 and Met 72 in the N-terminal part of the cen-

tral helix, nucleates the formation and stabilization of the

central a-helix because it leads to the stacking of hydropho-

bic residues Phe 68, Met 72, and Met 76 parallel to the axis

(Figure 2). The same effect can be described also in the C-ter-

minal part of our peptides. The importance of helix-nuclea-

tion upon Ca2þ binding was also reported by Siedlecka et al.

for a peptide encompassing the third Ca2þ-binding loop of

CaM.89

In addition, in our simulations for the peptide [54–106]

in the presence of Ca2þ, these ions are bound to the Ca2þ-

binding loops II and III in our peptide (1cll[54–106] and

1cll[54–106]pS81, Table I) even after 100 ns, showing that

the Ca2þ is stably bound.

Effect of Phosphorylation of Ser 81

The phosphorylation of Ser 81 has an important effect on

the a-helical content of Ca2þ-CaM[54–106] (Table II): phos-

phorylation of Ser 81 decreases the helical content by 5–15%

in comparison to the wild-type peptide, according to the

analyses of the far-UV CD spectra (Table II).

Quadroni et al. have measured and compared the CD

spectra of Ca2þ-CaM and Ca2þ-phosphorylated CaM.93 The

phosphorylation described in Ref. 93 was obtained by treat-

ment of CaM with CK2, a kinase that extensively phos-

phorylates Ser 81 in the central helix of CaM.44,46 Interest-

ingly, the intensity of the CD signal at 222 nm is stronger for

FIGURE 1 Near-UVCD spectra of peptide CaM[54–106] in 10 mM

MOPS, pH 7.5, 0.1M NaCl, 258C in the presence (dashed line) andabsence (continuous line) of 10 mM CaCl2.




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the wild-type Ca2þ-CaM in comparison to Ca2þ-phospho-

rylated CaM (Figure 5 in Ref. 93), as we observed for

CaM[54–106] and CaM[54–106]pS81 (Figure 6B in our pre-

vious study 47).

The lower intensity at 222 nm in the CD spectra seen for

Ca2þ-CaM[54–106]pS81 in comparison to Ca2þ-CaM[54–

106] is not correlated with weaker binding of Ca2þ to the

phosphopeptide in comparison to the wild-type peptide, but

results from the phosphorylation of Ser 81 because we have

confirmed, using radiolabeling,94 that both CaM[54–106]

and CaM[54–106]pS81 bind Ca2þ with similar affinity

(Figure 1 in the supplementary material section), in agree-

ment with published calcium binding studies on the wild-

type and phosphorylated CaM.

93,95

To investigate the structural effects correlated to the phos-

phorylation of Ser 81, we performed MD simulations on

CaM fragments encompassing the central a-helix of CaM,

having serine or phosphoserine in position 81 (pSer 81). To

explore as much as possible all the possible states in which

the central helix can exist, we performed MD simulations

starting with different fragments taken from several experi-

mentally determined structures of CaM (Table I; see Methods

section). We considered both crystal structures and also a

structure determined by NMR, since the middle part of the

central a helix of CaM is very flexible in solution. We calcu-

lated the CD spectra for snapshots corresponding to the

structures of the peptides being simulated for each trajectory

using the matrix method80 and compared the calculated

spectra with the experimental curves reported previously

(Figure 6 in Ref. 47). The matrix method for predicting CD

spectra54 is a quantum mechanical method in which the per-

turbation matrix is diagonalized and the eigenfunctions are

used to calculate the electric and magnetic dipole transition

moments, and the eigenvalues give the transition energies.

Simulations Done With Fragments of a Crystal Structure of CaM (PDB Code 1cll Starting Structures). Initially, we per-

formed 60-ns simulations for the fragment [54–106], wild-type and with phosphoserine (including Ca2þ) starting from

the structure solved by Chattopadhyaya et al.6 (PDB code:

FIGURE 2 Structure showing the nucleation of the a-helix pro-vided by the binding of Ca2þ in the N-terminal half of the fragment

54–106 taken from the structure of CaM (PDB code: 1cll). Back-

bone is represented as tube colored in green; carbon atoms colored

in gray; Ca2þ in the Ca2þ-binding loop II colored in orange. The

coordination of the Ca2þ atom is displayed by black dashed lines.

FIGURE 3 Secondary structure analysis (by DSSP) for the simulation of 1cll[64–93] (a) and

1cll[64–93]pS81 (b) as a function of the simulation time.

378 Settimo et al.



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1cll) using the GROMOS 96 force field (43a1).65,66 We

observed the formation of p-helix during both simulations

(data not shown). It has been reported that some force-fields

(e.g. GROMOS 96) generate p-helix during the MD simula-

tions, but this has been shown to be an artifact not supported

by experimental data.96,97 Upon changing the force-field to

OPLS and rerunning the simulations (1cll[54–106] ,1cll[54–

106]pS81,1cll[64–93] and 1cll[64–93]pS81 in Table I), we

did not observe the formation of p-helix (Figure 3).

CD spectra averaged over the whole trajectory (see Meth-

ods section for details) of 1cll[64–93] and 1cll[64–93]pS81

are very similar to the CD spectra averaged over the whole

trajectory for 1cll[54–106] and 1cll[54–106]pS81, respec-

tively. In addition, we observed that the removal of Ca2þ-

binding loops II and III do not affect the structure of the cen-

tral a-helix during the MD simulation. Therefore, the CD

signal is mainly generated by the central a-helix of CaM and

not by the Ca2þ-binding loops, so we decided to simulate

only the central a-helix 64–93 of CaM to study the local

effect of the phosphorylation of Ser 81.

The structure reported by Chattopadhyaya et al.6 has a

straight a-helix, which is unlikely to be observed in solution.11

However, there is some experimental evidence for a straight

central a-helix in at least a fraction of the molecules in solu-

tion.19 Interestingly, the central helix of 1cll[64–93] bends in

its middle between 6 and 15 ns in the simulation (Figures 3a

and 4), in agreement with the NMR structure reported for the

full-length CaM.11 Surprisingly, this bending is not seen for

1cll[64–93]pS81 (Figures 3b and 4), where a stable interaction

between the phosphate group of pSer 81 and the amino group

of Lys 77 rigidifies the central a-helix of CaM, hindering the

bending. However, the calculated CD spectra over the whole

trajectory for the simulations of 1cll[64–93] and 1cll[64–

93]pS81 have similar ellipticity at 222 nm (Figure 2 in the sup-

plementary material section) and therefore differ from the ex-

perimental data (Figure 6 in Ref. 47).

Simulations Done With Fragments of a Crystal Structure of CaM (PDB Code 1prw Starting Structures). If a phosphate

group is added to the hydroxyl group of Ser 81 in the crystal

structure of CaM determined by Fallon and Quiocho,7 there

is a destabilizing interaction between the negative charges of

the phosphate group and the negative partial charge ass-

ociated with the helix-dipole moment in the C-terminal of

the helix 64–76 (Figure 5). This destabilizes the helical portion

64–76 in 1prw[64–93]pS81, in agreement with the fact that

the phosphorylated peptide of CaM possesses less helical

content than the wild-type peptide.

The a-helix is flexible in the middle during the simulation

(see the large changes in interhelical angle in Figure 6) in

agreement with the experimental evidence mentioned before

for the central a-helix of the full-length CaM. In 1prw[64–

FIGURE 4 Flexibility of the central a-helix of CaM. Change in the interhelical angle (angle

between the helical segment 64–74 and 83–93) as a function of the simulation time for 1cll[64–

93](continuous line) and 1cll[64–93]pS81 (dashed line).




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93]pS81, the central helix is interrupted by a region lacking

secondary structure (white strip in Figure 7). The calculated

CD curves (average over the whole trajectory) (Figure 8) are

in agreement with the experimental curves that show higher

ellipticity for wild-type than for the phosphorylated peptide.

In 1prw[64–93], the hydroxyl group of Ser 81 donates ahydrogen bond to the main-chain oxygen of Asp78 throughout

the trajectory as captured in the snapshot of the structure

seen at 10 ns (Figure 9a). In 1prw[64–93]pS81, the hydroxyl

oxygen cannot donate a hydrogen bond to the main-chain

oxygen (Figure 9b). Instead, the phosphate group of phos-

phoserine 81 can interact with the sidechain of Lys 75, with

consequent rigidification of the structure (seen in Figure 6

as mentioned above) and stabilization of a loop (Figure 9b)

that is seen throughout the simulation (white strip in Fig-

ure 7).

Simulations Done With Fragments of a Model From an En-

semble of CaM Structures Solved by NMR (PDB Code 1cff Starting Structures). Elshorst et al.9 determined by NMR

the structure of a complex of Ca2þ-CaM and reported an en-

semble of 26 models (PDB code 1cff).

We calculated the theoretical CD spectra for the fragment

54–106 of each of these 26 models. The fragment [54–106]

of model 4 from 1cff produces a CD spectrum very similar to

that of Ca2þ-CaM[54–106] reported by us47 and has favor-

FIGURE 5 The structure of 1prw[64–93]pS81 at the beginning of

the simulation. The addition of a phosphate group to the fragment

1prw[64–93] (coordinates from the structure having PDB code: 1prw),

gives an unfavorable interaction (as indicated by the asterix ‘‘*’’) with

the C-terminal negative charge provided by the dipole moment of helix

[64–76] (helix colored in blue). The helical dipole moments (arrows)

with their partial charges are indicated for the N-terminal helix (in

blue) and C-terminal helix (in red) of fragment [64–93].

FIGURE 6 Flexibility of the central a-helix of CaM. Change in the interhelical angle (angle

between the helical segment 64–74 and 83–93) as a function of the simulation time for 1prw[64–

93] (continuous line) and 1prw[64–93]pS81 (dashed line).

380 Settimo et al.



9/13

able F and C angles in the Ramachandran plot (Figure 3 in

the supplementary material section).

The secondary structural elements of 1cff_4[64–93] do

not change significantly during the simulation, except for the

formation of a bend that is not persistent (Figure 10a). By

contrast 1cff_4[64–93]pS81 forms a stable bend in the mid-

dle of the central a-helix (green strip in Figure 10b). This

bend is stabilized by an interaction between the side chain of

phosphoserine 81 and Arg 86 throughout the simulation.

In this case, the calculated CD curves over the whole tra-

jectory (Figure 11) are in good agreement not only with the

intensities but also with the shapes of the experimental

curves (Figure 6 in Ref. 47).

In agreement with the results of our simulations, one pos-

sible reason for the shape of the experimental curves (more

intense peak at 205 nm in comparison to the peak at 222

nm) is that the main-chain oxygen atoms in the middle of

the central a-helix of CaM are accessible to the solvent and

FIGURE 7 Secondary structure analysis (by DSSP) for the simulation of 1prw[64–93] (a) and

1prw[64–93]pS81 (b) as a function of the simulation time.

FIGURE 8 Average of the theoretical curves for the whole trajectory of the peptide 1prw[64–93]

(continuous line) and 1prw[64–93]pS81 (dashed line).




10/13

establish hydrogen bonds to water molecules in addition to

main-chain i? i þ 4 hydrogen-bonds.98,99

Comparison of the Results From the MD Simulations. The

validity of our MD simulations was assessed by different

methods,100 e.g., by running simulations on different starting

conformations and for different simulation times, as can be

seen in Table I.

Both the simulations done on the fragments taken from

1prw and 1cff predicted correctly the stronger intensity at

222 nm of the dichroic signal for the wild-type peptide rela-

tive to the phosphopeptide. However, the averaged CD spec-

tra calculated from the simulations of the fragments of 1cff

showed the best agreement. Even the shape of the curves,

with a more intense peak at 205 nm in comparison to the

peak at 222 nm, was predicted correctly. In addition, the per-

centages of the secondary structure elements seen during this

simulation (Figure 10) agree quite well with the datareported in Table II. The improved results with this starting

structure may be due to the fact that the structure was deter-

mined in solution.

Conversely, the simulations of the peptide from the struc-

ture 1cll (having the continuous central a-helix) give results

that agree poorly with experiment. We have extended the sim-

ulation of 1cll[54–106] to 100 ns and we observed results very

similar to those for the shorter fragment 1cll[64–93] (e.g. even

at 100 ns 1cll[54–106]pS81 had a straight central a-helix as in

the starting structure). However, this fact could be consistent

with the finding reported by Qin and Squier that there is a

population of Ca2þ-CaM having a straight central a-helix.19

FIGURE 9 Frame-snapshots observed at 10 ns of simulation for

1prw[64–93] (a) and 1prw[64–93]pS81 (b). Key hydrogen bonds

established by the side-chain of residue 81 are shown.

FIGURE 10 Secondary structure analysis (by DSSP) for the simulation of 1cff_4[64–93] (a) and

1cff_4[64–93]pS81 (b) as a function of the simulation time.

382 Settimo et al.



11/13

In all the MD simulations of the phosphopeptide, we

observed the formation of an interaction between the side

chain of one lysine or arginine residue and the phosphate

group of phosphoserine 81. In general, this interaction rigidi-

fies the central a-helix of CaM, decreasing the flexibility of

the central a-helix and most likely affecting the recognition

and activation to the CaM-dependent targets as seen experi-

mentally.42 An additional experimental result supports this

model. We have reported47 that CaM[54–106]pS81 cannot

be phosphorylated at Thr 79, despite the fact that a phosphor-

ylated side-chain at n þ 2 is a strong positive determinant

for CK2 phosphorylation of short peptides.49 In our simula-

tion, the negatively charged phosphate group of phosphoser-

ine 81 interacts with a positively charged residue (Lys or

Arg), which would preclude the interaction of phosphoserine

81 with CK2. The phosphate group of pSer 81 accepts a

hydrogen bond from the Lys/Arg residue rather than inter-

acting with the basic residues near the active site of CK2

where the central helix of CaM is predicted to bind.48 In

addition, the basic residues of CK2 that are thought to recog-

nize the acidic residues in the consensus sequence of phos-

phorylation48 would interact unfavorably with the Lys/Arg

that forms a salt bridge with pSer 81. As a consequence, the

binding of CaM[54–106]pS81 to CK2 would be impaired,

with a consequent decrease of the phosphorylation of Thr

79. We ran some MD simulations with fragments containing

pThr 79, and found also in this case a similar phenomenon

in which the phosphate group interacts with Lys or Arg resi-

dues. This agrees with the absence of peptides bis-phos-

phorylated in positions 79 and 81.47 The rigidifying stabiliza-

tion of helical structure given by a salt bridge between a

phosphoserine and lysine residues has been reported in the

literature51; interestingly, also in our simulations done with

1cll fragments having pSer 81 or pThr 79, we observe similar

interactions in which the phosphorylated residue interacts

with the side chain of the lysine located at i-4 position, with

the formation of a salt bridge between Lys 75 and pThr 79 or

between Lys 77 and pSer 81.

CONCLUSIONS

The synthetic fragment encompassing the central helix of

CaM and the Ca2þ-binding loops II and III gives far-UV and

near-UV CD spectra similar to those of CaM when (i) Ca2þ

is bound and (ii) it is phosphorylated in position 81, a resi-

due highly phosphorylated by CK2. We have explained with

modeling and MD simulations the structural reasons forthese effects. In particular, when Ca2þ binds to loops II and

III, near-UV CD indicates that there is rigidification of the

structure, similar to that seen for the full-length CaM, and

we suggest that important interactions between hydrophobic

residues nucleate the formation of a-helix. Introduction of a

phosphate at Ser 81 affects the stabilization of a-helix by

influencing the hydrogen-bond network established by the

FIGURE 11 Average of the theoretical curves for the whole trajectory of the peptide 1cff_4[64–

93] (continuous line) and 1cff_4[64–93]pS81 (dashed line).




12/13

side-chain of residue 81. Quantum-mechanical calculations

of the CD spectra on snapshots during the MD simulations

give results in agreement with the experimental intensities

that we reported for the peptide CaM[54–106] and CaM[54–

106]pS81. It is very important to run different MD simula-

tions on different starting structures for CaM, since the cen-tral part of the central a-helix of CaM has a peculiar flexibil-

ity in solution.

The conformational changes that take place in this pep-

tide upon Ca2þ binding and phosphorylation might also

occur in the full-length CaM. The advantage of using a rela-

tively small fragment of CaM to perform these structural

investigations is that it is easier to study with molecular

modelling and MD simulations in comparison to the full-

length protein.

We thank Prof. Lorenzo A. Pinna for useful discussions about the

study and to Dr. Dan Price for providing the parameters for the

construction of the topology for phosphoserine for the simulationwith OPLS. Also thanks to Dr. Alice Glättli for additional discus-

sions on the formation of p-helix using GROMOS 96 force field.

Access to the software was provided by the Centre for Scientific

Computing (CSC, Espoo, Finland).

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