Matti Myllykoski

Krzysztof Kuczera

Dr Petri Kursula
email: petri.kursula@oulu.fi

Complex formation between calmodulin and a peptide from the intracellular loop of the gap junction protein connexin43: Molecular conformation and energetics of binding

Keywords: calmodulin, gap junction, connexin43, solution structure, calorimetry, regulation

Gap junctions are formed by a family of transmembrane proteins, connexins.

Connexin43 is a widely studied member of the family, being ubiquitously expressed in a variety of tissues and a target of a large number of disease mutations. The intracellular loop of connexin43 has been shown to include a calmodulin binding domain, but detailed 3-dimensional data on the structure of the complex are not available. In this study, we used a synthetic peptide from this domain to reveal the conformation of the calmodulin-peptide complex by small angle X-ray scattering.

Upon peptide binding, calmodulin lost its dumbbell shape, adopting a more globular conformation. We also studied the energetics of the interaction using calorimetry and computational methods. All our data indicate that calmodulin binds to the peptide from cx43 in the classical 'collapsed' conformation.

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Introduction

Gap junctions are tight cell contacts, formed by transmembrane proteins of the connexin family, which allow for the diffusion of small molecules between adjacent cells. These intercellular channels are crucial for the transfer of, for example, metabolites, ions, and second messengers. Each gap junction is composed of two hemichannels, and each hemichannel in turn is formed of six connexin protein subunits. Connexins are a family of structurally related integral membrane proteins that share a similar architecture, having 4 transmembrane domains, two extracellular loops, and 3 cytoplasmic domains. The most ubiquitous connexin is connexin43 (cx43), which is expressed in a large number of different tissues and cell types [START_REF] Laird | Life cycle of connexins in health and disease[END_REF].

A number of disease-linked mutations in cx43 have been reported. While the mutations are spread throughout the protein, a large fraction of characterized mutations in oculodentodigital dysplasia (ODDD) is found in the intracellular loop [START_REF] Laird | Closing the gap on autosomal dominant connexin-26 and connexin-43 mutants linked to human disease[END_REF][START_REF] Paznekas | GJA1 mutations, variants, and connexin 43 dysfunction as it relates to the oculodentodigital dysplasia phenotype[END_REF]. Also other connexins are disease-linked; for example, cx32 mutations cause Xlinked Charcot-Marie-Tooth disease that severely affects the myelin sheath in the nervous system [START_REF] Bergoffen | Connexin mutations in Xlinked Charcot-Marie-Tooth disease[END_REF][START_REF] Abrams | Mutations in connexin 32: the molecular and biophysical bases for the X-linked form of Charcot-Marie-Tooth disease[END_REF].

Since calmodulin (CaM) binding to gap junctions was discovered [START_REF] Peracchia | Functional modulation of cell coupling: evidence for a calmodulin-driven channel gate[END_REF][START_REF] Van Eldik | Interaction of calmodulin and other calcium-modulated proteins with mammalian and arthropod junctional membrane proteins[END_REF][START_REF] Peracchia | Is calmodulin involved in the regulation of gap junction permeability?[END_REF][START_REF] Welsh | Calmodulin binds to chick lens gap junction protein in a calcium-independent manner[END_REF], a vast amount of studies have indicated that CaM is, indeed, involved in the calciumdependent regulation of gap junction function. CaM has even been shown to be able to pass through a gap junction, possibly due to its elongated shape [START_REF] Brooks | Calmodulin transmitted through gap junctions stimulates endocytic incorporation of yolk precursors in insect oocytes[END_REF][START_REF] Curran | Passage of 17 kDa calmodulin through gap junctions of three vertebrate species[END_REF]. Cx43 has been shown to interact with CaM, and a specific binding site was recently [START_REF] Zhou | Identification of the calmodulin binding domain of connexin 43[END_REF] localised to the cytoplasmic loop, between residues 136-158. Similar sites have been characterized or suggested to be present in other connexins [START_REF] Dodd | Calmodulin association with connexin32-derived peptides suggests trans-domain interaction in chemical gating of gap junction channels[END_REF][START_REF] Peracchia | Calmodulin directly gates gap junction channels[END_REF][START_REF] Peracchia | Slow gating of gap junction channels and calmodulin[END_REF][START_REF] Sotkis | Calmodulin colocalizes with connexins and plays a direct role in gap junction channel gating[END_REF][START_REF] Zhou | Calmodulin Mediates the Ca2+-Dependent Regulation of Cx44 Gap Junctions[END_REF] target. Here, we set out to study the molecular conformation of the CaM-cx43 complex, for which no detailed 3D structural information has been available. Smallangle X-ray scattering (SAXS) and isothermal titration calorimetry (ITC) both strongly suggest that CaM adopts the collapsed conformation when it is bound to the CaM-binding domain of cx43.

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Materials and methods

Protein expression and purification

The synthetic 15-residue peptide representing the predicted CaM-binding site of human cx43, KVKMRGGLLRTYIIS (residues 144-158), was purchased from SBS Genetech, China. Calmodulin was expressed in E. coli from the pETCM vector [START_REF] Hayashi | An expression system of rat calmodulin using T7 phage promoter in Escherichia coli[END_REF] and purified using calcium-dependent affinity chromatography on phenyl sepharose, as previously described [START_REF] Kursula | A structural insight into lead neurotoxicity and calmodulin activation by heavy metals[END_REF].

Small-angle X-ray scattering

Synchrotron SAXS data for CaM and the CaM-cx43-CBD complex were collected on beamline I711 at MAX-Lab, Lund, Sweden [START_REF] Cerenius | The crystallography beamline I711 at MAX II[END_REF]. Data processing was done using the in-house Bli711 software, and programs from the ATSAS package [START_REF] Konarev | ATSAS 2.1, a program package for small-angle scattering data analysis[END_REF] were used for data analysis and molecular modeling.

Isothermal titration calorimetry

The interaction between CaM and the cx43 peptide was studied by ITC, using the VP-ITC equipment (Microcal Inc.). The experiment was carried out similarly under several different temperatures; the concentrations of CaM (in the cell) and the peptide (in the syringe) were 10.8 µM and 110 µM, respectively. Prior to the experiment, the protein and peptide were extensively dialyzed against the assay buffer (10 mM HEPES pH 7.5, 100 mM NaCl, 10 mM CaCl 2 ). Data analysis was carried out in Microcal Origin.

Molecular modeling

An initial model of the CaM-cx43-CBD complex was built using the sequence homology to DRP-1, and the corresponding crystal structure of CaM complexed to a mutant DRP-1 peptide (PDB entry 1ZUZ). The programs Coot [START_REF] Emsley | Coot: model-building tools for molecular graphics[END_REF] and Swiss-PdbViewer [START_REF] Guex | SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling[END_REF] were used for model construction and initial energy minization,

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respectively. Thereafter, the complex was subjected to molecular dynamics (MD) simulations (see below).

Computational methods

The simulation followed the protocol earlier described [START_REF] Zoete | Protein-ligand binding free energy estimation using molecular mechanics and continuum electrostatics. Application to HIV-1 protease inhibitors[END_REF]. Harmonic constraints were applied to the protein, using force constants of 10 kcal/(mol Å 2 ) for atoms within 6 Å, 10 kcal/(mol Å 2 ) for atoms within 6-11 Å and 15 kcal/(mol Å 2 ) for atoms further than 15 Å from the peptide. The CaM:cx43 peptide complex was energy minimized using 1000 steps of the ABNR method and next underwent 50 ps MD equilibration at 300 K. Finally, a 100-ps MD trajectory was generated at 300 K, with coordinates saved every 0.1 ps. The calculations did not include solvent; solvation was taken into account by using a distance-dependent dielectric constant = 1/4r.

The 1000 saved structures were used to calculate quantities of the type

∆X = X(CaM:cx43) -X(CaM) -X(cx43)
where X(CaM:cx43) refers to the value of X in the complex, and X(CaM) and X(cx43) to the corresponding values for CaM alone and cx43 alone, respectively. All three quantities are obtained from the same structure, with only the selected atoms considered (single trajectory approximation). The trajectory averages were used to estimate binding free energies using two approximate formulas (values in kcal/mol):

∆G = 0.0335<∆G PB > + 0.0016<∆SASA> -5.04 (a) ∆G = -0.0028<∆E elec > + 0.0399<∆E vdW > -7.63 (b)
Here, ∆E elec and ∆E vdw are the CHARMM electrostatic and van der Waals energies, respectively, ∆SASA is the change in solvent accessible surface area, and ∆G PB is the change in electrostatic free energy calculated with the Poisson-Boltzmann continuum electrostatic method. The optimized parameters in the above equations were obtained by fitting to experimental binding free energies of six CaM:peptide complexes with an RMS deviation of ca. 1 kcal/mol (P. Kursula, K.Kuczera, in preparation). All simulations were performed using the program CHARMM [START_REF] Brooks | CHARMM: the biomolecular simulation program[END_REF][START_REF] Brooks | CHARMM: A program for macromolecular energy, minimization, and dynamics calculations[END_REF] and the version 22 protein topology and parameters [START_REF] Mackerell | All-Atom Empirical Potential for Molecular Modeling and Dynamics Studies of Proteins †[END_REF].

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Results and discussion

Binding of CaM to the 15-residue peptide from the cx43 intracellular loop

Previously, a longer peptide of 23 residues was used to detect a CaM-binding domain within the intracellular loop of cx43 [START_REF] Zhou | Identification of the calmodulin binding domain of connexin 43[END_REF]. Similar peptides were also studied in context of cx44/cx46 [START_REF] Zhou | Calmodulin Mediates the Ca2+-Dependent Regulation of Cx44 Gap Junctions[END_REF]. We decided to further map the binding domain on cx43 and used a much shorter peptide of 15 residues, immediately before the 3 rd transmembrane helix of cx43. Compared to the peptide previously used, this shorter peptide is missing, for example, two glycine and two glutamate residues, which can be assumed to be of little relevance to CaM binding. It should be noted that within the CBD and in the immediate vicinity of it, several characterized ODDD mutations are present, which could also affect CaM binding (Figure 1A) [START_REF] Paznekas | GJA1 mutations, variants, and connexin 43 dysfunction as it relates to the oculodentodigital dysplasia phenotype[END_REF].

Using isothermal titration calorimetry (Figure 1B-D), it became obvious that the cx43-CBD bound to CaM in a 1:1 ratio (the fitted stoichiometry for the binding reaction was between 0.87 and 1.38 for all individual titrations), with an affinity in the low micromolar range (K d between 2 and 12 µM for all experiments); the longer peptide was previously shown to have an affinity of approximately 1 µM [START_REF] Zhou | Identification of the calmodulin binding domain of connexin 43[END_REF]. We carried out the experiment at several different temperatures between +9 and +37°C;

the binding reaction was endothermic at low temperatures and exothermic at high temperatures. At low temperatures, the reaction is driven by a large favourable entropy.

From the analysis of enthalpy at different temperatures, we could estimate the heat capacity of the binding reaction. We, thus, can compare the cx43-CBD result to those obtained using other CaM-binding peptides in earlier studies. The estimated ∆Cp for cx43, -3.6 kJ/mol (-850 cal/mol), is in the same range as average values, -3.2 kJ/mol, observed for peptides that are known to induce the so-called collapsed conformation of CaM around the peptide [START_REF] Brokx | Energetics of target peptide binding by calmodulin reveals different modes of binding[END_REF]. For peptides that mainly interact with only one lobe of CaM, the corresponding heat capacity is approximately half of that of a collapsed complex, -1.6 kJ/mol [START_REF] Brokx | Energetics of target peptide binding by calmodulin reveals different modes of binding[END_REF]. On the other hand, the complex between a peptide from MBP and CaM, which remains in an extended conformation, has a ∆Cp in the range of -0.5 kJ/mol [START_REF] Majava | Interaction between the C-terminal region of human myelin basic protein and calmodulin: analysis of complex formation and solution structure[END_REF], indicative of an even smaller buried surface upon binding, and/or a different type of interaction. The ITC data strongly argue in favour

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of the option that CaM binds to the cx43-CBD in a closed conformation, enveloping the peptide tightly between its N-and C-terminal lobes.

Determination of the 3D solution structure of the CaM-cx43-CBD complex

In order to analyze the structural features of the CaM-cx43-CBD complex, we carried out a SAXS experiment for CaM in both the presence and absence of the cx43-CBD peptide. Already the raw scattering curves (Figure 2A) indicated that the structure of the complex is significantly different from that of unliganded CaM. The calculated distance distributions (Figure 2B) further indicated that the dumbbell shape of CaM is lost upon peptide binding, and the distribution indicates the presence of a more globular particle. This is also supported by the calculated radii of gyration and maximum particle diameter (Table I). Ab initio modeling of the 3D structure was carried out for CaM and the complex using DAMMIN [START_REF] Svergun | Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing[END_REF]. The results confirmed the presence of a dumbbell-shaped elongated unliganded CaM, and a collapsed conformation for the peptide complex (Figure 2C).

Crystal structures of CaM and its respective peptide complexes could be superimposed on the ab initio structures with good fits. The result clearly indicates that CaM fully collapses around the cx43 peptide, in a 'classical' mode of binding.

The best fit (Figure 2D) was obtained for the complex between CaM and a peptide from CaM-dependent kinase II (CaMKII) [START_REF] Meador | Modulation of calmodulin plasticity in molecular recognition on the basis of x-ray structures[END_REF], but in essence, all available collapsed CaM-peptide complexes fit to the SAXS data very well. For CaM without the peptide, a good fit to the corresponding crystal structure [START_REF] Rupp | Crystallization and preliminary X-ray analysis of two new crystal forms of calmodulin[END_REF] was also obtained (data not shown), further confirming the conformational difference between the liganded and unliganded forms in solution.

Modeling of the cx43 structure

Using the structure of the complex of CaM with the DRP-1 S308D mutant peptide (PDB entry 1ZUZ), a model of the cx43-CBD bound to CaM was obtained (Figure 3A). The structure shows the presence of residues Ile156 and Met147 in the hydrophobic pockets of the N-and C-terminal lobes of CaM, respectively. This binding mode is analogous to that seen for peptides from CaM-dependent protein kinases [START_REF] Meador | Modulation of calmodulin plasticity in molecular recognition on the basis of x-ray structures[END_REF][START_REF] Bertini | Accurate solution structures of proteins from X-ray data and a minimal set of NMR data: calmodulin-peptide complexes as examples[END_REF]. In addition, residues Lys144, Lys146, Arg148, and Arg153 are most

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likely involved in salt bridge interactions with acidic side chains from CaM. Unlike many other CaM-peptide complexes, it seems that there is no basic residue in cx43 that would interact with the acidic groups Asp80 and Glu84 at the bending region of the central helix. An analysis of the electrostatic and surface area contributions of key residues after the MD simulation is given in Table II.

Considering the gap junction as a whole, the size of CaM is relatively large, taking also into account that a total of six CaM molecules could theoretically bind onto the cytoplasmic face of the hexameric cx43 hemichannel. Modeling of the Cα trace of the cx32 hemichannel suggested that it is transmembrane domain 3 which is mainly forming the inner face of the channel [START_REF] Fleishman | A Calpha model for the transmembrane alpha helices of gap junction intercellular channels[END_REF]; thus, the CaM-binding site studied here, being located immediately at the cytoplasmic side of this transmembrane domain in cx43, could, indeed be involved in gap junction regulation and conformational changes of cx43 related to it. A model of a CaM-bound gap junction built based on the above considerations is shown in Figure 3B.

Concluding remarks

We have obtained 3-dimensional structural information on the complex between the cytoplasmic loop of C. An example of ITC titration and curve fitting of the CaM-cx43-CBD complex.

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The experiment was carried out at +9.2°C; at low temperatures, the reaction is endothermic.

D. Fitted ITC curves for all used temparatures, indicating a stoichiometry of 1:1.

The temparatures are, from top (endothermic) to bottom (exothermic), +9.2, +16, +21, +30, +35, and +37°C.

  , indicating a putative general mechanism of gap junction regulation by CaM. Currently, no highresolution data on the CaM-cx43 complex exist, and the sequence homology between cx43 and structurally characterized CaM target proteins is low. CaM binds to its hundreds of target proteins usually in a calcium-dependent manner. The classical collapsed binding mode involves the wrapping of the peptide by the N-and C-terminal lobes of CaM, while the central linker helix is bent. During recent years, a growing number of divergent CaM-peptide recognition modes have been detected. The relationships between CaM conformation upon target peptide binding and function have been poorly characterized; thus, it is important to determine the 3D conformation of a CaM complex for any newly identified CaM A C C E P T E D M A N U S C R I P T

  cx43 and CaM, by means of synchrotron SAXS. Combined with our calorimetric and simulation data, our results unequivocally indicate that CaM collapses around its target sequence in cx43. Considering the gap junction as a whole, the CaM binding site is in a position that may allow for the regulation of gap junction function by calcium signals via CaM. In the absence of high-resolution structural data for the complex, our data give a low-resolution glimpse into the complex and the likely location of bound CaM with respect to the whole gap junction, and provide a framework for further determining structure-function relationships between CaM and cx43.

Figure 1 .

 1 Figure 1. The used peptide and its binding to CaM. A. Alignment of the cx43-CBD with CBDs from selected other proteins exhibiting a collapsed mode of binding. Known cx43 mutations within or near the CBD are also indicated in italics above the sequence. The peptide used in this study is underlined, and basic residues are highlighted in bold. B. The observed enthalpy as a function of temperature in ITC. The slope of the curve for ∆H allows the estimation of the heat capacity.

Figure 2 .

 2 Figure 2. SAXS of CaM and the CaM-cx43-CBD complex.A. Scattering curves. CaM is shown in green and the complex in black.B. Distance distributions from GNOM. Colouring as in A.C. Ab initio model for the complex obtained from DAMMIN (green), with the superimposed structure of the CaM-CaMKII-CBD complex (PDB entry 1CDM; CaM in blue and the peptide in red). D. Fitting of the theoretical scattering curve from 1CDM (red) to the measured curve (black) for the complex.

Figure 3 .

 3 Figure 3. Models of the CaM-cx43 interaction.A. Model of the CaM-cx43 complex. In this view, the cytoplasmic leaflet of the plasma membrane would be on the right hand side.B. The putative location of the CaM binding site (orange) with respect to the entire gap junction (cyan). In theory, 6 CaM molecules (green) could be bound at the cytoplasmic face of cx43; the size of CaM is in the same range as the channel diameter. Helix 3 from one monomer is shown in magenta, with the orange CBD preceding it in sequence.

Table I . Results from SAXS and comparisons to theoretical values.

 I

			1UP5, apo-
	CaM [32]; 1CDM, CaM complexed with a peptide from CaMKII [31]; 1WRZ, CaM
	complexed with a peptide from DRP-1 kinase [33].
	Sample	Rg (nm)	Dmax (nm)
	SAXS:
	CaM	2.16	6.5
	CaM+cx43	1.76	5.5
	Crystal structures:
	1CDM	1.61	5.2
	1UP5	2.29	7.0
	1WRZ	1.70	5.4

Table II . The calculated interaction free energy and buried surface area contributions from selected residues.

 II

	Electrostatic contributions (kcal/mol)	Buried surface area (Å 2 )
	Calmodulin
	GLU 7	-1.5	GLU 7	45.1
	GLU 11	-3.6	ALA 10	19.4
	LYS 13	1.5	GLU 11	75.4
	GLU 14	-3.4	GLU 14	36.7
	GLN 41	1.1	ALA 15	25.6
	ASP 80	2.9	LEU 18	10.4
	GLU 84	1.2	PHE 19	43.2
	GLU 114	-1.4	MET 36	32.3
	GLU 120	-2.2	LEU 39	40.6
	GLU 123	-2.0	GLN 41	44.5
	GLU 127	-1.6	MET 71	15.2
	LYS 148	2.1	MET 72	41.4
			LYS 75	31.9
			MET 76	81.0
			ASP 80	22.0
			GLU 84	16.9
			GLU 87	23.1
			ALA 88	42.5
			PHE 92	45.1
			LEU 105	14.2
			MET 109	23.2
			LEU 112	29.2
			GLU 114	17.5
			GLU 120	22.8
			MET 124	58.9
			GLU 127	12.3
			PHE 141	17.4
			MET 144	31.0
			MET 145	94.0
			LYS 148	59.7
	peptide
	LYS 144	-3.2	LYS 144	115.1
	LYS 146	2.3	VAL 145	70.2
	ARG 148	-7.7	LYS 146	140.7
	ARG 153	1.1	MET 147	156.2
	THR 154	1.1	ARG 148	74.6
	TYR 155	2.8	GLY 149	14.6
	SER 158	11.7	GLY 150	30.7
			LEU 151	62.6
			LEU 152	61.3
			ARG 153	165.9
			THR 154	85.6
			TYR 155	140.6
			ILE 156	135.9
			ILE 157	154.5
			SER 158	74.4

Acknowledgements

This study has been supported by grants from the Academy of Finland, the Sigrid Juselius Foundation, and the Department of Biochemistry, University of Oulu. Data collection at MAX-Lab was supported by the European Community -Research Infrastructure Action under the FP6 "Structuring the European Research Area" Programme (through the Integrated Infrastructure Initiative "Integrating Activity on Synchrotron and Free Electron Laser Science"), contract RII3-CT-2004-506008 (IA-SFS). We thank Noora Löytynoja for helping in protein purification, and the staff of beamline I711 at MAX-Lab for excellent support during SAXS data collection.

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