The hydrophobic contacts between the center of the bI domain and the a1/a7 helices are crucial for the low-affinity state of integrin a4b7 Jie Liu1,*, Ting Fu2,*, Bo Peng1, Hao Sun1, HuiYing Chu2, GuoHui Li2 and JianFeng Chen1 1 State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China 2 Laboratory of Molecular Modeling and Design, State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, China
Keywords affinity; cell adhesion; hydrophobic contacts; integrin; molecular dynamic simulation Correspondence G. Li, Dalian Institute of Chemical Physics, 457 ZhongShan Road, Dalian 116023, China Fax: +86 0411 84675584 Tel: +86 0411 84379593 E-mail: [email protected]
J. Chen, Institute of Biochemistry and Cell Biology, 320 YueYang Road, Shanghai 200031, China Fax: +86 21 54921658 Tel: +86 21 54921142 E-mail: [email protected]
*These authors contributed equally to this work (Received 11 February 2014, revised 21 April 2014, accepted 1 May 2014)
Integrin a4b7 mediates both rolling and firm adhesion of lymphocytes by modulating its affinity to the ligand: mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Integrin activation is associated with allosteric reshaping in the b subunit I (bI) domain. A prominently conformational change comprises displacement of the a1 and a7 helices in the bI domain, suggesting that the location of these helices is important for the change in integrin affinity. In the present study, we report that the hydrophobic contacts between the center of the b7I domain and the a1/a7 helices play critical roles in keeping a4b7 in a low-affinity state. Using molecular dynamics simulation, we identified nine hydrophobic residues that might be involved in the critical hydrophobic contacts maintaining integrin in a low-affinity state. Integrin b7I domain exhibited a lower binding free energy for ligand after disrupting these hydrophobic contacts by substituting the hydrophobic residues with Ala. Moreover, these a4b7 mutants not only showed highaffinity binding to soluble MAdCAM-1, but also demonstrated firm cell adhesion to immobilized MAdCAM-1 in shear flow and enhanced the strength of the a4b7–MAdCAM-1 interaction. Disruption of the hydrophobic contacts also induced the active conformation of a4b7. Thus, the findings obtained in the present study reveal an important structural basis for the low-affinity state of integrin.
Introduction Integrins comprise a family of heterodimeric adhesion molecules that mediate cell–cell, cell–extracellular matrix and cell–pathogen interactions, as well as transmit signals bidirectionally across the plasma membrane. The biological functions of integrin depend on the dynamic regulation of the affinity of integrin [1–3]. A good example is integrin a4b7, a lymphocyte homing
receptor that has important roles with respect to lymphocytes homing to the intestine and gut-associated lymphoid tissues [4,5]. In the resting state, integrin a4b7 supports the rolling adhesion of lymphocytes via a low-affinity interaction with mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Upon activation, a4b7 binds to MAdCAM-1 with high affinity and
Abbreviations ADMIDAS, adjacent to MIDAS; MAdCAM-1, mucosal addressin cell adhesion molecule-1; MD, molecular dynamics; MIDAS, metal iondependent adhesion site; MM/GBSA, molecular mechanics/generalized born surface area; SyMBS, synergistic metal ion-binding site; WT, wild-type.
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mediates firm cell adhesion. The dynamic regulation of a4b7 affinity allows this integrin to mediate both the rolling and firm adhesion of lymphocytes, representing two critical steps during lymphocyte homing, which plays a critical role in gut immune homeostasis and the pathogenesis of intestinal inflammatory disorders . Integrin affinity can be regulated by either ‘inside– out’ signals from the cytoplasm or extracellular divalent cations via their binding to three metal ionbinding sites, the metal ion-dependent adhesion site (MIDAS), the adjacent to MIDAS (ADMIDAS) and the synergistic metal ion-binding site (SyMBS), in the bI domain [1,7–13]. Compared with the low-affinity state in Ca2+ and Mg2+, the removal of Ca2+ or the addition of Mn2+ markedly increases the ligand-binding affinity of most integrins [7,14]. Studies have revealed that integrin affinity regulation is closely associated with its global and local conformational changes. The crystal structures of integrins aVb3, aIIbb3, a4b7 and targeted molecular dynamic (MD) studies have indicated a series of conformational changes in the integrin headpiece during its transition from a low-affinity to a high-affinity state, most notably reshaping in the bI domain [15–18]. Upon activation, integrins adopt an open conformation through a swing out of the hybrid domain, which is accompanied by conformational changes within the bI domain that propagate through the a7 helix C-terminus. The C-terminal of the a7 helix moves axially toward the hybrid domain, causing the hybrid domain to swing out. In addition, the downward motion of the a7 helix leads to the breaking of contact interactions between the b6a7 loop and the a1 helix N-terminus, which results in a1 helix straightening and internal rearrangements of the specificity determining loop, followed by the movement of the b1-a1 loop towards the MIDAS, which is important for ligand binding [19–21]. The displacement of a1 and a7 helices also lead to the local rearrangements of MIDAS and ADMIDAS metal ion-binding sites, allowing them to adopt a geometry for highaffinity ligand binding . Notably, most integrins are not constitutively activated but exist in an inactive state under physiological conditions, which is very important for their normal biological functions . Integrin activation is associated with displacement of the a1 and a7 helices in the bI domain, suggesting the important roles of the a1 and a7 helices with respect to maintaining integrin in a low-affinity state, whereas the exact mechanism remains elusive. Based on the structural analysis of integrin a4b7 , we found that remarkable hydrophobic contacts are formed between the hydrophobic side chains at the inner face of the a1/a7 helices and the 2916
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hydrophobic residues from the opposite b sheets at the center of the bI domain. It is tempting to suggest that these hydrophobic contacts may restrain the flexibility of the a1 and a7 helices and keep integrin in low-affinity state. In the present study, we report that the hydrophobic contacts between the center of the b7I domain and the a1/a7 helices are crucial for stabilizing the low-affinity state of integrin a4b7. Using computational dynamics simulation, we identified nine hydrophobic residues that might be involved in the critical hydrophobic contacts maintaining integrin in a low-affinity state. Integrin a4b7, with Ala substitution of each of the nine hydrophobic residues to disrupt the hydrophobic contacts, showed high-affinity binding to soluble MAdCAM-1. In addition, these mutants mediated firm cell adhesion to immobilized MAdCAM-1 and enhanced the strength of the a4b7–MAdCAM-1 interaction compared to wild-type (WT) a4b7-mediated cell adhesion. Moreover, mutants in the a1 and a7 helices induced the active conformation of a4b7. The findings of the present study demonstrate that these hydrophobic contacts between the center of b7I domain and the a1/a7 helices are crucial for the low-affinity state of integrin a4b7.
Results Hydrophobic contacts between the center of the b7I domain and the a1/a7 helices in the lowaffinity state of integrin a4b7 Because the structure of the open headpiece of integrin a4b7 has not been solved, homology modeling was performed to construct the open b7I domain using the ORCHESTRAR module in SYBYLX1.1 (Tripos Associates, St Louis, MO, USA) with aIIbb3 crystal structure (Protein Data Bank code: 3FCU) as template (Fig. 1A). Compared with the structure of the closed b7I domain, the open b7I domain model structure showed downward motion of the a7 helix and straightening of the a1 helix similar to that observed in the aIIbb3 crystal structure. It is noteworthy that hydrophobic residues in the middle of a1 helix appeared to move inward in the open conformation and the hydrophobic residues at the inner face of the a7 helix showed downward displacement (Fig. 1B). In the closed conformation of the a4b7 headpiece, the side chains of hydrophobic residues at the inner face of a1 helix (Met145, Leu149, Val152, Leu159, Leu163) point towards the center of the b7I domain and are structurally contiguous to the hydrophobic side chains of Tyr137, Met139 and Ile172 in b1/b2 sheets at the b7I domain center (Fig. 1B, left). FEBS Journal 281 (2014) 2915–2926 ª 2014 FEBS
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Fig. 1. The hydrophobic contacts between the center of the b7I domain and the a1/a7 helices. (A) Superimposition of the closed (light blue) and open (wheat) structures of b7I domain. The striking feature of these two structures was the conformational change of the a1 and a7 helices. (B) Residues might involved in hydrophobic contacts formed by the a1 helix (left) and the a7 helix (right) with their opposite hydrophobic faces in the b7I domain. L155, V161, V360 and M364 were selected as a negative control. The selected amino acid residues are shown in stick form, carbon atoms are colored in light blue (closed) and wheat (open), nitrogen, oxygen and sulfur atoms are colored in blue, red and yellow, respectively. Images produced using PYMOL (http://www.pymol.org). (C) Sequence alignment of integrin b subunit I domain. The hydrophobic residues presented in (B) are shown in red.
Therefore, it is tempting to suggest that hydrophobic contacts are formed between the a1 helix and the center of the b7I domain. Similarly, hydrophobic contacts are also predicted to form between the hydrophobic residues Leu362, Ile363 and Leu370 at the inner face of the a7 helix and the hydrophobic residue (Ile326) in the opposite b5 sheet (Fig. 1B, right). In addition, these hydrophobic residues are conserved in all integrin b subunits (Fig. 1C) and the orientation and position of these residues are rearranged in the open conformation of integrin (Fig. 1B). Taken together, we hypothesized that these hydrophobic contacts might restrict the motion of the a1 and a7 helices to maintain the b7I domain in the closed conformation. Disruption of the hydrophobic contacts decreases b7-LDTS binding free energy To confirm our hypothesis, we first performed computational simulation of an Ala substitution of these hydrophobic residues to diminish the hydrophobic interactions. As a negative control, we also included Ala mutations of two hydrophobic residues (Leu155 and Val161) in the a1 helix and two hydrophobic residues (Val360 and Met364) in the a7 helix, which point to solvent and are unable to form hydrophobic con-
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tacts with the b7I domain center (Fig. 1B). The molecular mechanics/generalized born surface area (MM/ GBSA) method was applied to estimate the binding affinity of the LDTS peptide, a4b7-binding motif in MAdCAM-1 , to the b7I domain of WT or mutant integrin a4b7. Two thousand snapshots were taken from the last 10 ns of MD trajectories for analysis of the binding free energy (Table 1). The binding free energy was shown as the sums of molecular mechanics (DEele and DEvdw) and solvation energies (DGnp and DGele). Compared with WT a4b7, nine mutants (M139A, L149A, V152A, L159A, I172A, I326A, L362A, I363A and L370A) showed a lower binding free energy for LDTS, suggesting that these mutations increased the ligand-binding affinity of b7 integrin (Table 1). By contrast, mutants Y137A, M145A and L163A showed a higher binding free energy, suggesting the decreased ligand-binding affinity of b7 integrin. In addition, all of the negative control residue mutations (L155A, V161A, V360A and M364A) showed increased b7-LDTS binding free energy. Further analysis of the energy components of binding free energy revealed that the strength of ligand binding was mainly a result of the electrostatic energy in the gas phase (DEele), with a slight contribution from the van der waals interactions (DEvdw) and the nonpolar solvation 2917
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Table 1. The predicted binding free energies (neglecting the configurational entropy; kcalmol 1). The predicted binding affinity of LDTS peptide, a4b7-binding motif in MAdCAM-1, to the b7I domain of WT or mutant integrin a4b7 was computed by the MM/GBSA method. The 2000 snapshots were taken from the last 10 ns of MD trajectories for binding free energy prediction. The binding free energy can be approximately expressed as the sum of molecular mechanics (DEele: electrostatic energy in the gas phase; DEvdw: van der Waals energy) and solvation energies (DGnp: nonpolar solvation energy; DGele: polar solvation energy). The hydrophobic residue mutations that lead to lower binding free energy for LDTS peptide than WT a4b7 were shown in bold. Data are shown as the mean SD (n = 2000). P-values were calculated using a two-tailed Student’s t-test compared to WT. DEele WT Y137A M139A M145A L149A V152A L155A L159A V161A L163A I172A I326A V360A L362A I363A M364A L370A
159.94 203.19 187.99 181.22 159.61 129.12 112.26 121.61 137.17 161.91 130.06 218.38 132.83 167.43 169.98 196.14 121.49
22.67 22.70 14.70 19.56 19.71 13.33 21.84 14.67 22.41 24.66 14.77 30.36 25.91 22.20 34.59 29.08 14.62
14.97 20.97 17.86 11.72 22.56 21.73 18.93 22.30 16.10 19.55 20.14 25.37 13.58 21.39 21.12 19.21 20.71
3.55 4.07 3.99 4.20 3.85 3.27 4.48 3.20 3.60 4.35 3.77 4.17 3.89 4.03 3.88 3.71 3.41
3.53 4.17 4.21 3.83 4.24 3.87 3.92 3.82 3.65 3.92 3.72 5.13 3.78 4.30 4.13 4.08 3.80
energy (DGnp), although the strength was serious impaired by the polar solvation energy (DGele). Taken together, nine hydrophobic residues are predicted to form hydrophobic contacts linking the a1 and a7 helices to the center of the b7I domain, which is important for the low-affinity state of integrin. Disruption of the hydrophobic contacts decreases b7LDTS binding free energy, suggesting an increased ligand-binding affinity of integrin. Hydrophobic residue mutations in b7 enhance soluble MAdCAM-1 binding to a4b7 To confirm the above prediction, we mutated each of these hydrophobic residues in human b7 subunit to Ala, and then transiently co-expressed with human a4 subunit in 293T cells, which do not express endogenous a4b7. Cell surface expression of the mutant and WT a4b7 was determined via antibody FIB504 against b7 using immunofluorescence flow cytometry (Table 2). Of the b7 mutants, all expressed at a comparable level to WT b7, except for I326A expressing at approximately 60% of WT levels. To examine the effects of these mutations on the activation state of integrin a4b7, the binding of soluble MAdCAM-1–His protein complexed with Alexa Fluor 488-anti-His IgG to a4b7 293T transient transfectants was determined by immunofluorescence flow cytometry. Soluble MAdCAM-1 2918
0.22 0.26 0.26 0.51 0.24 0.18 0.39 0.19 0.30 0.41 0.18 0.19 0.20 0.22 0.25 0.23 0.22
153.09 203.66 182.59 180.22 158.03 125.56 118.00 119.23 136.29 162.18 127.26 222.47 137.29 161.82 167.15 198.01 117.36
21.99 20.28 13.35 16.99 19.21 12.50 22.20 13.75 21.00 23.29 13.69 27.77 22.62 21.29 32.89 26.99 13.31
25.34 24.68 27.46 16.55 28.37 29.16 17.11 28.50 20.63 23.19 26.66 26.41 12.90 31.30 28.08 21.42 28.64
3.51 5.08 3.46 5.24 4.08 3.22 4.49 3.30 3.42 4.85 3.51 4.64 4.41 4.10 3.97 3.46 3.43