http://informahealthcare.com/grf ISSN: 0897-7194 (print), 1029-2292 (electronic) Growth Factors, 2015; 33(1): 40–49 ! 2014 Informa UK Ltd. DOI: 10.3109/08977194.2014.964868

RESEARCH PAPER

Investigation of alanine mutations affecting insulin-like growth factor (IGF) I binding to IGF binding proteins Xin Chen, Danhui Duan, Shuyan Zhu, and Jinglai Zhang

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Institute of Environmental and Analytical Sciences, College of Chemistry and Chemical Engineering, Henan University, Kaifeng, Henan, P.R. China

Abstract

Keywords

Binding properties of wild type (WT) and six single amino acid substituted variants (E3A, E9A, D12A, D20A, F23A, and E58A) of insulin-like growth factor I (IGF-I) were analyzed with respect to their binding details to IGF binding proteins (IGFBPs) by molecular dynamics (MD) simulations. The binding sites and binding interactions on IGF-I and IGFBPs are screened and compared with the static X-ray structure. Electrostatic interaction is the primary driving force of the interaction between IGF-I and IGFBPs. Mutation may cause the rearrangement of binding sites, however, the unfolding of protein induced by mutation is not obvious in this work. We also provide the detailed picture of binding factors. And the results show that, whether the unfolding of helix occurs or not, the Ala mutation will change the molecular atmosphere of the binding interface by the rearrangement of conformation, and further affects the binding residues and binding interactions.

Binding factors, insulin like growth factor (IGF), molecular dynamics (MD) simulation, mutation effect, structural and conformational changes

Introduction The insulin-like growth factors (IGFs) are widely expressed polypeptides that promote cell proliferation and differentiation. Aberrant regulation of the IGF system is implicated in many diseases, such as diabetes, cancer, and growth disorders. IGF actions are finely regulated by a family of six highaffinity IGF binding proteins (IGFBP-1 to -6) (Bach et al., 2005; Carrick et al., 2005; Clemmons, 2001; Firth & Baxter, 2002; Guo et al., 2013; Hwa et al., 1999). These proteins not merely carry the IGFs and prolong the half-life of IGFs, but have influences on both the bioactivity and distribution of IGFs in the extracellular environment. For instance, IGFBPs may localize IGF molecules to the receptor-abundant cell surface and promote the subsequent release of IGFs (Firth & Baxter, 2002). Targeting the IGFs-IGFBPs interaction may therefore provide understanding of their biological functions and novel therapeutic opportunities for many common diseases. IGFBPs consist of three domains of approximately equal length, with highly conserved C- and N-terminal domains joined by a variable linker L-domain. The proposed IGF binding sites on the IGFBPs are believed to be located in the highly conserved C- and N-domains (Baxter, 2000; Carrick et al., 2002; Clemmons, 2001). The L-domain appears to

Correspondence: Xin Chen and Jinglai Zhang, Institute of Environmental and Analytical Sciences, College of Chemistry and Chemical Engineering, Henan University, Kaifeng, Henan 475001, P.R. China. E-mail: [email protected] (X. Chen) [email protected](J. Zhang)

History Received 6 May 2014 Revised 8 September 2014 Accepted 9 September 2014 Published online 26 September 2014

contribute significantly to the structural integrity and consequently the IGF binding potential of recombinant N-domain (Carrick et al., 2001; Hashimoto et al., 1997). Substantial deletion of the L-domain of IGFBP4 did not significantly lower its affinity for IGF (Qin et al., 1998). Thus it suggests that the L-domain dose not interact directly with IGF but plays a role in maintaining structural integrity. Threedimensional structures of C- and N-domains have been solved (Headey et al., 2004; Klaus et al., 1998;Z_eslawski et al., 2001). The L-domain is not conserved and is not directly involved in high-affinity IGF binding. Because of the high flexibility of L-domain, the whole three-dimensional structures of IGFBPs are still unknown. Despite the differences in IGF-binding affinities and specificities between the part and whole structures of IGFBPs, these structures of N- and C-domains are probably representative of all IGFBPs (Bach et al., 2005; Baxter, 2000; Imai et al., 2000). In this respect, it is noteworthy that the IGF binding properties on N- and C-domains of IGFBPs are studied. The IGF binding sites on C- and N-domains of IGFBPs, as well as the IGFBP binding regions on IGFs, have been mapped using various approaches (Clemmons, 2001; Headey et al., 2004; Horney et al., 2001; Shand et al., 2003; Skolnick et al., 2000; Yan et al., 2004). Alanine-scanning mutagenesis (ASM) is one of the most popular techniques to study the protein-protein interaction. ASM is also the popular method for mapping functional epitopes, as alanine substitutions remove side-chains atoms past the b-carbon without introducing additional conformational freedom (Buckway et al., 2001; Li & Zheng, 2011; Moreira et al., 2007). However, Jansson et al. (1997) has found that the altered affinities are resulted not only in the

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DOI: 10.3109/08977194.2014.964868

removal of specific interactions through amino acid substitution, but also in changing the global structure of protein. The reduced a-helix content in the IGF-I variant is assessed by far-UV circular dichroism spectral analysis. The mutation effect in the complex of IGFs and IGFBPs is still unclear. In our previous study, the structural evolution of IGF-I and IGFBP4 resulted by the opposite polarity mutagenesis was investigated (Chen et al., 2013b). Yet the Ala-substitution may reduce the contacts with binding proteins, moreover, it may break-up the hydrogen bonding (H-bond) and salt bridge (SB) interactions formed by the side chains of the replaced residue. Thus the unfolding of helix or sheet may appear around the mutation site. To get more insight into the molecular basis of the mutation effect in IGF variants, molecular dynamics (MD) simulations on a series of sitedirected ASM of IGF-I and IGFBPs are performed in this paper. Recently, MD simulation has been widely used to study protein-protein interactions (Barducci et al., 2013; Noskov et al., 2013; Zoete et al., 2005). It can provide structural details, dynamics behaviors, energy landscape and other properties that experiments either cannot provide or have difficulty offering. In this paper, we present the MD studies of the binding dynamics of the wild-type (WT) and the six single amino acid substituted variants of IGF-I on the surface of IGFBPs. Sitedirected ASM was performed on the key binding sites screened in WT structure. In addition to the analysis of the conformational and structural changes of the mutation sites, the relationship of the binding factors between IGF-I and IGFBPs was also mapped. The results are helpful to promote the understanding of the mutation effect and the interaction mechanism of IGF-I and IGFBPs at the atomic level.

Materials and methods System setup The interactions between IGF and IGFBP have been extensively studied during the past decades, yet there is still little structural information from crystallography spectroscopy or nuclear magnetic resonance (NMR) available for the complexes. The X-ray structure of IGF-I and IGFBPs is obtained from the protein data bank (PDB) and the PDB entry is 2DSQ (Sitar et al., 2006). It is a ‘‘hybrid’’ ternary complex of the C-terminal domain fragment of IGFBP1 (CBP1), N-terminal domain fragment of IGFBP4 (NBP4) and IGF-I. Figure 1 shows the tertiary structure of the complex. IGF-I is composed of three a-helices which are denoted as H1 (Ala8 – Cys18), H2 (Ile43 – Arg50) and H3 (Leu54 – Met59), respectively. IGF-I inserts into the pocket of CBP1 and NBP4 and binds on the IGFBPs surface with electrostatic and van der Waals (vdW) interactions. Especially, the H1 ‘‘thumb’’ inserts into the IGFBPs pocket and the other two helices locate at the top of the pocket. Then the complex was immersed in a rectangular period box of TIP3P water molecules (Jorgensen et al., 1983). The size of water box was set to be large enough to accommodate the structural changes of proteins during the MD simulations. And the ˚ 3. The volume of the water box is 73.65  71.10  70.12 A average distance left between the protein and the walls of ˚ . We added two sodium ions to neutralize the water box is 20 A

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Figure 1. Representation of the three-dimensional structure of IGF-I (green), C-terminal fragment of IGFBP1(CBP1, yellow) and N-terminal fragment of IGFBP4 (NBP4, blue) adapted from the X-ray structure. The mutation residues are shown with CPK model.

system. And the neutral pH was obtained by altering the protonation state of Lys and His. IGF-mutant systems were created by using the VMD package (Humphrey et al., 1996) based on the equilibrated WT system. For each mutant system, we performed energy minimization and three parallel 50 ns-MD simulations without any restraining potential applied to relax the complex structure. MD preparation In this study, all MD simulations were performed with NAMD version 2.7 (Kale et al., 1999) using Charmm27 force field (MacKerell et al., 1998). The production MD phase was ˚ for carried out with a time step of 2 fs. A cutoff of 12 A nonbonded interactions was used with switching vdW poten˚ with the SHAKE algorithm applied to tial beginning at 10 A bonds that involved hydrogen. We calculated the long-range electrostatic interactions with the Particle Mesh Ewald (PME) summation scheme. And we used periodic boundary conditions with the isothermal-isobaric ensemble (NpT) at 1 atmosphere and 310 K. Energy minimization optimized the geometry of the protein molecules and MD simulation equilibrated each system for three parallel 50 ns. Analytical methods Time evolutions of root mean square deviation (RMSD), potential energy, solvent accessible surface areas (SASA)

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(Chen et al., 2013a, 2013b; Chiu et al., 2008), and the H-bond number were analyzed. SASA is calculated with the probe ˚ . The time-dependent interface area (Ainter ), radius of 1.4 A interaction energy (Einter ), for the systems investigated is defined as follows: 1 Ainter ¼ ðSASAIGFI þ SASAIGFBPs  SASAIGFIþIGFBPs Þ ð1Þ 2

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Einter ¼ PIGFIþIGFBPs  PIGFI  PIGFBPs

ð2Þ

where SASAIGFBPs is the molecular surface area for IGFBPs, SASAIGFI is the corresponding quantity for IGF-I, and SASAIGFIþIGFBPs is the surface area of the IGFBPs-IGF-I complex. And where Einter is the total interaction energy between IGFBPs and IGF-I, and PIGFBPs, PIGF-I, PIGF-I+IGFBPs are the potential energy of IGFBPs, IGF-I and the complex, respectively (Chen et al., 2008; Shen et al., 2008). Here the H-bond is defined by the following criterion: given a heteroatom A attached to an H-atom and another heteroatom B not bonded to A, an H-bond is formed only if ˚ the distance between two heavy atoms is smaller than 3.5 A  and the A-H-B angle is smaller than 30 . For the salt-bridge (SB) interaction, the distance from the anionic carboxylate (RCOO) of either aspartic acid or glutamic acid and the cationic ammonium (RNHþ 3 ) from lysine or the guanidinium (RNHCðNH2Þþ ) of arginine is defined. A distance cutoff of 2 ˚ is used as a discrimination criterion for the presence of 5A salt-bridge.

Results and discussion Equilibration for WT complex The crystal structure provides very valuable information for understanding the molecular basis of IGF-I binding to IGFBPs. However, being an X-ray structure that is averaged over many copies in the crystal and over a long time, a static protein structure dose not easily show dynamics effects caused by thermal motions. Three parallel 50 ns-MD

Figure 2. (a) The root mean square deviation (RMSD) and (b) Potential energy of wildtype (WT) system with respect to molecular dynamics (MD) simulation time.

simulations were performed on the initial X-ray structure of IGF-I, CBP1 and NBP4 to relax and equilibrate the complex. In Figure 2(a), we show RMSD traces for the backbone atoms of IGF-I and IGFBPs during the three 50 ns-long MD simulations. In general, these data indicate that the IGF-I and IGFBPs complex is preserved during MD relaxation given ˚ . The their modest RMSD value between 0.5 and 3.5 A proteins seem well equilibrated after 10 ns for all the three MD runs. We further show potential energy of the complex proteins during the MD processes in Figure 2(b). The potential energy in Run 1 is gradually decreased from about 1158 to 1490 kcal mol1 and keeps stable around around 1345 kcal mol1 after 25 ns. All the three curves converged, which shows the equilibration of the proteins. Comparison of equilibration and X-ray structures Table 1 shows the electrostatic interaction type of binding couples, the occupancy of binding couples in three MD simulations, and the distance of the binding couples in the equilibration states. Three parallel MD runs produce different binding states of IGF-I to IGFBPs. Glu3 and Asp12 are active in the first and third MD runs. Glu9, Asp20, Phe23, Arg55 and Glu58 work in all the three MD runs. Yet the binding of Asp45 to the IGFBPs only appears in the second MD run. The cationic ammonium (RNHþ 3 ) from Lys and the guanidinium (RNHCðNH2Þþ ) of Arg interact with the anionic carboxylate 2 (RCOO) of Asp and Glu to form multiple SB interactions at the same time. The multiple electrostatic interactions between IGF-I and IGFBPs usually possess longer lifetime. For example, the occupancies of the SB interactions formed between Glu3 and Lys67 are more than 70%. The same results also occur in the SBs of Asp12:Arg221, Asp20:Arg28, Asp20:Arg52, and Glu58:Arg58. It is also found that the SB interactions formed between same residues are various in different MD runs. There are eight possible binding couples between Glu9 and Arg190. Yet the numbers of binding couples of the two residues in the three MD runs are 4, 8 and

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Table 1. Binding details of wild-type (WT) IGF-I and IGFBPs. WT Binding couples IGF-I Glu3-OE1 Glu3-OE2 Thr4-O Gly7-HN Gly7-O Glu9-OE1

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Glu9-OE2

Asp12-OD1 Asp12-OD2 Asp20-OD1

Asp20-OD2

Phe23-O Phe23-HN Asp45-OD1 Arg55-HH12 Arg55-HH21 Glu58-OE2 Glu58-OE1

IGFBPs Lys67-HZ2(NBP4) Lys67-HZ1(NBP4) Lys67-HZ2(NBP4) Lys67-HZ3(NBP4) Hse70-HE2(NBP4) Pro179-O(CBP1) Asn180-OD1(CBP1) Leu178-HN(CBP1) Arg190-HH21(CBP1) Arg190-HH22(CBP1) Arg190-HH11(CBP1) Arg190-HH12(CBP1) Arg190-HH21(CBP1) Arg190-HH22(CBP1) Asn180-HD21(CBP1) Arg190-HH11(CBP1) Arg190-HH12(CBP1) Arg221-HH21(CBP1) Arg221-HH22(CBP1) Arg221-HH21(CBP1) Arg221-HH22(CBP1) Arg28-HH11(NBP4) Arg28-HH12(NBP4) Arg28-HH21(NBP4) Arg28-HH22(NBP4) Arg52-HH11(NBP4) Arg52-HH12(NBP4) Arg52-HH21(NBP4) Arg52-HH22(NBP4) Arg28-HH11(NBP4) Arg28-HH12(NBP4) Arg28-HH21(NBP4) Arg28-HH22(NBP4) Arg52-HH11(NBP4) Arg52-HH12(NBP4) Arg52-HH21(NBP4) Arg52-HH22(NBP4) Ile4-HN(NBP4) Ile4-O(NBP4) Arg152-HH21(CBP1) Arg152-HH22(CBP1) Gln89-OE1(NBP4) Gln89-OE1(NBP4) Arg58-HH11(NBP4) Arg58-HH21(NBP4) Gln89-HE21(NBP4) Arg58-HH11(NBP4) Arg58-HH12(NBP4) Gln89-HE21(NBP4) Gln89-HE22(NBP4)

Equilibration state Occupancy (%) 74.91(1); 80.56(1); 76.11(1); 78.76(1); 15.19(1) 32.38(1) 37.11(2) 26.45(3) 77.66(1); 90.35(1); 88.10(2); 66.44(2); 72.26(1); 89.71(1); 5.15(1) 92.10(2); 87.65(2); 44.63(1); 44.83(1); 42.83(1); 19.39(1); 86.21(1); 84.81(1); 84.51(1); 81.06(1) 90.17(3) 86.29(3) 89.77(3) 83.67(3) 86.71(1); 86.81(1); 98.10(1) 84.01(1) 88.56(3) 80.27(3) 90.00(3) 86.57(3) 100(1); 100(1); 38.97(2) 20.12(2) 21.69(1); 30.11(2) 90.30(1); 94.15(1); 57.34(3) 90.43(2) 88.88(2) 93.67(3) 89.97(3)

80.03(3) 87.06(3) 76.98(3) 79.87(3)

89.17(2) 75.21(2) 79.88(3) 73.28(3) 90.12(2); 79.46(3) 80.45(2) 89.14(3) 90.20(3) 58.19(3) 60.47(3) 45.15(3) 46.57(3) 79.30(2) 93.12(2) 82.73(2)

79.23(2) 85.37(2)

100(2); 100(3) 100(2); 100(3) 30.55(3) 94.01(2) 90.65(2); 93.20(3)

˚) Distance(A 3.75(1); 3.30(1); 1.64(1); 3.12(1); 2.35(1) 2.97(1) 2.21(2) 3.43(3) 3.70(1); 3.53(1); 2.75(2); 4.30(2); 4.76(1); 3.72(1); 3.26(1) 1.52(2); 3.18(2); 3.32(1); 3.29(1); 3.67(1); 4.49(1); 2.75(1); 4.42(1); 1.77(1); 3.27(1) 1.75(3) 3.28(3) 2.65(3) 3.31(3) 3.07(1); 4.40(1); 2.92(1) 4.13(1) 2.65(3) 3.31(3) 1.66(3) 3.26(3) 2.13(1); 1.94(1); 2.33(2) 3.97(2) 3.49(1); 3.32(2) 3.38(1); 3.91(1); 2.99(3) 2.54(2) 4.21(2) 1.82(3) 3.44(3)

3.26(3) 2.47(3) 1.96(3) 2.55(3)

1.73(2) 3.15(2) 3.50(3) 3.71(3) 2.56(2); 3.58(3) 4.21(2) 1.90(3) 3.04(3) 2.97(3) 2.36(3) 4.78(3) 3.99(3) 3.26(2) 1.68(2) 4.98(2)

4.58(2) 2.93(2)

3.49(2); 1.82(3) 3.15(2); 2.12(3) 3.42(3) 1.69(2) 4.51(2); 2.87(3)

Binding type SB SB SB SB H-bond H-bond H-bond H-bond SB SB SB SB SB SB H-bond SB SB SB SB SB SB SB SB SB SB SB SB SB SB SB SB SB SB SB SB SB SB H-bond H-bond SB SB H-bond H-bond SB SB H-bond SB SB H-bond H-bond

H-bond and SB represent hydrogen bond and salt bridge interactions, respectively. The N-terminal of IGFBP4 and the C-terminal of IGFBP1 are denoted by NBP4 and CBP1, respectively. And the data in three molecular dynamics (MD) runs are distinguished by numbers 1, 2 and 3 in brackets.

5, respectively. At the same time, Glu9 also interact with Asn180 to stabilize the interaction network in the first MD run. Moreover, Arg28 and Arg52 interact with Asp20 alternatively in the three MD runs. The various binding forms enhance the binding affinity of IGF-I and IGFBPs. For IGF-I, 10 electrostatic binding residues are screened in the MD simulations, and the carboxyl-rich residues play most important roles in the binding of IGF-I. More binding couples are found in NBP4 domain, which is consistent with the experiment results (Sitar et al., 2006; Siwanowicz et al., 2005). Among all the binding interactions listed in Table 1, three binding couples in NBP4 are also found in the static

crystal structure, and they are Ile4:C¼O to Phe23:NH, Ile4:NH to Phe23:C¼O, and Arg28:HH11 to Asp20:OD1. Interactions formed by Gly7, Glu9 and Asp12 appeared in the crystal structure are also active in the dynamics structure of the WT complex. However, the IGF-binding residues Ala8, Cys43 and Phe44 indicated in the X-ray structure are not active in the MD process. We further show per-residue RMSF of the proteins in the three MD runs in Figure 3. Moreover, we also compare the simulation-derived RMSF plots to the protein B-factors obtained from the X-ray structures. Our MD-generated RMSF of the structures of IGF-I and IGFBPs agrees

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reasonably well with the published X-ray data. Although there are differences among the three MD runs, the shapes of the three MD-curves are similar to the curve of B-factor. Selection of the mutation sites

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As the site-directed Ala mutation will change the side chain of the substituted residue, the residues with active side chain are considered as the preference. Then the residues with ˚ and the interaction occupancy binding distance within 5 A which is greater than 40% are selected. Six single amino acid substituted variants (E3A, E9A, D12A, D20A, F23A, and E58A) of IGF-I are investigated in this paper, and they are named as A1, A2, A3, A4, A5 and A6, respectively. Five acidic amino acids (3 Glu (E) and 2 Asp (D)) and one nonpolar amino acid (Phe (F)) are mutated by Ala. Especially, Phe23 is one of the most important binding sites to IGFBPs

Figure 3. Comparison of three molecular dynamics (MD)-generated root mean square fluctuation (RMSF) and experimental B-factor data.

Figure 4. The average root mean square deviation (RMSD) for mutants (a) A1, (b) A2, (c) A3, (d) A4, (e) A5 and (f) A6 with respect to the simulation time. The blue lines represent the mean RMSD values for each mutant in the three molecular dynamics (MD) runs. And the error bars represent the standard deviation.

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and IGFIR that mentioned in experimental studies (Beisel et al., 2001; Cascieri et al., 1988; Siwanowicz et al., 2005), which is also highlighted in the dynamics structure. Compared to the other five mutants, although the binding interaction of Phe23 is produced by the backbone atom, F23A is also introduced in this work to study the mutation effect. Equilibration for mutation systems MD simulations for 6 IGF-I mutants with IGFBPs are performed with 5000 steps energy minimization and 50 ns equilibration. Figure 4 shows average RMSDs of the six mutants in the three parallel MD runs during the production simulations. The preserved mean RMSD values of the six mutants show the equilibration of the systems. Distribution of the binding sites in mutants Table 2 and Tables S1–S5 show the binding details of the six mutant systems in the three MD runs, respectively. As shown in the tables, the site-directed mutation result in the rearrangement of the binding sites with both the elimination of original binding couples and the formation of new binding couples. In all the six mutants, the original binding sites are successfully eliminated by the ASM. Simultaneously, the mutation also causes more elimination of original binding sites and the formation of new binding sites. For example, The A1 mutant (E3A) cancelled the Glu3, Thr4 and Gly7 binding residues. Meanwhile, Gly19 and Gln15 work as new binding sites in A1 system. The substitution of Glu3 changed the binding atmosphere of IGF-I to IGFBPs, and dismisses the other two neighboring binding residues. The whole binding structure is also affected by the mutation, and the distribution of binding couples is rearranged. In A2 system, the binding of Thr4 on IGFBPs is broken by the E9A mutation. At the same time, Gln15 appears as a new binding site in the equilibrium state of IGF-I variant and IGFBPs. Similar rearrangements of

DOI: 10.3109/08977194.2014.964868

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Figure 5. The mean number of binding couples (a) and binding residues (b) in C-terminal of IGFBP1 (CBP1, red) and N-terminal of IGFBP4 (NBP4, blue) of wild-type (WT) and mutation systems.

the binding sites occur in A3 and A5 systems. However, D20A mutation does not bring more elimination of the original binding sites in A4 system. In other words, Asp20 is the only residue that is dismissed by the mutation. And Asn26 is the newly added binding site in A4 mutant. Especially, there is no new binding residues appear in A6 system. E58A mutation cancels the bindings of Thr4 and Glu58 with IGFBPs. Analyses of the elimination residues in all the six mutants show that the binding of Thr4 is most easily destroyed. And the binding of Gln15 is the primary newly added interaction in the IGF-I variants. We also find that the Glu3 can form SB interactions with Lys67 in NBP4 domain and Lys183 in CBP1 domain. Glu3 locates at the joint of NBP4 and CBP1 domains, and it is close to the two basic residues Lys67 and Lys83. Then the two residues can interact with Glu3 at the same time or alternatively. Similarly, Asp20 binds to IGFBPs with Arg28 and Arg52. The multiple binding sites between IGF-I and IGFBPs stabilize the binding affinity of the two proteins. Figure 5 shows the distribution of the binding couples on CBP1 and NBP4 of all the 7 systems. The average numbers of binding couples and binding residues are displayed, respectively. It is clearly shown in the graphs that the NBP4 possesses more binding sites than CBP1, which show the predominant binding ability of IGF-I on NBP4 domain. And this theoretical finding agrees well with the experiments. Mutation experiments have shown that the N-terminal hydrophobic pocket is the primary site of high affinity binding of IGF with IGFBPs (Bach et al., 2005; Buckway et al., 2001; Imai et al., 2000). The distribution of the binding sites also shows the difference and specificity of each mutation. For example, A2 mutation dismisses the binding of Glu9 (IGF-I) with Arg190 in CBP1 domain. Thus the number of binding couples in CBP1 is lessened. For A4 system, the bindings of Asp20 (IGF-I) with Arg28 and Arg52 in NBP4 domain are cancelled by the mutation. Then the number of binding sites of NBP4 in A4 system is less than that of WT system. Binding interactions in mutants To understand the binding interactions more deeply, we broke down the contributions to the interaction between IGF-I and

Figure 6. The total (black line), electrostatic (red line) and van der Waals (vdW, blue line) interaction energies of each mutation system during molecular dynamics (MD) simulations.

IGFBPs into the electrostatic and vdW parts. The electrostatic part represents the classic long-range columbic interaction, and the vdW part is the ‘‘contact energy’’. The two terms represent the major part of nonbonding interactions in MD simulations. Figure 6 shows the total, electrostatic and vdW interaction energies of IGF-I mutants and IGFBPs in one MD run. And the distributions of the interaction energies in the other two MD runs are shown in Figures S1 and S2. It is obvious that the total interaction energy (black line) and electrostatic interaction energy (red line) curves have similar trends and values in all the three MD simulations, which shows that the electrostatic interaction is the main driving force of the interaction between IGF-I and IGFBPs. The vdW energy (blue line) is much smaller than electrostatic energy. In A1 system, the total, electrostatic and vdW interaction energies at the final sub-stable states are 615.98, 497.46 and 118.50 kcal mol1, respectively. The contribution of the electrostatic interaction to the total in A1 is about 80%. H-bond and SB are the two main electrostatics interactions in the protein system. Table 3 shows the mean numbers of the two interactions formed by WT and mutated IGF-I with IGFBPs in the final equilibration state. The number of SB is larger than that of H-bond in all the systems.

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Structural and conformational changes of mutants

Table 2. Binding details of the A1 (E3A) mutation system.

Table 3 details the helix sequences in the WT and mutation systems in the final MD states. Unfolding of the helix appears in A1, A2, A4 and A6 mutants. The A2 and A4 mutants produce more unfolding cases of the helices. Moreover, the helices of WT IGF-I also unfold in two MD simulations. One H2-terminal and one H3-terminal are unfolded in the second and third MD runs, respectively. It shows that the unfolding of the helix terminal is common, and it may directly resulted by the higher flexibility of the terminal residues. In A1 mutant, one terminal of H3 is unwound during one 50 ns-MD simulation. The sequence of H3 changed from 54–60 to 54–58, and two terminal residues deviated from H3. However, the mutation site of E3A is far from the unfolding residues, and then we conclude that the unfolding may be mainly caused by the flexibility of helix terminus, but not by the mutation. Similar unfolding mechanism occurs in the A4 and A6 mutants. Surprisingly, in the A2 mutant, all the three helices unfold in one equilibration state. Except for H1, the other two helices are not close to the mutation site. And no direct evidence shows that the unfolding of H2 and H3 is caused by the mutation. However, the E9A mutation is directly involved in the unfolding of H1 in A2 mutant. To get more insight into the molecular basis of the mutation effect, Table 4 depicts the electrostatic interaction details nearby the mutation site. The elongation of the interaction distance is tagged in red, and the decreased ones are marked in blue. The E9A substitution ˚ increase of the interaction distance with introduces +0.13 A Gly7. The distance between Gly7 and Val11 also increases ˚ . Simultaneously, the H-bond distance from 2.75 to 3.44 A ˚ . Conversely, the of Ala8 and Asp12 increases by +0.33 A H-bond distance between Ala9 and Ala13 decreases. It discerns that the mutation effect of E9A is broken down two parts. One is the positive effect to stabilize the H1 from Ala9 to Ala13. And the other part is the negative effect to facilitate the unfolding of Gly7 and Ala8 from the helix. In addition to the mutation effect, the unfolding of H1 in A2 is also induced by the flexibility of the terminal residues.

A1 IGF-I

Relationship of the binding factors

Glu58-OE2

To get direct insight into the mutation effect, the binding factors listed in Table 3 are graphed in Figure 7. The interaction area curve (black line) is the most even one among all the five curves, which shows that the mutation do not have great impact on the contact area of IGF-I and IGFBPs. As depicted in Table 3, A3 possesses the largest Ainter of 16.23 nm2, which is 0.65 nm2 larger than that of WT system. The smallest Ainter of 14.87 nm2 appears in A2 mutant. Interestingly, the trends of the other four curves of interaction energy (green line), number of binding couples (red line), H-bond (blue line) and SB (cyan line) agree reasonably well with each other. The descending order of the interaction energy is same to the order of the number of electrostatic binding couples. This finding supports the conclusion that the electrostatic interaction is the primary driving force for IGF-I binding on IGFBPs. Especially, the H-bond curve maintains a relatively larger fluctuation than the other curves, which shows that the H-bond interaction is easy to be disturbed by

Gly7-HN Glu9-OE1

Glu9-OE2

Asp12-OD1 Asp12-OD2 Gln15-O Gln15-HE22 Gln15-HE21 Gly19-O Asp20-O Asp20-OD1

Asp20-OD2

Phe23-HN Phe23-O Asp45-OD1 Arg55-HH11 Arg55-HH12 Arg55-HE Glu58-OE1

Binding couples IGFBPs Pro179-O(CBP1) Arg190-HH11(CBP1) Arg190-HH12(CBP1) Arg190-HH21(CBP1) Arg190-HH22(CBP1) Arg190-HH11(CBP1) Arg190-HH12(CBP1) Arg190-HH21(CBP1) Arg190-HH22(CBP1) Arg221-HH21(CBP1) Arg221-HH22(CBP1) Arg221-HH22(CBP1) Tyr49-HH(NBP4) Gly31-HN(NBP4) Gly31-O(NBP4) Met196-O(CBP1) Hse5-HE2(NBP4) Hse5-HE2(NBP4) Arg28-HH11(NBP4) Arg28-HH12(NBP4) Arg28-HH21(NBP4) Arg28-HH22(NBP4) Arg52-HH11(NBP4) Arg52-HH12(NBP4) Arg52-HH21(NBP4) Arg52-HH22(NBP4) Arg28-HH11(NBP4) Arg28-HH12(NBP4) Arg28-HH21(NBP4) Arg28-HH22(NBP4) Arg52-HH11(NBP4) Arg52-HH12(NBP4) Arg52-HH21(NBP4) Arg52-HH22(NBP4) Ile4-O(NBP4) Ile4-HN(NBP4) Tyr156-HH(CBP1) Ile88-O(NBP4) Gln89-OE1(NBP4) Ile88-O(NBP4) Gln89-OE1(NBP4) Gln89-OE1(NBP4) Arg58-HH21(NBP4) Arg58-HH22(NBP4) Arg58-HH11(NBP4) Arg58-HH12(NBP4) Arg58-HH21(NBP4) Arg58-HH22(NBP4) Arg58-HH11(NBP4) Arg58-HH12(NBP4)

Equilibration state ˚) Distance (A 2.93(1);2.01(3) 1.81(1);3.40(2); 3.38(1);4.81(2); 2.99(1);2.39(2); 4.69(1);3.59(2); 2.56(1);1.88(2); 4.26(1);3.53(2) 1.75(1);2.01(2); 3.32(1);3.67(2); 4.33(2) 3.73(2) 4.53(2) 1.74(2);1.59(3) 3.47(1);4.45(2) 2.78(1) 2.31(2);2.27(3) 2.02(1);2.37(3) 3.27(1);3.97(2) 1.70(1);3.56(3) 3.01(1);1.84(3) 3.08(1);3.16(2) 4.62(1);1.70(2) 2.60(2) 4.25(2) 1.70(2) 3.33(2) 2.32(1);4.43(3) 4.00(1);2.79(3) 1.66(1) 3.34(1);3.81(2) 1.77(2) 3.27(2) 2.85(2) 4.54(2) 2.06(1);2.41(2); 3.21(1);2.46(2); 1.65(2) 2.34(1) 3.31(1);1.71(3) 3.04(1) 1.77(1);2.49(3) 2.45(1) 3.98(1);1.91(2); 4.76(1);3.56(2); 1.81(2);2.08(3) 3.44(2);3.72(3) 1.96(1);3.35(2); 3.20(1);4.47(2); 3.40(1);3.45(2); 4.90(1);4.72(2)

3.36(3) 4.92(3) 1.85(3) 4.49(3) 3.88(3) 1.84(3) 2.95(3)

2.02(3) 2.11(3)

1.97(3) 3.76(3)

2.85(3) 4.15(3) 4.07(3)

Binding type H-bond SB SB SB SB SB SB SB SB SB SB SB H-bond H-bond H-bond H-bond H-bond H-bond SB SB SB SB SB SB SB SB SB SB SB SB SB SB SB SB H-bond H-bond H-bond H-bond H-bond H-bond H-bond H-bond SB SB SB SB SB SB SB SB

H-bond and SB represent hydrogen bond and salt bridge interactions, respectively. The N-terminal of IGFBP4 and the C-terminal of IGFBP1 are denoted by NBP4 and CBP1, respectively. And the data in three molecular dynamics (MD) runs are distinguished by numbers 1, 2 and 3 in brackets.

the mutation. We also note that two peaks appear in A1 and A3 systems. Comparison of all the binding factors in Table 3, Table 2 and Table S2 shows that both the two mutations cause the large rearrangement of the binding sites, so that the Ainter , the numbers of H-bond and SB interactions increase, resulting in the increase of the interaction energies in these two mutation systems. Simultaneously, A2 and A4 are the two notable low points in the graph. Table 3 also discern that the unfolding of helix is readily to occur in the two systems. The site-directed

Investigation of alanine mutations on IGF-I

DOI: 10.3109/08977194.2014.964868

47

Table 3. Binding factors of wild-type (WT) and mutated IGF-I with IGFBPs. System 2

Ainter (nm ) H-bond Number SB Number Einter (kcal mol1) Helices sequence after MD in Run 1 Helices sequence after MD in Run 2 Helices sequence after MD in Run 3

WT

A1

A2

A3

A4

A5

A6

15.58 6 21 540.94 7–18 43–50 54–60 7–18 43–48 54–60 7–18 43–50 54–59

15.77 9 23 614.67 7–18 43–50 54–58 8–18 43–50 55–60 7–18 43–50 54–60

14.87 7 22 550.13 8–18 43–49 56–58 8–18 43–50 54–60 8–18 43–50 55–59

16.23 10 25 672.27 7–18 43–50 54–60 7–18 43–50 54–60 7–18 43–50 54–60

15.37 6 20 498.11 7–18 43–49 54–59 9–18 43–48 54–60 7–18 43–50 54–59

15.47 7 23 593.22 7–18 43–50 54–60 7–18 43–50 54–60 7–18 43–50 54–60

15.78 7 23 604.77 7–18 43–50 54–60 7–18 43–48 54–60 8–18 43–50 54–60

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The Ainter and Einter represent the mean interaction area and the interaction energy of IGF-I and IGFBPs in the three molecular dynamics (MD) runs, respectively. The H-bond number and SB number represent the average numbers of hydrogen bond and salt bridge interactions between IGF-I and IGFBPs. The sequences of the three a-helices at the equilibration state of the three MD simulations are also displayed. And the unfolding cases are highlighted with underlines.

Table 4. Interaction pairs nearby E9A in wild-type (WT) and A2 mutation systems. WT Gly7:O-Ala8:HN Ala8:O-Glu9:HN Glu9:O-Leu10:HN Gly7:O-Glu9:HN Gly7:O-Val11:HN Ala8:O-Asp12:HN Glu9:O-Ala13:HN

A2 3.20 3.14 3.15 3.42 2.75 2.07 2.11

Gly7:O-Ala8:HN Ala8:O-Ala9:HN Ala9:O-Leu0:HN Gly7:O-Ala 9:HN Gly7:O-Val11:HN Ala8:O-Asp12:HN Ala9:O-Ala13:HN

3.11 3.07 3.24 3.55 3.44 2.40 1.70

(0.09) (0.07) (+0.09) (+0.13) (+0.69) (+0.33) (0.41)

This is the results in one MD simulation.

mutations in these two points result in both the rearrangement of binding sites and the unfolding of local structure. Thus the contact areas of IGF-I and IGFBPs are decreased and the interaction energies are also reduced. Briefly, both the structural evolution and the rearrangement of the interaction network in the mutation systems produce the difference to the WT system. The analyses show that the Ala mutagenesis has both positive and negative effects on the interaction of IGF-I and IGFBPs. The single Ala substituted mutant can remove the original binding interactions at the mutation site. Moreover, it may also eliminate other binding sites. If newly added binding sites formed in the mutation system are not many, the interaction energy of IGF-I and IGFBPs will be decreased, such as A2, and A4. Conversely, the mutation may also increase the binding sites with the conformational changes, and further increase the interaction energy, such as A1 and A3. Some studies have also found that T4H in IGF-I resulted in a 7-fold and 4-fold increase in the affinity for insulin receptor A (IRA) and IRB, respectively (Shooter et al., 1996). And T31A mutation in IGF-I also resulted in a small but significant 2-fold increase in human placental insulin receptor binding (Bayne et al., 1990). Glu3, Glu9, Asp12 and Glu58 residues are also active in the binding experiments of IGF-I on IGFBPs, which is consistent with the findings in this paper. A charge reversal of E9K in IGF-I caused decrease in IGFBP-2 and -6 binding affinity of 140- and 30-fold, respectively (Magee et al., 1999).

Figure 7. The relative value of interaction area (black), interaction energy (green) of IGF-I and IGFBPs, number of IGF binding couples (red), H-bond (blue) and salt bridge (SB, cyan) interactions formed between IGF-I and IGFBPs with respect to all the systems. The value of wild-type (WT) system is used as a reference value.

The Ala mutation of residues Asp12, Gln15 and Phe16 yielded decreases in IGFBP-1 binding of 3- to 50-fold relative to WT IGF-I (Jansson et al., 1998). E3A IGF-I and F49A IGF-I exhibit an 8000-fold decrease in IGFBP-1 affinity but only a 15-fold decrease in IGFBP-3 affinity relative to WT IGF-I (Dubaquie et al., 2001). Some studies have also shown that mutations of IGF-I may preclude high affinity binding to IGFBPs (Bayne et al., 1988; Clemmons et al., 1990; Magee et al., 1999). However, the binding epitopes on the IGFs are different for IGFBPs. Then the binding results in this work provide a new perspective on the understanding of the binding mechanisms of IGF-I and hybrid IGFBPs.

Conclusions By performing all-atom MD simulations, we have investigated the atomic level structural variations for WT IGF-I and IGF-I with site-directed Ala mutations on IGFBPs to understand the mutation effect in the protein-protein complex.

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X. Chen et al.

IGF-I inserts deep into the pocket composed by NBP4 and CBP4. The electrostatic interaction constitutes the majority of the total interaction energy of IGF-I and IGFBPs. We discussed the key electrostatic binding residues and binding interactions on IGF-I and IGFBPs in this paper. Especially, the binding sites in dynamics structure were compared with the static X-ray data. Six typical binding residues were selected to perform the site-directed Ala mutations. It was found that Ala mutation had both positive and negative effects on the binding of IGF-I on IGFBPs. The results from the multiple simulations confirmed that the mutation would bring the rearrangement of the binding sites. However, it might not certainly bring the local unfolding of helix. In this work, most unfolding of helix was mainly resulted by the flexibility of terminus. The positive and negative mutation effects show that the biochemical or biological properties of Ala substituted IGF-I mutants cannot be used in a straightforward way to dissect the direct involvement in binding of individual residue since conformational and structural changes may be involved. Our simulations additionally provide a detailed picture of binding mechanisms of IGF-I and IGFBPs governed by multiple binding factors, such as the contact area, the numbers of binding couples, H-bond and SB interactions. These findings provide a perspective view to understand the mutation effect and the binding interactions in the dynamics structure of IGF-I and hybrid IGFBPs. It may provide more insight into the interface interaction mechanism of protein-protein system and be helpful for the structure based drug design. As IGFs have been implicated in the growth of various tumors, the details interaction of IGF on the surface of IGFBP obtained in this work may also be helpful in the development of molecular probes to assist in diagnostic screening.

Declaration of interest This work is financially supported by the National Natural Science Foundation of China (Grant No. 21003037 and No. 30900236) and the National Science Foundation of the Education Department of Henan Province (13A150085).

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Supplementary material available online Supplementary Figure S1–S2 and Table S1–S5