Molecular modeling of the complex between Torpedo acetylcholine receptor and anti-MIR Fab198

Biochemical and Biophysical Research Communications 356 (2007) 569–575 www.elsevier.com/locate/ybbrc

Molecular modeling of the complex between Torpedo acetylcholine receptor and anti-MIR Fab198 Maria Konstantakaki a, Socrates J. Tzartos a

b,c

, Konstantinos Poulas

b,* ,

Elias Eliopoulos

a,*

Department of Agricultural Biotechnology, Agricultural University of Athens, 75 Iera Odos, Votanikos, GR11855 Athens, Greece b Department of Pharmacy, University of Patras, GR26504, Rio-Patras, Greece c Department of Biochemistry, Hellenic Pasteur Institute, 127 Vas. Sofias Avenue, GR11521, Athens, Greece Received 23 February 2007 Available online 9 March 2007

Abstract Myasthenia gravis is a neuromuscular disorder caused by an antibody-mediated autoimmune response to the muscle-type nicotinic acetylcholine receptor (AChR). The majority of monoclonal antibodies (mAbs) produced in rats immunized with intact AChR compete with each other for binding to an area of the a-subunit called the main immunogenic region (MIR). The availability of a complex between the AChR and Fab198 (Fab fragment of the anti-MIR mAb198) would help understand how the antigen and antibody interact and in designing improved antibody fragments that protect against the destructive activity of myasthenic antibodies. In the present study, ˚ resolution structure of the Torpedo AChR. In order we modeled the Torpedo AChR/Fab198 complex, based primarily on the recent 4 A to computationally dock the two structures, we used the ZDOCK software. The total accessible surface area change of the complex compared to those of experimentally determined antigen–antibody complexes indicates an intermediate size contact surface. CDRs H3 and L3 seem to contribute most to the binding, while L2 seems to contribute least. These data suggest mutagenesis experiments aimed at validating the model and improving the binding affinity of Fab198 for the AChR.  2007 Elsevier Inc. All rights reserved. Keywords: Nicotinic acetylcholine receptor; Automated docking; Antibody–antigen complex; Molecular modeling

The muscle nicotinic acetylcholine receptor (AChR) is a transmembrane glycoprotein with a molecular weight of 290 kDa. It consists of a ring of five homologous subunits in the stoichiometry a2bcd (adult form) and a2bed (embryonic form) and is found in post-synaptic membranes at the neuromuscular junction [1]. Each AChR subunit consists of an N-terminal extracellular domain (ECD) of about 210 residues, four transmembrane segments (M1–4), a

Abbreviations: AChBP, acetylcholine-binding protein; AChR, acetylcholine receptor; ASA, accessible surface area; CDR, complementarity determining region; ECD, extracellular domain; MIR, main immunogenic region; MG, myasthenia gravis; mAb, monoclonal antibody. * Corresponding authors. E-mail addresses: [email protected] (K. Poulas), [email protected] (E. Eliopoulos). 0006-291X/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.02.161

large cytoplasmic domain between M3 and M4, and a short extracellular C-terminal tail. Our knowledge of the three-dimensional structure of the AChR is incomplete, being mainly derived from electron microscopy studies on two-dimensional crystals of Torpedo electric organ AChR. Recently, the structure of the ˚ resolution was determined by elecTorpedo AChR at 4 A tron microscopy, allowing a more detailed description of the AChR [2]. Attempts to obtain high quality X-ray diffraction crystals have met with little success. However, our knowledge of its structure has been advanced by the determination of the crystal structures of molluscan acetylcholine-binding proteins (AChBPs) [3–5], which share structural similarities with the ECDs of the AChR subunits. The AChR plays an important role in the autoimmune disease myasthenia gravis (MG), which is caused by

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autoantibodies against AChRs located post-synaptically at the neuromuscular junction [6]. The majority of anti-AChR monoclonal antibodies (mAbs) derived from rats immunized with intact AChR and of serum antibodies in MG patients bind to a region on the a-subunit of the AChR named the main immunogenic region (MIR) [7]. A major epitope of the MIR has been found to include residues a67 76. The a67 76 sequence of Torpedo AChR is WNPADYGGIK, while that of human AChR is WNPDDYGGVK [8]. mAb198 is an anti-MIR mAb which binds to muscle AChR from various mammalian species and to AChR from Torpedo and Electrophorus electric organs. While intact mAb198 causes antigenic modulation (i.e. accelerated internalization and degradation rate) of AChRs in muscle cell cultures, the Fab or Fv fragment does not cause modulation, but inhibits the pathogenic modulatory activity of intact anti-AChR antibodies [9,10]. The crystal struc˚ resolution ture of Fab198 has been determined at 2.8 A [11], while the structure of the MIR decapeptide a67–76, both free and bound to the single chain Fv fragment of mAb198, has been studied by two-dimensional NMR spectroscopy [12]. The present study is an attempt to predict the threedimensional structure of the complex formed between Torpedo AChR and Fab198 in order to provide a structural basis for binding site analysis and the rational design of antibody fragments of improved affinity for use as efficient protectors of the AChR. It is well established that the antigen-binding site of antibodies is formed by six loops (complementarity determining regions—CDRs) supported on a structurally highly conserved b-sheet framework region. These CDRs adopt a limited number of canonical conformations [13], determined by their loop length and certain ‘‘structure determining’’ residues. Fab198 CDR-L2, CDR-H1, and CDR-H2 are consistent with the canonical structure classification approach. In our study, the antibody–antigen interactions were drawn by analogy with information about other antigen–antibody interactions, as deposited in the Benchmark database [14]. For the AChR-Fab198 complex, we assume a lock and key model of extensive complementarity between two rigid bodies.

Materials and methods The structures for free Torpedo AChR (pdb code: 2BG9) and Fab198 (pdb code: 1FN4) [2,11] were used. The control structures were taken from the same database. The pdb files were processed using the ZDOCK [15] blocking function. Fab198 CDRs and framework residues, both shown by site-directed mutagenesis to be involved in determining the binding affinity of humanized scFv198 for Torpedo AChR [16], were allowed to interact with the AChR. Docking of Fab198 to the Torpedo AChR was performed using the automated initial stage docking algorithm implemented by ZDOCK [15]. The (a)b (the a-subunit next to b) and (a)c (the a-subunit next to c) interfaces of the AChR subunits were both tested. ZDOCK identifies near-native complexes using a scoring function The resulting decoys were ranked using the ZDOCK scoring

function, based on pairwise shape complementarity, desolvation, and electrostatics. To facilitate comparisons with available experimental data, we used the Benchmark database [14], which contains the experimentally determined complexes. Reference complexes were selected by their similarity in length to Fab198 CDR-H3 (15 amino acids). Since no X-ray structure is available for an antibody-antigen complex with a 15 amino acid CDR-H3, we selected four complexes: (a) vascular endothelial growth factor (pdb code:1BJ1) complexed with the murine neutralizing antibody, A.4.6.1, with a 14 amino acid CDR-H3 [17], (b) the influenza virus hemagglutinin Thr31Ile mutant complexed with a neutralizing antibody (pdb code: 2VIS), with a 14 amino acid CDR-H3 [18], (c) the human factor IX Gla domain complexed with the inhibitory antibody, 10C12 (pdb code: 1NL0), with an 11 amino acid CDR-H3 [19], and (d) influenza virus N9 neuraminidase complexed with NC41 Fab (pdb code: 1NCA), with an 11 amino acid CDR-H3 [20]. QUANTA-CHARMM was used for structure refinement [21,22]. Specifically, the protein forcefield for CHARMM was used with a steepest descend energy minimization algorithm. Further analysis of the selected complexes was performed by calculating the accessible surface area (ASA) change. The total ASA was calculated for the unbound structures and for the complex using the DSSP server (http://swift.cmbi.ru.nl/gv/dssp) [23]. The ASA change in the complex was calculated by subtracting the total ASA of the complex from the sum of the ASAs of the unbound structures. The ASA change of the interacting residues in the complex was calculated using the protein–protein interaction server [24], available at http:// www.biochem.ucl.ac.uk/bsm/PP/server. This was followed by examina˚ , using VMD [25]. tion of interface interactions within a range of 4 A Superimposition of Fab198-(a)b subunit complex on the Fab198-(a)c subunit complex or 1F3R.pdb was performed using SPDBviewer [26] or QUANTA, respectively. The contribution of the interacting residues to the stability of the complex was analyzed by computational alanine scanning mutagenesis, using ROBETTA software [27]. The ClusPro server [28] was used as a different scoring method for ranking the complexes by clustering analysis ˚. of the ZDOCK output, with the clustering radius set at 5 A

Results and discussion Docking with Fab198 was performed on both the (a)b and (a)c AChR subunits to obtain all putative complexes. The ZDOCK program gives a ranking of the best complexes for the top ten predictions. In order to perform exhaustive sampling of Fab198 rigid body rotations with respect to the AChR, we used the dense rotational sampling function. We visually inspected 100 complexes for each of the (a)b and (a)c interfaces and selected those with the Fab198 CDRs oriented towards the receptor. From these complexes, we selected those with the highest total ASA change and MIR buried surface area and eliminated those in which there were clashes between Fab198 and any AChR subunit. We obtained one optimal solution for Fab198 interacting with either subunit (a)b (Fig. 1A and B) or subunit (a)c (data not shown). The two models, when superimposed, follow the same heavy to light chain orientation and are closely matched. As a result only data for the Fab198-(a)b complex is reported here. These two Fab198-(a)b and Fab198-(a)c subunit complexes were analyzed by computational techniques and by comparison with experimentally determined complexes. Initially, we analyzed the total ASA change and the ASA change of the MIR epitope and the CDRs. Amino acid res-

M. Konstantakaki et al. / Biochemical and Biophysical Research Communications 356 (2007) 569–575

571

Fig. 1. The Torpedo AChR-Fab198 complex. (A) Torpedo AChR (a)b and b-subunit dimer: ribbon representation of the AChR a-subunit (pink) and b-subunit (wheat) and sphere representation of the MIR (red). (B) Spacefilling view of the Fab198-AChR complex model. AChR a-subunit (pink), AChR b, c, d subunits (wheat), MIR (red), Fab198 variable light chain (cyan), and variable heavy chain (blue). (C) Stereo view of the CDRs contributing most to the binding of Fab198 to the MIR. The AChR a-subunit is indicated in pink and the MIR in red. CDR-L3 is shown in dark green, CDR-H2 in cyan, and CDR-H3 in dark blue. (For interpretation of the references in color in this figure legend, the reader is referred to the web version of this article.)

Fab198 heavy chain-( ) subunit complex

ASA c hange for Light c hain

140 120 100 80 60

FW

40

FW

20 0 11

21

31

41

51

61

100

CDR-L3

CDR-L1

80 FW

CDR-L2

60 40 20 1

D

11

21

31

41

51

60

30 20 10

80 70 50 40

W67

(67-76)

81 91 101 Fab198 Light chain

ix

N68

MI

71

60

Y72 P69

ix

61

Complex Fab198-( ) subunit vs Fab198 light chain 90

s ubunit

s ubunit ASA c hange

50 40

120

71 81 91 101 Fab198 Heavy chain

Complex Fab198-( ) subunit vs Fab198 heavy chain 90 80 70

140

0 1

C

Fab198 light chain-( ) subunit complex

B

ASA c hange

ASA c ha n g e fo r H e a v y c h a in

A

30 20 10 0

0 1

11

21

31

41

51

61

71

81

91

101 111

1

11

21

31

41

51

61

71

81

91

101 111

MI

Fig. 2. ASA change for Fab198-(a)b subunit complex. The plots indicate the ASA change of the Fab198 antibody residues (A,B) and the AChR ASA change of the receptor residues on formation of the Fab198-(a)b subunit complex (C,D). The Fab198 ASA change is calculated separately for heavy chain (A, CDR-H1: 49.2, CDR-H2: 193.8, CDR-H3: 312, Total heavy chain CDR ASA change: 555) and for light chain (B, CDR-L1: 134.5, CDR-L2: 24.9, CDR-L3: 210.5. Total light chain CDR ASA change: 370), respectively. On the AChR ASA change plots (C,D) two main interacting regions are indicated, the MIR and the a1-helix, which is an N-terminal region known to be structurally important. Numbers indicate the ASA change between the unbound ˚ 2. molecules and Fab198-(a)b subunit complex in A

idues participating significantly in the binding interfaces were identified and their contribution to binding examined (Fig. 2).

The interfaces for the two proposed models consist of three protein chain segments (Fig. 1B), each of which include more than twenty interacting residues from both

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Table 1 Interface hydrogen bonds in the Fab198-(a)b subunit complex Fab198 (1FN4) CDR L1 Light chain framework CDR L3 Heavy chain framework CDR H2 CDR H3

Torpedo AChR (2BG9)

Distance ˚) (A

Tyr95 (OH) Arg50 (NH1)

Arg66 (NH2) Asn10 (ND2) His3 (NE2) Arg6 (NE) Trp67 (O) Tyr72 (OH)

2.5 3.5 3.3 3.7 3.6 4.9

Tyr53 (OH) Tyr97 (O) Tyr97 (OH)

Gly74 (O) Asn68 (OD1) Asp71 (OD2)

2.7 2.4 4.0

Asn30 (OD1) Tyr32 (OH) Tyr49 (OH)

The residue and atom pairs participating in the hydrogen bond are indicated.

a-subunit and Fab198 (Fig. 1C). The interfaces were first studied and validated in detail by identifying hydrophobic patches and by calculating potential hydrogen bonds between interacting residues. The number of potential hydrogen bonds at the complex interface was nine for the Fab198-(a)b subunit complex (Table 1). In both models, more than 10 interface contacts involved tyrosine residues. This is in agreement with previous data showing that a large number of aromatic residues, especially tyrosines, are found at antigen–antibody contact surfaces [18,20]. AChR-Fab198 hydrophobic patches In the present study, we identified two hydrophobic patch pairs formed at the interface and examined the amino acid composition of the patches, together with their size and shape in the complex. The first hydrophobic patch pair is formed between Fab198 CDR-H2 Trp47, CDR-H2 Trp52, CDR-H2 Tyr53, and CDR-H2 Tyr56, and the a-subunit AChR MIR amino acids, Tyr72, Gly73, Gly74, and Tyr112. The

two surfaces are roughly equal in size but, in the complex, the ASA change of the CDR amino acids is higher than that of the AChR amino acids (Table 2). The second patch pair is formed between the concave surface of Fab198 CDR-H3 Leu96, CDR-H3 Tyr97, CDR-H3 Gly98, CDR-L1 Tyr32, CDR-L3 Tyr91, and CDR-L3 Tyr95 and the convex surface of the AChR a-subunit MIR amino acids, Trp67, Pro69, Ala70, Tyr72, Gly73, Gly74, Tyr112, and Thr113. The Fab198 hydrophobic patch surface is larger than the complementary patch on the AChR a-subunit. Tyrosine is the most common amino acid in both patch pairs and the sum of the ASA changes for tyrosines is greater than that for any other amino acid (Table 2).

Fab198 ASA change on binding to Torpedo AChR The ASA change for each CDR is shown in Fig. 2A and B. For both proposed complexes, the total ASA change is ˚ 2, which is comparable to that for an higher than 750 A intermediate size-binding interface for antigen–antibody complexes. All six CDRs seem to make contacts with the AChR, but the total ASA change for the heavy chain CDRs is greater than that for light chain CDRs, indicating a greater contribution of the former to the binding. The order of the ASA change for each of the CDRs upon complex formation is H3 > L3 P H2 > L1 > H1 > L2. This is in agreement with the general notion that L2 is often not required for binding, while L3 and H3 are almost always involved [17,18], and with Kleinjung et al. [12], who suggested that the main interacting loops for scFv198 are H3, H2, and L3. The Fab198 amino acid residue ASA change for heavy and light chain variable domains in the Fab198-(a)b model complex, is shown in Fig. 2A and B. The Fab198 amino acid ASA change plots, for both the heavy and light antibody chain variable domains, show three peaks, as seen

Table 2 The ASA change of the two hydrophobic patch pairs formed between Fab198 and AChR in the Fab198-(a)b subunit complex ˚ 2) ˚ 2) Fab198 CDR ASA change (A AChR MIR ASA change (A PATCH 1

PATCH 2

CDR-H2

CDR-H3

CDR-L1 CDR-L3

Residue No. Trp47 Trp52 Tyr53 Tyr56 ˚ 2) Total CDR ASA change (A

3.9 83.3 57.8 38.1 183.1

Leu96 Tyr97 Gly98 Tyr32 Tyr91 Tyr95

74.1 190.0 36.9 71.3 30.9 84.1

˚ 2) Total CDR ASA change (A

487.3

Residue No. Tyr72 Gly73 Gly74 Tyr112 ˚ 2) Total AChR ASA change (A

77.3 39.1 29.0 15.1 160.5

Trp67 Pro69 Ala70 Tyr72 Gly73 Gly74 Tyr112 Thr113 ˚ 2) Total AChR ASA change (A

46.0 102.1 22.0 77.3 39.1 29.0 15.1 14.9 345.5

˚ 2. Numbers indicate the ASA change between the unbound molecules and Fab198-(a)b subunit complex in A

M. Konstantakaki et al. / Biochemical and Biophysical Research Communications 356 (2007) 569–575

in the ASA change plot for the heavy chain amino acids of the four experimentally determined complexes with a similar length CDR-H3 selected from the protein–protein Benchmark database (see Materials and methods). Overall, in the Fab198-(a)c subunit complex, an ASA change is seen for 11 of the 27 heavy chain CDR amino acid residues and 9 of the 26 light chain CDR residues. In the Fab198-(a)b subunit complex, an ASA change is seen in 14 heavy chain and 12 light chain CDR residues. Amino acid residues with a high ASA change in both complexes are CDR-H2 Trp52 ˚ 2), CDR-H3 Tyr97 (>180 A ˚ 2), and heavy chain (>80 A 2 ˚ framework residue Arg50 (>37 A ). Computational mutagenesis alanine scanning analysis of the two proposed Fab198-AChR complexes indicated that substitution of CDR-H3 Tyr97 with alanine is destabilizing for the binding interfaces. These residue positions are also involved in antibody–antigen interactions in the four experimentally determined complexes. MIR ASA change on binding to Fab198 The decapeptide a67–76 is thought to be the linear region of the larger discontinuous MIR epitope and has been suggested to be a major binding site for anti-AChR antibodies [7]. In the complexes proposed, the ASA changes indicate MIR involvement in complex formation, as shown in Figs. 1C and 2C and D. Residues a67–71 show a similar ASA change upon binding to the heavy or light chain. Of these, Asn68 shows the greatest ASA change, suggesting that this amino acid residue contributes to the Fab198–AChR interaction. This is in agreement with the antigenic characterization of the MIR performed by Papadouli et al. [29], which showed that Asn68 is indispensable for the binding of anti-MIR mAbs and that the shortest peptide capable of significant antibody binding is the pentapetide a67–71. Further, the ASA change for a72–76 is approximately 5· greater for heavy chain binding than for light chain binding, confirming that the heavy chain is more important for the binding of Fab198 to MIR region 72–76. Furthermore, according to Papadouli et al. [30], MIR residues Tyr72, Gly74, and Ile75 are important for the specificity of binding of mAbs, which is in agreement with our finding that the heavy chain variable region, especially the non-canonical CDR-H3, interacts strongly with MIR region a72–76. Computational mutagenesis suggests that substitution of Tyr72 by alanine is destabilizing for the internal energy of the system, while substitution of Ile75 has a neutral effect on the stability of the complex, indicating that these two amino acids could potentially participate in the binding with Fab198. Clustering analysis indicated that the top ranked complexes for both (a)b and (a)c subunits have the same orientation with the two Fab198-Torpedo AChR complexes ˚ 2 and three-peak ASA selected, with total ASA > 800 A change plots for Fab198 amino acid residues. For an accurate prediction of how anti-MIR Fab198 binds to the AChR, it might be useful to perform clustering analysis

573

after initial stage docking with a detailed potential algorithm, such as that used by the ClusPro server. Furthermore, this metascore clustering function could be useful for predicting the binding mode of other anti-MIR antibodies to the AChR.

Specific interactions between Fab198 CDRs and AChR residues Biomolecular association is thought to occur in two steps: (i) the encounter step, driven by non-specific, longrange, electrostatic forces and hydrophobic forces, followed by collision to form an intermediate complex and (ii) the recognition docking step, with the formation of specific interactions and non-covalent bonds [31]. Electrostatic interactions would therefore be expected to directly influence both the initial association and the strength of the docked complex, due to the formation of salt bridges and hydrogen bonds. It has been suggested that antibodies form high electrostatic interactions with their antigens [31]. This may explain the agreement between the complexes automatically generated using Cluspro on the ZDOCK output and the predicted complexes using forcefield calculations and ASA change analysis of the ZDOCK output. Although all 10 MIR amino acid residues exhibit ASA change, only 5 or 6 of them were calculated by alanine scanning to be either hotspot (that destabilize the complex) or neutral (that do not destabilize the complex but could cause partial binding energy loss) mutations for the complex formation free energy of, respectively, the Fab198-(a)c or Fab198-(a)b subunit complex. This implies that the interactions at the binding interface of the AChR and Fab198 could be improved to obtain energetically more favorable complexes. It would be feasible to obtain a more tightly bound complex by introducing mutations that will increase ASA change (amino acids with larger side chains, of appropriate length and type). Arg50 heavy chain framework residue of Fab198 is a suitable candidate to validate the selected models, since it has been shown to interact with the MIR by NMR studies [12], and to test its contribution to the binding affinity of Fab198 for the AChR. In the majority of the sampled decoys for the Fab198– AChR complex, residue CDR-L2 Asn50 is related to the orientation of the binding. Notably, this residue has a significant ASA change and is neutral for the stability of both model complexes. In the influenza virus N9 neuraminidase/ NC41 Fab complex, the corresponding residue participates in the binding interface by forming hydrogen bonds and Van der Waals contacts with the antigen. This residue in Fab198 seems to be one of the few CDR-L2 residues possibly contributing to the stability of the Fab198-AChR complex, and it would be interesting to examine how this residue is involved in stabilizing the complex. Mutagenesis experiments could be performed to investigate the stereo-

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chemical importance of this residue and how the binding orientation influences the stability of the complex. The interacting antigen and antibody surfaces are complementary both structurally, as in other known antigen– antibody interactions, and chemically, with polar residues from the Fab198 interacting with polar residues from the AChR. Many of the surface amino acids of the antibody at the interface are aromatic, and thus present large areas of hydrophobic surface to the antigen. In addition, some residues, such as CDR-H2 Tyr53 and CDR-H3 Tyr97, participate in hydrogen bonding with the antigen via hydroxyl groups. The contact surfaces of the antigen and antibody fit each other remarkably well, with a few small unoccupied spaces between the interacting molecules, such as the cavity formed by the CDR-L1 residues 30–32. In conclusion, the antibody-binding site appears as a relatively concave surface with small protuberances and depressions formed by the side chains of the CDRs of the variable heavy chain and light chain regions, as commonly observed for non-flat antigen–antibody surfaces. There is an intermediate size cleft between CDRs H3 and L3, possibly corresponding to the binding site. Although, this cleft seems to be the geometrical center of the antigen–antibody interface, all six CDRs interact with the antigen. CDRs H3, L3, H2, and H1 make more interactions than CDRs L1 and L2. The minor contribution of CDR-L2 is in agreement with the general notion that CDR-L2 contributes the least to antigen binding. The geometrical center of the interface, which seems to lie near CDR-H3, is occupied by the side chain of TYR97. Finally, it may be possible to improve the binding affinity of Fab198 for Torpedo AChR by increasing the charge pairing between interacting Fab198-AChR residues. This could be achieved by substituting Fab198 residues forming hydrogen bonds in the model complexes (Table 1) with residues able to form a salt bridge with AChR residues.

[5]

[6] [7]

[8]

[9]

[10]

[11]

[12]

[13]

[14] [15]

Acknowledgements M.K. thanks Dr. Antoine Taly and Dr. Brian Pierce for their help and suggestions in docking. Part of the work was supported by grants of the Muscular Dystrophy Association of USA and by Pythagoras EPEAEK programme, EU and Greek Ministry of Education.

[16]

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