The solvent at antigen-binding site regulated C3d–CR2 interactions through the C-terminal tail of C3d at different ion strengths: insights from molecular dynamics simulation

    The solvent at antigen-binding site regulated C3d - CR2 interactions through the C-terminal tail of C3d at different ion strengths: insights from molecular dynamics simulation Yan Zhang, Jingjing Guo, Lanlan Li, Xuewei Liu, Xiaojun Yao, Huanxiang Liu PII: DOI: Reference:

S0304-4165(16)30131-3 doi: 10.1016/j.bbagen.2016.05.002 BBAGEN 28467

To appear in:

BBA - General Subjects

Received date: Revised date: Accepted date:

13 July 2015 16 March 2016 2 May 2016

Please cite this article as: Yan Zhang, Jingjing Guo, Lanlan Li, Xuewei Liu, Xiaojun Yao, Huanxiang Liu, The solvent at antigen-binding site regulated C3d - CR2 interactions through the C-terminal tail of C3d at different ion strengths: insights from molecular dynamics simulation, BBA - General Subjects (2016), doi: 10.1016/j.bbagen.2016.05.002

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ACCEPTED MANUSCRIPT The solvent at antigen-binding site regulated C3d - CR2 interactions through the C-terminal tail of C3d at different ion strengths: insights from molecular

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dynamics simulation

Yan Zhang1, 2, Jingjing Guo1, Lanlan Li2, Xuewei Liu2, Xiaojun Yao2, and Huanxiang

School of Pharmacy, Lanzhou University, Lanzhou, 730000, P. R. China, 2State Key

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Liu1, 2*

Laboratory of Applied Organic Chemistry and Department of Chemistry, Lanzhou

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University, Lanzhou, 730000, P. R. China.

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Corresponding Author: E-mail: [email protected]. Tel: +86-931-8915686. Fax:

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+86-931-8915686.

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ACCEPTED MANUSCRIPT Abstract

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Background: The interactions of complement receptor 2 (CR2) and the degradation

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fragment C3d of complement component C3 play important links between the innate

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and adaptive immune systems. Due to the importance of C3d-CR2 interaction in the design of vaccines and inhibitors, a number of studies have been performed to

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investigate C3d-CR2 interaction. Many studies have indicated C3d-CR2 interactions

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are ionic strength-dependent.

Methods: To investigate the molecular mechanism of C3d-CR2 interaction and the

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origin of effects of ionic strengths, molecular dynamics simulations for C3d-CR2

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complex together with the energetic and structural analysis were performed.

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Results: Our results revealed the increased interactions between charged protein and ions weaken C3d-CR2 association, as ionic strengths increase. Moreover, ion

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strengths have similar effects on antigen-binding site and CR2 binding site. Meanwhile, Ala17 and Gln20 will transform between the activated and non-activated states mediated by His133 and Glu135 at different ion strengths.

Conclusions: Our results reveal the origins of the effects of ionic strengths on C3d-CR2 interactions are due to the changes of water, ion occupancies and distributions.

General significance: This study uncovers the origin of the effect of ionic strength on C3d-CR2 interaction and deepens the understanding of the molecular mechanism of 2

ACCEPTED MANUSCRIPT their interaction, which is valuable for the design of vaccines and small molecule

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inhibitors.

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Keywords: C3d; complement; CR2; molecular dynamics simulation; MM-GBSA

1. Introduction

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Complement protein C3 is the central component of the complement system

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because it supports the activation of all the three pathways of complement activation including the classical, alternative, and lectin pathways. C3 plays an essential role in

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linking the innate and adaptive immunity [1]. The degradation product C3d of

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complement C3 represents the minimal ligand for complement receptor 2 (CR2，

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CD21), which has full binding activity [2-3]. C3d is dual-functional since C3d can covalently attach to pathogen cell surfaces through a highly reactive thioester bond

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(antigen binding site) and simultaneously bind to CD21/CD19/CD81 complex in order to form B-cell co-receptor complexes. The forming of co-receptor complexes has a profound molecular adjuvant effect, reducing the threshold of antigen required for B cell activation by 1000-10000 fold [4]. The molecular adjuvant effect is beneficial to the host under normal circumstances, however, it can also be detrimental when the body has autoimmune disease [5]. CR2 is the regulators of complement activation family of proteins, which is a transmembrane protein containing 15 or 16 short consensus repeats (SCRs). The neighboring SCR domains are linked through short linkers composed of 3–8 residues

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ACCEPTED MANUSCRIPT [6-9]. CR2 plays an important role in B cell activation and the generation of normal immune responses, primarily found on mature B cells and follicular dentritic cells

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(FDCs) [4, 10-11]. The ligands for CR2 include the C3 activation fragments iC3b,

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C3dg, and C3d [5, 12], the low affinity IgE receptor CD23 [13], the Epstein-Barr virus surface glycoprotein gp350/220 [14] and the cytokine interferon α (IFN-α) [15]. Several cellular responses have similar nature of the C3d-CR2 interaction, which have

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been observed when gp350/220 binding to CR2, specifically, gp350/220 can induce B

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cell proliferation. The N-terminal two SCR domains of CR2 is the binding site for iC3b, C3d and gp350. Moreover, the viral protein competes with iC3b and C3d for

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the interaction with CR2 [16]. Thus, the studies of C3d-CR2 interactions are helpful

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for understanding the binding mechanisms of CR2 and its natural ligands.

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Due to the importance of C3d-CR2 interaction and its role in reducing the threshold of antigen required for B cell activation, a number of studies have been

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performed to investigate C3d-CR2 interaction for the design of vaccines and inhibitors [17-20]. For example, many studies have indicated that the interaction between C3d and CR2 depends on ionic strengths and pH [21-23]. The SCR domains are important for regulation of complement activation and the conformational changes of interdomain contact region can adapt to different ligands [24-26]. Despite the main progress in understanding CR2-C3d interaction, several critical questions remain unknown: (1) what is the origin of the influence of ionic strengths on C3d-CR2 interaction? (2) How do antigen binding site and CR2-binding surface link? (3) What changes the conformation Ala17 and Gln20 involving the thioester bond undergoes? 4

ACCEPTED MANUSCRIPT In this study, to elucidate the detailed mechanism of the interaction between C3d and CR2 and to seek the origin of the effect of ionic strength on the interaction of C3d

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and CR2, all-atom molecular dynamics (MD) simulations of the C3d-CR2 complex

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were performed. Based on the trajectories of MD, the binding free energy calculation combined with the analysis of solvent occupancies, correlation and cluster analysis for C3d-CR2 complex was performed to identify the origins of ionic strengths and

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investigate the correlation of antigen binding site and CR2-binding surface. In

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addition, the conformational changes of Ala17 and Gln20 involving the thioester bond are studied. This valuable information obtained in the study can provide theoretical

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autoimmune circumstances.

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guidance in vaccine design under normal circumstances and in design of inhibitors in

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2. Materials and methods

2.1 Structure preparation

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The structural complex of complement receptor CR2 and C3d was taken from the Protein Data Bank (PDB ID: 3OED). This structure contains two copies of the C3d - CR2 complex. We retained chain A (C3d), chain C (CR2), crystal waters and deleted other chains, with a total of 424 residues. Hydrogen atoms were added using AMBER10 package [27]. C3d fragment is an α-barrel structure (Figure. 1A) and has two important functional sites. One site is antigen-binding site, which is a convex and somewhat basic surface. The site contains the thioester-constituting residues and C3d can covalently attach to target antigen through ester linkages (Figure. 1B). The other site is CR2-binding surface, which is a more concave and acidic surface. The site is 5

ACCEPTED MANUSCRIPT involved in the interaction with the N-terminal two SCR domains of CR2. The two SCR domains interact with each other through the linkers and interdomain contact

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region (Figure. 1C-D).

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2.2 Molecular Dynamics Simulation

The molecular dynamics simulations including energy minimization, systems equilibration and production protocols, were performed with the AMBER10 package

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[27]. The proteins were described by the AMBER99SB force field [28]. Counterions

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were added to keep the whole system neutral. The systems were then solvated using atomistic TIP3P water [29] in rectangular box with at least 10 Å distance around the

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structures. The simulations of the system were performed at salt concentrations

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around 50, 100 and 150 mM in order to investigate the effects of ion concentration on

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the interactions of C3d and CR2(SCR1-2). Each simulated system was carried out with periodic boundary conditions.

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We first carried out the energy minimization in AMBER10 for 4 steps with the gradually reduced force constants of 5.0, 2.0, 0.1 and 0 kcal/mol/Å2 applied to proteins, respectively. During the energy minimization process, the steepest descent method of 2500 steps were performed firstly and then switched to conjugate gradient for the following 2500 steps. The particle mesh Ewald (PME) summation method [30] was applied to treat Coulombic interactions. The equilibration and subsequent production runs were performed. The equilibration was simulated with force constants of 2.0, 1.0, 0.1 and 0 kcal/mol/Å2 applied to the complex for 100, 100, 100 and 500 ps, respectively. The SHAKE algorithm [31] was applied on all atoms covalently bonded 6

ACCEPTED MANUSCRIPT to hydrogen atoms with an integration time step of 2 fs. All the systems were annealed from 0 to 310 K in a 50 ps timescale using a force constant of 2 kcal/mol/Å2 to the

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complex. All subsequent equilibration and the production phases were performed in

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the isothermal isobaric (NPT) ensemble that used a Berendsen barostat [32] in a target pressure of 1 bar with a coupling constant of 2.0 ps. Coordinate trajectories of all the equilibration and production runs were recorded every 1 ps. Three parallel trajectories

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for the C3d-CR2 complex at each ion concentration were simulated for 50.0 ns in the

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NPT ensemble at 310 K. 2.3 Binding Free Energy Calculation

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To explore the interaction of C3d - CR2 from the energetic perspective, the

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binding free energy calculation based on the trajectories of molecular dynamics

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simulation were performed by using MM-PBSA and MM-GBSA methods. MM-PBSA and MM-GBSA methods have been widely applied to calculate binding

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free energies of macromolecules and their ligands[33-38]. In addition, a special advantage of MM-GBSA is that it can decompose the total binding free energy into atomic/group contributions in a structurally nonperturbing formalism using a fully pairwise potential[39-40]. The first step to apply MM-PBSA and MM-GBSA methods is to generate the multiple snapshots from an MD trajectory of the protein-protein complex by stripping water molecules and counterions. Snapshots were extracted at intervals of 10 ps from the last 20ns of MD trajectory. The free energy is calculated for complex, complement receptor CR2 and its ligand C3d of each snapshot. The binding free energy is 7

ACCEPTED MANUSCRIPT computed as follows:

Gbind  Gcomplex  GCR 2  GC 3d

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The free energy G can be calculated by the following scheme based on

G  Egas  Gsol

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MM-PBSA and MM-GBSA methods[41-42]:

Egas  Eint  Eele  EvdW

Eint  Ebond  Eangle  Etorsion

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Gsol  GPB (GB )  GSA GSA  SAS

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Egas, Eint, Eele, EvdW, Ebond, Eangle and Etorsion are the gas-phase energy, internal

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energy, the Coulomb energies, van der Waals energies, the bond energies, the angle

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energies and the torsion energies, respectively. The conventional molecular mechanics force fields such as AMBER are used to calculate van der Waals and

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electrostatic energies[43]. Gsol is the solvation free energy which can be decomposed into the polar and nonpolar contributions. GPB(GB) is the polar solvation contribution

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calculated by solving PB or GB equation[41-42]. Dielectric constant for solvent is set to 80. For solute, the dielectric constants of 1-4 are set to calculate binding free energies of C3d-CR2 complex in different ionic strengths, respectively. Compared with other dielectric constants, binding free energies of C3d-CR2 complex for parallel trajectories are more stable when the dielectric constant is set to 4. Moreover, the binding surface of C3d and CR2 is more hydrophilic and the dielectric constant of 4 for solute is suitable for the hydrophilic system[3]; Thus dielectric constant for solute is set for 4. GSA is the nonpolar solvation contribution that is estimated by the solvent accessible surface area (SAS) determined with a water probe radius of 1.4 Å. The 8

ACCEPTED MANUSCRIPT surface tension constant γ was set to 0.0072 kcal/mol/Å2 [44]. S and T are the total solute entropy and the temperature, respectively.

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To obtain the detailed interaction profile of CR2 and C3d, the MM-GBSA method

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was applied to decompose the whole binding energy to each residue by combining molecular mechanics energies and solvation energies without considering the contribution of entropies.

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2.4 Cluster Analysis

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To study the conformational changes of Pro130-Gln168 and Gly270-Pro294 of C3d, clustering analysis of MD trajectories was carried out by using the SOM

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algorithms from the PTRAJ module of the AMBER10 [45]. Snapshots every 2 ps

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were collected for the cluster analysis.

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2.5 Dynamic cross-correlation map analysis The dynamic cross-correlation map (DCCM) analysis was performed to study

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the correlation motion between protein domains over the last 20ns MD trajectories [46-47]. The cross-correlation of the atomic displacements of atoms i and j is given by:

Ri  R j

C (i, j ) 

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 R  R  R  R  2 i i j j   Ri and R j are the displacement from the mean position of atoms i and j,

respectively. The more C (i, j ) deviates from 1 (or −1), the motions of i and j are less correlated (or anti-correlated). C (i, j ) =1 (or -1) implies that the motions of i and j are completely correlated (or anti-correlated). 9

ACCEPTED MANUSCRIPT 2.6 Calculation of interdomain angles The interdomain angles were calculated to study the relative intermodular

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position of two SCR domains, which was useful for understanding conformational

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changes of CR2. Three angles including tilt, twist and skew angles were calculated according to the reference [25]. The tilt angle describes the depth of the angle between the adjacent SCR domains, and its decrease directly means the open of the

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V-shaped conformation of CR2. The skew angle describes the direction where the

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C-terminal domain is tilted relative to the N-terminal domain. An initial step to open the structures of CR2 was the changes of twist angle. In other words, the changes of

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twist angle can indicate the open or close of the V-shaped conformation of CR2 [18].

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2.7 Calculation of solvent occupancies

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To study the effects of solvent on C2d-CR2 interactions and to further seek the origins of the influence of ion strengths on these interactions, the solvent occupancies

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were calculated over the last 20 ns parallel trajectories for every system using the PTRAJ module of the AMBER10[45]. All the trajectories were first imaged and fit to the first frame by RMS of the backbone atoms of C3d-CR2 complex. Then, the solvent occupancies of water were calculated using the grid command with a 0.5 Å * 0.5 Å * 0.5 Å spacing over the whole box. 3. Results and Discussion 3.1 The monitoring of MD trajectories and the overall structural features To monitor if molecular dynamics simulations are up to the equilibration, the root mean square deviations (RMSDs) of backbone atoms of different domains were 10

ACCEPTED MANUSCRIPT calculated firstly and the obtained results were shown in Figure 2. From Figure 2, it can be seen that the RMSDs of C3d and CR2 are very small during 50ns MD

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stimulations and remain stable in the last 20ns trajectories. To explore the differences

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in the flexibilities of these residues, we further calculated the RMSDs of the backbone atoms averaged for each residue of C3d and CR2 from the last 20 ns parallel MD trajectories (Figure 3). For C3d, the flexibilities of Asp36-Ala52, Phe76-Arg79 and

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Gly200-Lys216 regions are larger at high ion strengths whereas Ile115-Glu127,

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Ile132-Phe153, Glu167-Leu190, N-terminal and C-terminal tails of C3d exhibit the opposite changes (Figure S1). For SCR1, most of residues of SCR1 have the

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increased flexibilities at high ionic strengths. However, the flexibilities of N-terminus

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of SCR1 reduce. For SCR2, although high ionic strengths slightly increase the

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flexibilities of SCR2 except for C-terminus of SCR2, ionic strengths have smaller effects on the flexibilities of SCR2 than SCR1.

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3.2 The interaction energetic features of C3d-CR2 and the influence of ionic strengths

MM-PBSA and MM-GBSA methods were used to calculate the binding free energy of C3d-CR2 complex. Firstly, we extracted 2000 snapshots from the last 20 ns MD trajectory for the calculation of enthalpy. Solute entropy was calculated by using extracted 100 snapshots from the last 20 ns MD trajectory. The average binding free energies and the detailed contributions of various energy components were shown in Table 1. Moreover, the binding free energies from each MD trajectories are given in Table S1. From Table 1, it can be seen that the binding free energies of C3d-CR2 11

ACCEPTED MANUSCRIPT complex are obviously reduced when ion strengths increase, indicating that C3d-CR2 interactions depend on ion strengths and the changes are consistent with the

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experiments [18, 48-50]. The reduction of binding free energy is mainly from the

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decreased contributions of nonpolar interactions including van der Waals and nonpolar desolvation interactions. Although the direct electrostatic interactions also have significant decrease, these interactions were compensated by the polar

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desolvation free energies, leading that the electrostatic free energies of binding of

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C3d-CR2 complex were impacted by the ion strengths weakly. Although many studies [19, 23] have indicated electrostatic interactions play important roles in C3d-CR2

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interactions and in their functions and evolution, the nonpolar interactions including

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the van der Waals and the nonpolar solvation interactions have large contributions and

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thus provide driving forces in the binding process of C3d and CR2. To explore the effects of ion strengths on intra-molecular interactions of C3d and

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inter-molecular interactions between C3d and CR2, we further calculated the energy contributions of each section (Table s2). From Table s2, at high ion strengths, the electrostatic interactions in vacuum, van der Waals and nonpolar solvation energies of C3d, CR2 and C3d-CR2 complex will be weakened obviously. Conversely, by increasing ion strengths from 50mM to 150mM, polar solvation energies of C3d, CR2 and C3d-CR2 complex reduce, leading that the overall electrostatic interaction contribution of C3d, CR2 and C3d-CR2 complex reduce firstly from 50 mM to 100 mM and increase again from 100 mM to 150 mM. Consistent with Table 1, ion strengths primarily affect nonpolar contributions and thus the nonpolar contributions 12

ACCEPTED MANUSCRIPT of the residues with more than 1 kcal/mol contributions are given in Figure 4. For C3d, Asp36/Glu37/Glu39 and Ile164/Glu166 clusters have slightly increased nonpolar Gln50/Leu53,

Lys251/Asp252

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whereas

and

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contributions,

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Lys291/Asp292/Ala293/Pro294 clusters have reduced nonpolar contributions, especially Lys291/Asp292/Ala293/Pro294 cluster with the significant reduction. For CR2, high ion strengths lead that the reduced nonpolar contributions of Arg13, Tyr16,

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Arg28, Arg36 and Arg89. From the structural perspective, Ile164C3d and S42-L44CR2,

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Leu53C3d and Tyr16CR2 have increased and weak hydrophobic interactions, respectively. In addition, the occupancies of the hydrogen bonds or salt bridges

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formed between Asp36C3d-Arg13CR2, Glu39C3d-Arg13CR2, Glu167C3d-Lys57CR2 are

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increased at high ion strengths (Table S3). Whereas the occupancies of the hydrogen

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bonds or salt bridges formed between Glu37C3d-Arg13CR2, Asp163C3d-Lys41CR2, Glu166C3d-Arg83CR2, Glu167C3d-Lys108CR2, Glu54C3d-Tyr16CR2, Lys251C3d-Pro87CR2,

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Lys291C3d-Cys31CR2, Asp292C3d-Arg89CR2, Pro294C3d-Lys67CR2 are obviously reduced. In comparison with the work published by Mohan et al[51], many hydrogen bonds are similar. However, there are some different hydrogen-bonds interactions. For example, Ala204-Gly207 of C3d and Arg83-Ser85 of CR2 are involved in weak hydrogen bond interactions in their work whereas these hydrogen bonds are not formed in our work. Asp292 and Arg89 primarily form hydrogen bond in our work while Asp292 and Arg36 form strong hydrogen bond. To further explore the influence of ion strengths on electrostatic binding of C3d-CR2 complex, electrostatic free energies of binding of all ionizable amino acids 13

ACCEPTED MANUSCRIPT is given in Figure 5. For C3d, Asp36/Glu37/Glu39 and Lys291/Asp292 clusters are more favorable for electrostatic free energies of binding at high ion strengths.

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However, the changes of Lys162/Asp163/Glu166 cluster are the opposite. For CR2,

strengths.

However,

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Arg13/Lys57/Arg89 cluster has increased electrostatic contributions at high ion Arg28/Lys41/Lys67/Arg83/Lys108

cluster

has

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electrostatic contributions. Compared with results of a general alanine scan of all

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ionizable amino acids published by Mohan et al, either the electrostatic contributions

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of these amino acids are consistently favorable or unfavorable except for Glu73, Arg83 and Lys99 of CR2.

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3.3 The impact of ion strengths on the C3d-CR2 interaction from the structural

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perspective

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3.3.1 Correlation motions between C3d and CR2 The binding free energy analysis indicates that the increase of ion strength makes

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the interactions of C3d and CR2 become weak. The weakened interactions should be reflected in the structural changes. To reveal the structural changes, the DCCM analysis of C3d-CR2 complexes in the last 20 ns MD trajectories was firstly performed. The collective protein motions of C3d-CR2 complexes were shown in Figure 6. For C3d, the correlation motions of three continuous protein regions are affected by ionic strengths. These protein regions include Met1-Thr219, Thr220-Tyr269 (helices α10-α11) and Gly270-Pro294 (helix α12). The positive correlation motions between residues in the region Met1-Thr219 weaken as ionic strengths increase. Similarly, the positive correlation motions of the region 14

ACCEPTED MANUSCRIPT Met1-Thr219 and helix α12 are reduced. However, the positive correlation motions of helices α10-α11 with the region Met1-Thr219 and helix α12 are increased,

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respectively. These changes indicate that the interactions between helices α10-α11 and

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helix α12 enhance while helix α12 and the region Met1-Thr219 have impaired interactions. For CR2, the positive correlation motions inside SCR1 and SCR2 of CR2 are enhanced as ionic strengths increase. Interestingly, the ionic strengths

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mediate interdomain interactions of CR2 by strengthening the correlations motions of

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linker with Cys31-Ile38 and Thr86-Gly91 and reducing correlations motions of the linker with Cys112-Asn116 in high ion strengths as shown in Figure 6E. Consistent

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with our results, Lehtinen et al. also found that the linker and three loops

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(Thr35-Ile39、Pro88-Gly92 and Ala115-Trp119) were responsible for the interdomain

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contact regions [25]. For C3d-CR2 complex, the positive and negative correlation motions of the region Gln50-Thr219 of C3d with SCR1 and SCR2 of CR2 are

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weakened, respectively, which suggest the interactions of the region Gln50-Thr219 with SCR1 and SCR2 enhance. 3.3.2 Conformational changes of CR2 CR2 is a flexible molecule which can adapt to different ligands and mediate C3d-CR2 interactions through the conformational changes of interdomain contact region. Thus, it is important to study the conformational feature of CR2 at different ion strengths. Here, the conformational changes of CR2 can be characterized by tilt, skew and twist angles, which describe the relative spatial arrangement of SCR1 and SCR2. The intermodular angles averaged by the former 10ns and last 10ns parallel 15

ACCEPTED MANUSCRIPT trajectories are given in the Table 2. Compared with the former 10ns MD trajectories, it can be seen that twist angles from all simulations are obviously increased in the last

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10ns MD trajectories and other angles have no obvious changes. In the last 10ns MD

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trajectories, twist angles are slightly larger at the high ion strengths. Thus, the V-shape conformations of CR2 tend to open at all ion strengths and change more obviously at high ion strengths. Twisting is the initial step to open the V-shape

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conformations and belongs to the first slow motion mode [52]. Open-close motions

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are the second slow motion mode. Moreover, the solution models have indicated that CR2 is in a more extended conformation[48]. Consistent with previous studies, the

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V-shape structure of CR2 has obviously twisted and thus has open tendency at

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different ion strengths. Since to open the V-shape conformations is a slow process, it

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should need a long simulation time. Due to the simulation time scale, we did not observe the open of V-shape structures of CR2 but just observe that it begins to twist.

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3.3.3 Conformational changes of key domains in C3d Pro130-Gln168 (Helices α5-α6 and T3-T4) and Gly270-Pro294 (helix α12) locate at ends of the acidic pocket of C3d and they are important for the binding of C3d to CR2. Thus, the conformational changes of these protein regions are significant for understanding the correlation between C3d-binding surface and antigen-binding site. To show the conformational changes of these protein regions, clustering analysis was performed based on pairwise similarity measured by RMSDs using SOM algorithms. Specifically, only Cα atoms of the regions are considered during the clustering analysis. The first trajectory from the three parallel ones is selected for 16

ACCEPTED MANUSCRIPT clustering frames into 3 clusters based on pairwise similarity measured by RMSDs. Three representative structures (shown in Figure S2) are obtained by clustering the

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frames at each ionic strength. The representative structure with the largest clustering

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ratio from each trajectory at three ionic strengths is compared, respectively (Figure 7). The results show that the region Pro130-Arg141 (helices T3 and T4) exhibits large conformational changes and the region Lys291-Pro294 (the C-terminal tail of helix

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α12) also has obvious changes. However, helices α5-α6 undergo very small changes.

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Thus, the detailed interactions of Pro130-Arg141 and Lys291-Pro294 are analyzed, respectively.

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3.3.3.1 Conformational changes of helices T3-T4

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Due to the residues connecting helices T3 and T4 primarily being loops, the

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region exhibits large conformational changes. A further comparison of the representative structures with the highest ratio of clusters at different ionic strengths

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shows that several residues including His133, Gln134, Glu135, Arg141 and Tyr273 within antigen-binding site pack more compact at high ionic strengths and looser at low ionic strengths (Figure 7A-B). As a result, polar interactions of these residues are enhanced at high ionic strengths and are weakened at low ionic strengths, which are consistent with Gly138-Asn142 with obviously reduced RMSDs at high ionic strengths. More deeply, at high ionic strengths, Glu135 and His133 flip over to the above of helices T3 and T4 from below, especially with a great turning of Glu135. The conformational changes of Glu135 and His133 lead to further compact pack of these residues with Gln134, Arg141 and Tyr273. As a result, Glu19 and Gln20 deviate 17

ACCEPTED MANUSCRIPT from His133 and close to Ile132 at high ionic strengths. In fact, stable hydrogen bonds are formed between Glu19 and Ile132 at high ionic strengths. However, at low ionic

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strengths, Glu19 and Gln20 close to His133. The changes lead to the reduced ability

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of forming the acyl-imidazole intermediate (Gln20 and His133) and thus tend to form a nonactivated C3. As a result, Ala17 and Gln20 keep a closed state. Consistent with the cluster analysis, the further monitoring of the distances between Ala17 and Gln20

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shows that the distances are smaller at high ionic strengths (Figure 8). From Figure 8,

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it can be seen that the distances change significantly at all MD trajectories, especially at low ionic strengths and involve the transformations between two states. Some

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studies have indicated that the conformations of Ala17 and Gln20 transform between

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the activated states and non-activated states and thus mediate transacylation reaction

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triggering the activation of C3 [1, 3]. Overall, these results show that Ala17 and Gln20 affected by Glu135 and His133

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tend to keep a closed state and be unfavorable for forming the acyl-imidazole intermediate (Gln20 and His133) involving mediating C3d attaching to the antigen at high ionic strengths. 3.3.3.2 Conformational changes of the C-terminal tail of helix α12 From Figure 7A and 7B, the C-terminal tail of helix α12 at extremely high ionic strengths tends to form coiled helix and far away from CR2-binding surface, especially with the largest deviation from the linker. However, the region at extremely low ionic strengths is more inclined to from the extended helix and close to CR2-binding surface. As a result, Lys291-Pro294 and several loops of CR2 including 18

ACCEPTED MANUSCRIPT linker, Cys31-Ile38 and Thr86-Gly91 have smaller contact surface at high ionic strengths and larger contact surface at low ionic strengths. Accordingly, the

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interactions between Lys291-Pro294 and these loops of CR2 are weakened at high

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ionic strengths in comparison with that at low ionic strengths. Specifically, the electrostatic interactions of Pro294 with Arg36, Lys67 and Arg89 are also obviously weakened at high ionic strengths. In addition, the hydrophobic interactions of Pro294

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with Phe65 are also affected by ionic strengths.

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These conformational changes of Lys291-Pro294 further change the interaction mode of Cys112-Asn116 and the linker through the linker (Figure 9). Due to the

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weakened interactions of Lys291-Pro294 with linker at high ionic strengths, residues

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Asn66-Tyr68 of the linker have large conformational changes. Specifically, the side

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chains of Asn66 and Tyr68 flip over at high ionic strengths and further lead to slightly flipping of Cys112-Asn116 in the same direction, which primarily weaken hydrogen

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bonds between amide side chain of Asn66 and mainchain oxygen atom of Ala114. Moreover, the orientations of amide side chains from Gln113, Asn115 and Asn116 change and the interactions of these residues with Lys61 and Phe65-Asn66 are weakened, especially Asn115 and Asn66 with significantly weakened interactions. Accordingly, the electrostatic distributions of the linker and Cys112-Asn116 change (Figure 9B-D). From Figure 9B-D, it can be seen the regions within the linker and Cys112-Asn116 show greater electronegative characteristics at low ionic strengths. On the contrary, the electronegativity of the regions reduces as ionic strengths increase. It is well known that CR2 has excessive positive electrostatic potential and 19

ACCEPTED MANUSCRIPT thus the electrostatic distributions of the regions at low ionic strengths are favorable for the interaction of the regions and linker. However, the orientations of

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Cys112-Asn116 are unfavorable for the interaction between the regions and other

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residues inside SCR2 and thus slightly increase the flexibilities of Val76-Arg83, Val94-Ser109 and Leu123-Val129 of SCR2. Thus, Cys112-Asn116 and the linker with different interaction mode may have different mediated mechanism for CR2

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conformations.

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3.4 The origin of the influence of ion strengths on C3d-CR2 interactions To seek the origins of the influence of ion strengths on these interactions, the

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occupancies of the sodium and chloride ions are calculated (Figure 10). For CR2

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binding site, the attraction between the charged protein and ions makes the binding

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interface of C3d and CR2 is surrounded by the sodium and chloride ions. The strengthened interaction between the charged protein and ions weakens the interaction

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between C3d and CR2 as the ion strengths increase. But as the ion strengths further increase from 100mM to 150mM, the surface between CR2 and C3d is filled with the sodium and chloride ions. The attraction between sodium and chloride ions will reduce the electrostatic interaction between the charged protein and ions conversely on some degree, which further leads that the slight enhance of the electrostatic interaction between C3d and CR2 in direct. However, the C3d-CR2 association is still weakened. The distribution of electrostatic potential displays the consistent information with the distribution of the sodium and chloride ions (Figure 11). Thus, for CR2 binding site, the electrostatic interaction between C3d and CR2 reduce firstly 20

ACCEPTED MANUSCRIPT from 50 mM to 100 mM and increase again from 100 mM to 150 mM. As for antigen binding site, the C3d-antigen interaction has similar changes if we can consider the

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whole system including C3d, CR2 and antigen. However, due to the lack of antigen,

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the distributions of electrostatic potential for antigen binding site display different trend in the system. Specifically, as the ion strength increases, the positive electrostatic potential becomes weaker gradually due to the attraction between the

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binding site and counter ions.

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The ion distributions change interaction mode of ion and exposed charged residues and further influence the interactions between water and protein. The

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calculations of the water occupancies show that the ion strengths have large impacts

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on the solvent occupancy and distribution (Figure 12). The obvious water occupied

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sites are primarily located at Asp128-Glu145 region near antigen binding site. At extremely low ionic strengths, three water occupied sites locate between helix α3 and

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the region (named hereafter S3 site), between helix α5 and the region (S5 site), between helix α12 and the region (S12 site), respectively. At 100mM ionic strengths, S5 and S12 sites are kept and have significantly increased solvent occupancies. However, at 150mM ionic strengths, although the whole protein is covered by water, there is only an S3 site largely occupied by water. The large water cluster leads highly concentrated interactions of the Asp128-Glu145 region by water-mediated interactions. Correspondingly, the continuous basic surface of the region is smaller as the ionic strengths increase and the interactions are more dispersedly. 4. Conclusions 21

ACCEPTED MANUSCRIPT In this study, 50ns molecular dynamics simulations were performed for the C3d-CR2 complex at ionic strengths corresponding to 50, 100 and 150 mM. The

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origins of the effects of ionic strengths on C3d-CR2 interactions are analyzed from

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the viewpoints of the binding free energy and protein dynamics. When the ion strengths increase, the increased interaction between charged protein and ions will reduce C3d-CR2 association. For CR2 binding site, the electrostatic interaction

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between C3d and CR2 reduce firstly from 50 mM to 100 mM and increase again from

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100 mM to 150 mM. As for antigen binding site, due to the lack of antigen, the positive electrostatic potential becomes weaker gradually due to the attraction

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between the binding site and counter ions as the ion strength increases. The

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calculation of water occupancies further reveal that the interaction between antigen

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binding site is more dispersedly at high ion strengths. Moreover, Ala17 and Gln20 involving the thioester bond and tend to be close at high ionic strengths and far away

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at low ionic strengths under the mediation of His133 and Glu135 affected by the water and ion. The information obtained in this study can provide the guidance for design vaccines and small molecule inhibitors. Acknowledgment This work was supported by the National Natural Science Foundation of China (Grant No: 21375054) and the Fundamental Research Funds for the Central Universities (Grant No: lzujbky-2014-k21). References [1] A. Sahu, J. D. Lambris, Structure and biology of complement protein C3, a 22

ACCEPTED MANUSCRIPT connecting link between innate and acquired immunity. Immunol.Rev.180(2001) 35-48

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[2] K. Kalli, J. Ahearn, D. Fearon, Interaction of iC3b with recombinant isotypic and

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chimeric forms of CR2. J. Immunol. 147(1991) 590-594.

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thermodynamic integration methods. Biopolymers 68 (2003) 16-34. [36] Y. Duan, C. Wu, S. Chowdhury, M. C. Lee, G. Xiong, W. Zhang, R. Yang, P. Cieplak, R. Luo, T. Lee, A point‐ charge force field for molecular mechanics simulations of proteins based on condensed‐ phase quantum mechanical calculations. J. Comput. Chem. 24 (2003) 1999-2012. [37] T. Hou, J. Wang, Y. Li, W. Wang, Assessing the performance of the MM/PBSA and MM/GBSA methods. 1. The accuracy of binding free energy calculations based on molecular dynamics simulations. J. Chem. Inf. Model. 51 (2011) 69-82. [38] H. Liu, X. Yao, Molecular basis of the interaction for an essential subunit pa− 27

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free energy calculation and free energy decomposition for the ras–raf and ras–ralgds complexes. J. Mol. Biol. 330 (2003) 891-913.

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molecules: Combining molecular mechanics and continuum models. Acc. Chem. Res. 33 (2000) 889-897. [43] M. C. Lee, Y. Duan, Distinguish protein decoys by using a scoring function based on a new amber force field, short molecular dynamics simulations, and the generalized born solvent model. Proteins: Struct. Funct. Bioinform. 55 (2004) 620-634. [44] D. Sitkoff, K. A. Sharp, B. Honig, Accurate calculation of hydration free energies using macroscopic solvent models. The Journal of Physical Chemistry 98 (1994) 1978-1988. 28

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algorithms. J Chem. Theory Comput. 3 (2007) 2312-2334.

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methionyl-trna synthetase by molecular dynamics simulations and structure network analysis. Proc. Natl. Acad. Sci. 104 (2007) 15711-15716.

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[48] J. M. Kovacs, J. P. Hannan, E. Z. Eisenmesser, V. M. Holers, Mapping of the c3d

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ligand binding site on complement receptor 2 (cr2/cd21) using nuclear magnetic

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resonance and chemical shift analysis. J. Biol. Chem. 284 (2009) 9513-9520. [49] K. A. Young, A. P. Herbert, P. N. Barlow, V. M. Holers, J. P. Hannan, Molecular

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basis of the interaction between complement receptor type 2 (cr2/cd21) and epstein-barr virus glycoprotein gp350. J. Virol. 82 (2008) 11217-11227. [50] L. Zhang, D. Morikis, Immunophysical properties and prediction of activities for vaccinia virus complement control protein and smallpox inhibitor of complement enzymes using molecular dynamics and electrostatics. Biophys. J. 90 (2006) 3106-3119. [51] R. R. Mohan, R. D. Gorham Jr, D. Morikis, A theoretical view of the c3d:Cr2 binding controversy. Mol. Immunol. 64 (2015) 112-122. [52] H. Wan, J. P. Hu, X. H. Tian, S. Chang, Molecular dynamics simulations of wild 29

ACCEPTED MANUSCRIPT type and mutants of human complement receptor 2 complexed with C3d. Phys.

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Chem. Chem. Phys. 15 (2013) 1241-1251.

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ACCEPTED MANUSCRIPT Table 1．Binding free energy components of the C3d-CR2 complex averaged by all three parallel trajectories at different ionic strengths (kcal/mol). 100 mM

Contributions

C3d-CR2

C3d-CR2

Eele

-300.74

EvdW

-111.09

Egas

-411.84

GSA

-20.11

GPB

289.93

GGB

301.89

GPBSOL

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C3d-CR2

-107.88

-102.12

-394.98

-386.04

-18.91

-18.40

278.26

273.68

289.61

284.97

269.82

259.35

255.29

281.78

270.69

266.58

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-283.92

Gnonpolara

-131.20

-126.79

-120.50

Gele, bind, PB b

-30.92

-27.75

-28.63

Gele, bind, GBc

-18.96

-16.41

-17.34

Htotal, PB

-142.02

-135.63

-130.75

Htotal, GB

-130.05

-124.28

-119.46

TS

-75.29

-72.13

-72.74

ΔGtotal, PB

-66.73

-63.50

-58.01

ΔGtotal, GB

-54.77

-52.15

-46.72

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a

150 mM

-287.10

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GGBSOL

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50 mM

Gnonpolar= EvdW+GSA; b Gele, bind, PB = Eele + GPBSOL c Gele, bind, GB = Eele + GGBSOL

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ACCEPTED MANUSCRIPT Table 2 Tilt, skew and twist angles for CR2 averaged by the former and last 10ns parallel MD trajectories. Tilt angle (°)

Skew angle (°)

Twist angle (°)

35.15(9.31)

100 mM

141.05(2.94)

35.85(9.38)

150 mM

140.30(3.43)

29.76(8.28)

39.37(15.50)

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142.90(3.06)

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50 mM

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The mean values from the former 10ns MD trajectories

41.33(15.55) 48.63(15.83)

The mean values from the last 10ns MD trajectories

100 mM

140.00(3.54)

150 mM

140.07(3.74)

31.13(9.22)

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142.10(3.73)

48.34(15.55)

26.59(6.57)

57.30(7.76)

28.54(7.97)

53.47(12.68)

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50 mM

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ACCEPTED MANUSCRIPT Figure captions Figure 1 Structures of C3d, CR2 and C3d-CR2 complex (PDB ID, 3OED): (A) C3d

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structure viewed from the side of CR2-binding site; (B) C3d structure

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viewed from the side of antigen-binding site and the residues responsible for covalent attachment to antigen shown in sticks; (C) CR2 structure; (D) Structure of the C3d-CR2 complex.

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Figure 2 The RMSDs of backbone atoms of C3d, SCR1 domain and SCR2 domain

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of CR2 from three parallel trajectories at ionic strengths corresponding to 50 mM (A-C), 100 mM (D-F) and 150 mM (G-I) using energy-minimized

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structure as the reference. The averaged RMSDs from three parallel

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trajectories are shown in the blue color.

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Figure 3 The RMSDs of backbone atoms of each residue of C3d and CR2 from the last 20ns MD trajectories of three parallel trajectories at ionic strengths

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corresponding to 50 mM (A-B), 100 mM (C-D) and 150 mM (E-F). Figure 4 The nonpolar contributions of the key residues from C3d (A) and CR2 (B). Figure 5 The electrostatic interaction contributions of all ionizable amino acids from C3d (A) and CR2 (B). Figure 6 Domain cross-correlation map (DCCM) averaged by three parallel trajectories with Cα atom pairs of C3d-CR2 complex at different ion strengths: (A) Ionic strengths corresponding to 50 mM; (B) Ionic strengths corresponding to 100 mM; (C) Ionic strengths corresponding to 150 mM; (D) The three correlated regions inside C3d are colored in white, blue and 33

ACCEPTED MANUSCRIPT yellow; (E) The strong correlated regions between linker and several loops inside CR2 are colored in red and purple; (F) The strong anti-correlated

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regions between C3d and CR2 are colored in blue and purple, respectively.

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Figure 7 Clustering analysis of Pro130-Gln168 and Gly270-Pro294 from run1 at ionic strengths corresponding to 50 mM (green), 100 mM (cyan) and 150 mM (magenta): (A) The representative structures with the highest ratio of

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clusters at different ionic strengths; (B) The detailed interactions of antigen

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binding site; (C) The detailed interactions of Lys291-Pro294 of helix α12 with CR2.

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Figure 8 The distances between the center of mass of side chains of Ala17 and Gln20

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forming the thioester bond from run1 at different ion strengths.

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Figure 9 The detailed interactions and electrostatic potential distributions of the linker and Cys112-Asn116 of CR2 with the highest ratio of clusters from

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run1 at different ionic strengths: (A) The detailed interactions of the two regions at ionic strengths corresponding to 50 mM (green), 100 mM (cyan) and 150 mM (magenta); (B) Electrostatic potential distributions of the two regions at 50 mM ionic strengths; (C) Electrostatic potential distributions of the two regions at 100 mM ionic strengths; (D) Electrostatic potential distributions of the two regions at 150 mM ionic strengths. Isopotential surfaces are shown at ±1kT/e. Blue and red represent positive and negative electrostatic potentials, respectively. Figure 10

Ions occupancy map representing the distributions of sodium and 34

ACCEPTED MANUSCRIPT chloride ions around the CR2 binding site (A, C and E) and antigen binding site (B, D and F) of C3d is shown in solid surface from three parallel

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(C-D) and 150 mM (E-F), respectively.

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trajectiories at ionic strengths corresponding to 50 mM (A-B), 100 mM

Figure 11 The electrostatic potential distributions of CR2 binding site (A, D and G), antigen binding site (B, E and H) and CR2 (C, F and I) at ionic strengths

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corresponding to 50 mM (A-C), 100 mM (D-F) and 150 mM (G-I),

Figure 12

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respectively.

Water occupancy map representing the distribution of water around

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C3d-CR2 complex is shown in solid surface from three parallel trajectiories

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at ionic strengths corresponding to 50 mM (A), 100 mM (B) and 150 mM

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(C), respectively.

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Graphical abstract

48

ACCEPTED MANUSCRIPT Highlights •

The solvent occupancies and distributions at antigen-binding site are largely

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High ionic strengths can enhance the polar interactions of antigen-binding site

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•

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influenced by ionic strengths.

and leads to the deviation of Lys291-Pro294 from CR2-binding surface.

Lys291-Pro294 and 160s cluster can regulate the upper and lower regions of

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•

Ala17 and Gln20 will transform between the activated and non-activated states at

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different ion strengths mediated by His133 and Glu135.

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•

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V-shape structure of CR2.

49