To explore potential non-canonical disulfide linkages feasible in human IgG2 mAbs via molecular dynamics simulations of a model system, Hinge++.
Hinge++ is derived from the crystal structure of a full-length murine IgG2a antibody by replacing its core hinge region with human IgG2 hinge. Fv and CH3 domains were discarded to speed up calculations. Eight independent simulations, grouped in four sets, were performed. In the control set, disulfide bonding is identical to canonical human IgG2 mAb. Different numbers of disulfide bonds were broken in the remaining three sets.
Two Fabs move towards Fc asymmetrically repeatedly leading to spatial proximity of LC.Cys214 and HC.Cys128 residues in one Fab with Cys residues in the upper hinge region, which could initiate disulfide scrambling. Local dynamics place the eight hinge region Cys residues in a large number of proximal positions which could facilitate non-canonical inter- and intra- heavy chain disulfide linkages in the hinge region.
Consistent with experimental studies, our simulations indicate inter-chain disulfide linkages in human IgG2 mAbs are degenerate. Potential rational design strategies to devise hinge stabilized human IgG2 mAbs are gleaned.
This is a preview of subscription content, log in to check access.
Buy single article
Instant unlimited access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
fragment antigen binding
protein data bank
root mean squared deviation
Salfeld JG. Isotype selection in antibody engineering. Nat Biotechnol. 2007;25(12):1369–72.
Labrijn AF, Aalberse RC, Schuurman J. When binding is enough: nonactivating antibody formats. Curr Opin Immunol. 2008;20(4):479–85.
Winter G, Duncan A, Burton D. Altered antibodies. USA patent WO-88/07089. Sept. 22. 1988.
Jefferis R. Antibody therapeutics:isotype and glycoform selection. Expert Opin Biol Ther. 2007;7(9):1401–13.
Beck A, Reichert JM, Wurch T. 5th European antibody congress 2009: November 30–December 2, 2009, Geneva, Switzerland. mAbs. 2010; 2(2):108–28.
Labrijn AF, Buijsse AO, van den Bremer ETJ, Verwilligen AYW, Bleeker WK, Thorpe SJ, et al. Therapeutic IgG4 antibodies engage in Fab-arm exchange with endogenous human IgG4 in vivo. Nat Biotechnol. 2009;27(8):767–71.
Aalberse RC, Schuurman J. IgG4 breaking the rules. Immunology. 2002;105(1):9–19.
Dillon TM, Ricci MS, Vezina C, Flynn GC, Liu YD, Rehder DS, et al. Structural and functional characterization of disulfide isoforms of the human IgG2 subclass. J Biol Chem. 2008;283(23):16206–15.
Wypych J, Li M, Guo A, Zhang Z, Martinez T, Allen MJ, et al. Human IgG2 antibodies display disulfide-mediated structural Isoforms. J Biol Chem. 2008;283(23):16194–205.
Liu YD, Chen X. Enk JZ-v, Plant M, Dillon TM, Flynn GC. Human IgG2 antibody disulfide rearrangement in vivo. J Biol Chem. 2008;283(43):29266–72.
Martinez T, Guo A, Allen MJ, Han M, Pace D, Jones J, et al. Disulfide connectivity of human immunoglobulin G2 structural isoforms. Biochemistry. 2008;47(28):7496–508.
Zhang B, Harder AG, Connelly HM, Maheu LL, Cockrill SL. Determination of Fab-hinge disulfide connectivity in structural isoforms of a recombinant human immunoglobulin G2 antibody. Anal Chem. 2010;82(3):1090–9.
Yoo EM, Wims LA, Chan LA, Morrison SL. Human IgG2 can form covalent dimers. J Immunol. 2003;170(6):3134–8.
Pink JRL, Milstein C. Inter heavy-light chain disulphide bridge in immune globulins. Nature. 1967;214(5083):92–4.
Edelman GM, Cunningham BA, Gall WE, Gottlieb PD, Rutishauser U, Waxdal MJ. The covalent structure of an entire γG immunoglobulin molecule. Proc Natl Acad Sci U S A. 1969;63(1):78–85.
Milstein C, Frangione B. Disulphide bridges of the heavy chain of human immunoglobulin G2. Biochem J. 1971;121(2):217–25.
Frangione B, Milstein C. Variations in the S–S bridges of immunoglobins G: Interchain disulphide bridges of γG3 myeloma proteins. J Mol Biol. 1968;33(3):893–906.
Dillon TM, Bondarenko P, Wypych J, Allen M, Balland A, Ricci Margaret S, et al.; Homogeneous antibody populations. USA patent WO 2009/036209. March 19. 2009.
Allen MJ, Guo A, Martinez T, Han M, Flynn GC, Wypych J, et al. Interchain disulfide bonding in human IgG2 antibodies probed by site-directed mutagenesis. Biochemistry. 2009;48(17):3755–66.
Schmid N, Bolliger C, Smith LJ, van Gunsteren WF. Disulfide bond shuffling in bovine α-lactalbumin: MD simulation confirms experiment. Biochemistry. 2008;47(46):12104–7.
Allison JR, Moll G-P, van Gunsteren WF. Investigation of stability and disulfide bond shuffling of lipid transfer proteins by molecular dynamics simulation. Biochemistry. 2010;49(32):6916–27.
Harris LJ, Larson SB, Hasel KW, Day J, Greenwood A, McPherson A. The three-dimensional structure of an intact monoclonal antibody for canine lymphoma. Nature. 1992;360(6402):369–72.
Berman H, Henrick K, Nakamura H. Announcing the worldwide Protein Data Bank. Nat Struct Mol Biol. 2003;10(12):980–0.
Harris LJ, Larson SB, Hasel KW, McPherson A. Refined structure of an intact IgG2a monoclonal antibody. Biochemistry. 1997;36(7):1581–97.
Prabakaran P, Vu BK, Gan J, Feng Y, Dimitrov D, Ji X. Structure of an isolated unglycosylated antibody C(H)2 domain. Acta Crystallogr D Biol Crystallogr. 2008;64(10):1062–7.
Branden C, Tooze J. Introduction to protein structure: Garland Publishing; 1998.
Dorrington K. The structural basis for the functional versatility of immunoglobulin G. Can J Biochem. 1978;56(12):1087–101.
William LJ, Corky J. Temperature dependence of TIP3P, SPC, and TIP4P water from NPT Monte Carlo simulations: Seeking temperatures of maximum density. J Comput Chem. 1998;19(10):1179–86.
Phillips JC, Braun R, Wang W, Gumbart J, Tajkhorshid E, Villa E, et al. Scalable molecular dynamics with NAMD. J Comput Chem. 2005;26(16):1781–802.
Cheatham TEI, Cieplak P, Kollman PA. A modified version of the Cornell et al. force field with improved sugar pucker phases and helical repeat. J Biomol Struct Dyn. 1999;16(4):845–62.
Brandt JP, Patapoff TW, Aragon SR. Construction, MD simulation, and hydrodynamic validation of an all-atom model of a monoclonal IgG antibody. Biophys J. 2010;99(3):905–13.
Chennamsetty N, Helk B, Voynov V, Kayser V, Trout BL. Aggregation-prone motifs in human immunoglobulin G. J Mol Biol. 2009;391(2):404–13.
Stella L, Melchionna S. Equilibration and sampling in molecular dynamics simulations of biomolecules. J Chem Phys. 1998;109(23):10115–7.
Schneider WP, Wensel TG, Stryer L, Oi VT. Genetically engineered immunoglobulins reveal structural features controlling segmental flexibility. Proc Natl Acad Sci U S A. 1988;85(8):2509–13.
Sowdhamini R, Srinivasan N, Shoichet B, Santi DV, Ramakrishnan C, Balaram P. Stereochemical modeling of disulfide bridges. Criteria for introduction into proteins by site-directed mutagenesis. Protein Eng. 1989;3(2):95–103.
Lightle S, Aykent S, Lacher N, Mitaksov V, Wells K, Zobel J, et al. Mutations within a human IgG2 antibody form distinct and homogeneous disulfide isomers but do not affect Fc gamma receptor or C1q binding. Protein Sci. 2010;19(4):753–62.
We appreciate the anonymous referees for their constructive criticism of the research work and for suggestions to improve the manuscript. We thank Drs. Sandeep Nema, Sa V Ho, James Carroll, B. Muralidhara, Patrick Buck and Kevin King for several helpful discussions and for critical reading of this manuscript. A postdoctoral fellowship for Xiaoling Wang in Biotherapeutics Pharmaceutical Research and Development, Pfizer Inc. is gratefully acknowledged. High Performance Computing Support received from Pfizer Research Informatics played an essential role in this project.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Time series of total energy, temperature, volume and time average of pressure of the simulation system for the eight simulations in the production runs. (DOC 286 kb)
The evolution of radius of gyration (Rg) of Hinge++ with simulation time in all trajectories is shown. (DOC 54 kb)
Time courses for (a) sulphur atom distance for the Cys pair LC2.C214-HC2.C236 from the second set of simulations in the four disulfide-bonded conditions described in the manuscript. The solid black circle at time 0 indicates the initial distance; (b). sulphur atom distance for Cys pairs HC2.C128-HC2.C236 from the first set of simulations in the four disulfide-bonded conditions. (DOC 59 kb)
Contact maps for the eight Cys residues in hinge region. (a) Control simulation; (b) Four-reduced2 simulation; (c) Six-reduced2 simulation; (d) All-reduced2 simulation. Grayscale indicates the frequency of observing a given Cys-Cys contact. A contact is defined by sulphur atom distance for two Cys residues being < 5 Å. Note that the order of Cys residues along the X-axes is opposite to that along the Y axes. Hence, these are not traditional contact maps. Each quadrant is labeled according to the nature of contacts. Inter-heavy chain contacts (inter-HC) are located in upper right or lower left quadrants. The two quadrants are symmetric to each other. Intra-heavy chain contacts (Intra-HC1 and Intra-HC2) reside in the rest two quadrants. Each of the two quadrants is symmetric. (DOC 114 kb)
Average (μ), standard deviation (σ) and coefficient of variation (ρ=μ/σ) for the overall RMSD profiles in 5 ns simulation time intervals. (DOC 94 kb)
The video is from the production run of All-reduced1 simulation. In the video, the sulphur atoms from the six pairs of Cys residues involved in canonical inter-chain disulfide bonds are highlighted as CPK spheres. The sulphur atoms are colored the same as their respective heavy (green and blue) and light (purple and red) chains. Hinge++ molecular model represents only the middle portions of human IgG2 mAbs (see the text for details). During the course of the video, a sulphur atom from a light chain Cys 214 (purple) momentarily moves away from its canonical heavy chain (green) partner and becomes close to other sulphur atoms (green) in upper hinge region. (MPG 18949 kb)
About this article
Cite this article
Wang, X., Kumar, S. & Singh, S.K. Disulfide Scrambling in IgG2 Monoclonal Antibodies: Insights from Molecular Dynamics Simulations. Pharm Res 28, 3128–3144 (2011). https://doi.org/10.1007/s11095-011-0503-9
- molecular modeling