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Interaction between the antigen and antibody is controlled by the constant domains: Normal mode dynamics of the HEL–HyHEL-10 complex

¹ School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
² School of Science, Kitasato University, Kanagawa 228-8555, Japan

Reprint requests to: Hideaki Umeyama, School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan; e-mail: umeyamah{at}pharm.kitasato-u.ac.jp; fax: 81-3-3446-9553.

(RECEIVED March 30, 2003; FINAL REVISION June 13, 2003; ACCEPTED June 13, 2003)

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03100803.

	Abstract

TOP Abstract Introduction Results Discussion Materials and methods References

 
The antigen binding fragment (Fab) of a monoclonal antibody (HyHEL-10) consists of variable domains (Fv) and constant domains (CL–CH1). Normal modes have been calculated from the three-dimensional structures of hen egg lysozyme (HEL) with Fab, those of HEL with Fv, and so on. Only a small structural change was found between HEL–Fab and HEL–Fv complexes. However, HEL–Fv had a one order of magnitude lower dissociation constant than HEL–Fab. The C fluctuations of HEL–Fab differed from those of HEL–Fv with normal mode calculation, and the dynamics can be thought to be related to the protein–protein interactions. CL–CH1 may have influence not only around local interfaces between CL–CH1 and Fv, but also around the interacting regions between HEL and Fv, which are longitudinally distant. Eighteen water molecules were found in HEL–Fv around the interface between HEL and Fv compared with one water molecule in HEL–Fab. These solvent molecules may occupy the holes and channels, which may occur due to imperfect complementarity of the complex. Therefore, the suppression of atomic vibration around the interface between Fv and HEL can be thought to be related to favorable and compact interface formation by complete desolvation. It is suggested that the ability to control the antigen–antibody affinity is obtained from modifying the CL–CH1. The second upper loop in the constant domain of the light chain (UL2–CL), which is a conserved gene in several light chains, showed the most remarkable fluctuation changes. UL2–CL could play an important role and could be attractive for modification in protein engineering.

Keywords: Immunoglobulin; constant domain; protein interaction; normal mode analysis; affinity

Abbreviations: Fab, antigen binding fragment of immunoglobulin • Fv, variable domains of immunoglobulin • CL, constant domain of immunoglobulin light chain • CH1, first constant domain of immunoglobulin heavy chain • HEL, hen egg white lysozyme • VH, variable domain of immunoglobulin heavy chain • VL, variable domain of immunoglobulin light chain • CDR, complementary determining region • CDR1-VL, first CDR of the variable domain in the light chain • FR, lower loop as framework region • UL, upper loop of constant domain

	Introduction

TOP Abstract Introduction Results Discussion Materials and methods References

 
Over the past few decades, a considerable number of experimental studies, such as X-ray and NMR analyses, have been performed to acquire the three-dimensional coordinates of a biopolymer. The X-ray crystal structure of proteins includes very informative features statically; however, as far as the dynamics are concerned, more information will be required. Recently, computer simulation approaches have been performed to study those dynamics. There are two typical approaches that have been applied to study protein conformational dynamics: molecular dynamics calculation (MD) and normal mode analysis (NMA). MD is free from the harmonic approximation; however, it is limited to studying phenomena in the time range of 10^-9 sec or faster, and the larger the molecule, the more the time frame is limited.

NMA has been developed as another tool for the study of the dynamic behavior of biologic macromolecules (Levitt et al. 1985). The method includes some limitations, for instance, the harmonic approximation and conventional calculation in vacuo. Levitt et al. (1985) reported that the diffusing water molecules cannot be treated, as they do not move in a harmonic potential well, and that the presence of surrounding water would not be expected to have a major effect. Therefore, exclusion of water molecules in the calculation may not change the conclusion, and can avoid a cost of an additional six degrees of freedom per water molecule. NMA is still useful for understanding protein dynamics, especially for directly describing subtle fluctuations of rigid bodies (Soejima et al. 1999). Moreover, a more important usefulness of NMA is that it might functionally describe collective motions from the normal mode (Brooks and Karplus 1983; Go et al. 1983; Nishikawa and Go 1987; Hayward et al. 1995; Ishida et al. 1998; Jaaskelainen et al. 1998; Miller and Agard 1999; Nojima et al. 2002).

The three-dimensional structures of three monoclonal antibodies, D1.3, HyHEL-5, and HyHEL-10, have been studied during binding to different sites on the surface of hen egg white lysozyme (HEL). The antibodies bind to HEL specifically with six upper loops of variable domains (Fv), termed complementary determining regions (CDRs; Ward et al. 1989; Batra et al. 1990; Fishmann et al. 1991). In addition, the principles of the antigen–antibody binding were explored with mutagenesis around the antigen–antibody interface (Lavoie et al. 1992; Kam-Morgan et al. 1993; Tsumoto et al. 1994, 1996; Sundburg et al. 2000).

In the HyHEL-10 system, the crystal structure of HEL–Fv (PDB code: 1C08 [PDB] ), which is a complex of HEL and Fv, had no significant differences from that of HEL–Fab (PDB code: 3HFM [PDB] ), which is a complex of HEL and Fab. However, it was reported HEL–Fv had a one order of magnitude lower dissociation constant than HEL–Fab in the system: HEL–Fab and HEL–Fv had 450 x 10⁸ M^-1, 4.2 x 10⁸ M^-1, respectively (Smith-Gill et al. 1984; Lavoie et al. 1992; Kam-Morgan et al. 1993; Tsumoto et al. 1994). We focused on the dynamics of HEL–Fab and HEL–Fv, and attempted to explore the difference in the interaction between the antigen and the antibody.

Figure 1 shows the schematic structure of HEL–HyHEL-10 (Padlan et al. 1989). Fab is composed of the light and the heavy chains (L and H). The heavy chain cut between CH1 and CH2 has only one constant domain of CH1. Each chain folds into two domains: the variable and the constant domains. The variable domains (VL: residues 1–107, VH: residues 1–114) have four upper loops (CDRs and another) and three lower loops (FRs), respectively. The constant domains of each chain (CL: residues 108–214, CH1: residues 115–215) also have three upper (ULs) and three lower (no names) loops, respectively. It has been considered that six CDRs are placed between HEL and Fv for antigen binding. On the other hand, while six FRs and ULs in HEL–Fab are placed between Fv and the constant domain of CL–CH1, these functions are unclear.

View larger version (48K): [in this window] [in a new window]   Figure 1. Schematic structure of the HEL–HyHEL-10 complex (PDB code: 3HFM [PDB] ). HEL, red; VL, green; CL, violet; VH, yellow; CH1, cyan. FR1-L(8–17), CDR1-L(24–34), FR2-L(39–44), CDR2-L(50–56), FR3-L(76–83), CDR3-L(89–97), UL1-L(138–144), UL2-L(164–173), UL3-L(198–204), FR1-H(8–17), CDR1-H(31–35), FR2-H(40–44), CDR2-H(50–65), FR3-H(83–90), CDR3-H(95–102), UL1-H(147–152), UL2-H(166–174), UL3-H(197–206).

	Results

TOP Abstract Introduction Results Discussion Materials and methods References

In addition, 18 water molecules are found around the HEL–Fv interface of the HEL–Fv complex (1C08 [PDB] ), while only 1 is found around the interface of HEL–Fab (3HFM [PDB] ).

Fluctuation changes of HEL–Fab in the HyHEL-10 system due to Fab or HEL removal
Regarding all frequency modes, Figure 2 shows the fluctuation profiles of C atoms on Fv and HEL in HyHEL-10 complexes. On HEL removal from the complex, the fluctuation changes appeared mainly around all CDRs and another upper loop, whose residue number is around 70, in each chain as shown in Figure 2A. On the other hand, on Fab removal from the complex, the fluctuation changes appeared mainly around epitopes in HEL, as shown in Figure 2B. The above results indicated that the fluctuation changes observed in this study were in the interacting regions between HEL and Fab (Padlan 1977; Padlan et al. 1989; Kondo et al. 1999). Namely, our results show that NMA calculation without water molecules can provide the protein–protein interaction approximately. Hence, NMA may be useful for illustration of protein–protein interactions between the antigen and the antibody, and further suppression of atomic vibration may express stronger protein–protein interaction.

View larger version (39K): [in this window] [in a new window]   Figure 2. Fluctuation changes between HEL-Fab and HEL or Fab. (A) Fluctuation changes on Fv between Fab and HEL-Fab. (Red line) Mean fluctuation of six structures of Fab or HEL, (blue line) mean fluctuation of six structures of HEL-Fab, (green line) significance level of 5% by the Wilcoxon Rank Sum Test with six samples of each. (B) Fluctuation changes on HEL between HEL and HEL-Fab. (Thin line) Mean fluctuation of six structures of Fab or HEL, (broken line) mean fluctuation of six structures of HEL-Fab, (thick line) significance level of 5% by the Wilcoxon Rank Sum Test with six samples of each. (Gray circles) Epitopes of HEL (H15, G16, Y20, R21, W63, R73, L75, T89, N93, K96, K97, I98, S100, D101, G102).

View larger version (38K): [in this window] [in a new window]   Figure 3. Fluctuation changes between HEL-Fab and HEL-Fv due to CL-CH1 removal. (A) Fluctuation changes on Fv between two complexes. (Red line) Mean fluctuation of six structures of HEL-Fv, (blue line) mean fluctuation of six structures of HEL-Fab, (green line) significance level of 5% by the Wilcoxon Rank Sum Test with six samples of each. (B) Fluctuation changes on HEL between two complexes. (Thin line) Mean fluctuation of six structures of HEL-Fv, (broken line) mean fluctuation of six structures of HEL-Fab, (thick line) significance level of 5% by the Wilcoxon Rank Sum Test with six samples of each. (Gray circles) Epitopes of HEL (H15, G16, Y20, R21, W63, R73, L75, T89, N93, K96, K97, I98, S100, D101, G102).

View larger version (20K): [in this window] [in a new window]   Figure 4. Fluctuation changes between HEL-Fab and HEL-Fv with frequency modes. (Red line) Significance level of 5% with all frequency modes, (blue line) significance level of 5% under 50 cm-1.

View larger version (27K): [in this window] [in a new window]   Figure 5. Fluctuation changes on CL-CH1 between HEL-Fab and CL due to HEL-Fv removal. (Red line) Mean fluctuation of six structures of CL-CH1, (blue line) mean fluctuation of six structures of HEL-Fab, (green line) significance level of 5% by the Wilcoxon Rank Sum Test with six samples of each.

	Discussion

TOP Abstract Introduction Results Discussion Materials and methods References

Antigen–antibody binding is mainly thought to be a result of noncovalent interactions. It is compared to the relationship between "a key and a keyhole," and results in the almost complete desolvation of the interfaces in which hydrophobic clusters, hydrogen bonds, electrostatic interaction, and van der Waals interactions may contribute to the antigen–antibody induced fit (Chothia and Janin 1975; Bhat et al. 1994). An antibody recognizes the antigenic surface with the variable domains, and a favorable interface formation may be necessary for the antigen–antibody binding.

The parts of the schematic structure of HEL–Fv, which are influenced by the CL–CH1 domains, are illustrated in Figure 6.

View larger version (79K): [in this window] [in a new window]   Figure 6. Schematic structure of the parts of HEL-Fv that are influenced by the CL-CH1 domains. Green, orange, and red residues indicate those that are influenced by CL-CH1; green, A chain; yellow, B chain; red, C chain; small spacefill, residues between domains within 5 Å and interactions reported by Padlan et al. 1989 (N31A, Q53A, S91A, N92A, S31B, D32B, Y58B, R14C, G16C, D18C, R21C, R73C, L75C, T89C). (Large blue spacefill) 18 water molecules around the interfaces between HEL and Fv in HEL-Fv (1C08 [PDB] ), (large cyan spacefill) a water molecule around the interfaces between HEL and Fv in HEL-Fab (3HFM [PDB] ) superimposed on HEL-Fv (1C08 [PDB] ).

The RMSD results indicated that the three-dimensional structure of HEL-Fab is similar to that of HEL–Fv, although the domain superimpositions are different. Namely, there are tiny but significant conformational changes in the HEL–Fv complex. Considering the fluctuation differences around interfaces between HEL and Fv domains, the removal of constant domains from the HEL–Fab complex may lead to the insertion of water molecules because of the imperfect complementarity.

It was reported that eight water molecules may mediate the stabilization of interaction in the hydrogen bonds formation, and the buried surface area increase was 100 Ų at the interface in the HEL–Fv complex in comparison with HEL–Fab (Kondo et al. 1999). The increment of the buried surface area means that another induced fit may occur at the antigen–antibody interface compared with the HEL–Fab complex, and such exceptional conformational changes may bring about imperfect complementarity.

It is suggested that CL–CH1 may play important roles in the perfect antigen–antibody complementarity without some water molecules and the large suppression of induced local conformational changes at the antigen–antibody interface.

Kabat et al. compared the sequences of the constant domains of human and mouse immunoglobulin light chains, and predicted that the conserved residues of 160–175 may have unique functions (Kabat et al. 1975). The residues that Kabat et al. focused on correspond to UL2–CL in this study. UL2–CL shows considerable fluctuation changes on HEL–Fv removal, and it is naturally considered that UL2–CL is the most effective region in the CL–CH1 domains at the interface between HEL and Fv. Therefore, UL2–CL may play an important role for longitudinal HEL–Fv interactions.

In the HEL–HyHEL-10 system, we can reach the following conclusions.

First, fluctuation changes may explain antigen–antibody interactions, and the noncovalent interactions can be interpreted with the vibrational features. This is useful for illustrating the local interactions and distant interactions due to tiny dynamic changes.

Second, the constant domains of CL–CH1 may have important functions not only for supporting the variable domains as a framework, but also for conserving the favorable interface between HEL and Fv. Incomplete desolvation around the binding interfaces and excess induced local conformational changes may bring about affinity loss of the protein–protein interaction, and it may be compensated for by the energetically insufficient water-mediated hydrogen bonds formation.

Last, the second upper loop of the CL domain could play an important role in keeping the favorable binding interfaces as distant interactions.

These results have further implications. Regarding protein engineering, our results suggest that the antigen–antibody affinity could be controlled by CL–CH1 mutations without changing the antigen–antibody binding specificity, and, UL2–CL, especially, could be a most attractive region.

HEL–Fab and HEL–Fv complexes have an identical sequence of the Fv region and the RMSD around 0.6 with comparison between HEL–Fv moieties: The binding specificity may be maintained. On the other hand, it was reported that HEL–Fv had one order magnitude lower dissociation constant than HEL–Fab. In the other words, the affinity of HEL–Fv may differ without changing binding specificity, and could be controlled by constant domain mutation.

	Materials and methods

TOP Abstract Introduction Results Discussion Materials and methods References

 
The crystal structures of HEL–Fab and HEL–Fv in the HyHEL-10 system are available with pdb codes 3HFM [PDB] and 1C08 [PDB] , respectively, in the Brookhaven Protein Data Bank (PDB). Supposing that the coordinates of these are preserved after dissociation of the components, the original three-dimensional coordinates can be divided into Fab, Fv, CL–CH1, and so on, according to the situations.

First, energy minimization of the X-ray coordinates was performed with a slightly modified force field of AMBER, and NMA was done with torsion angles (Kamiya et al. 2003). We assumed that the complexes were in vacuo, and that the distance-dependent dielectric constant (r/Å) for electrostatic energy was maintained. The electrostatic potential and van del Waals potential were cut off at 9 Å and were smoothly switched to zero at 10 Å. A threshold of 0.04 kcal/mole Å for the maximal component of the gradients of atoms was used. Six energetically optimized structures on each were obtained under restriction conditions, and these were gradually relaxed near the experimental coordinates. The thermal fluctuations of the atoms were calculated under 300 K (Sumikawa et al. 1998; Soejima et al. 1999; Takeda-Shitaka et al. 1999). Eckart’s condition was applied to obtain the fluctuation of each structure (Eckart 1935). The significance level was calculated on couples of six optimized conformations by Wilcoxon’s rank sum test (Nojima et al. 2002). Refinements of the protein coordinates and applying the statistics may reduce the unexpected effects such as the difference of the X-ray resolution as much as possible.

	Acknowledgments

 
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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Adachi, M. Articles by Umeyama, H. Search for Related Content PubMed PubMed Citation Articles by Adachi, M. Articles by Umeyama, H. Social Bookmarking                   What's this?