Atomic Model Structure of the NIST Monoclonal Antibody (NISTmAb) Reference Material

Abstract

[...]the H and V chains have identical amino acid sequence, but slightly different glycans. [...]the radius of gyration of this model, 5.6 nm, agrees closely with available solution state measured values for NISTmAb from scattering methods [7]. Journal of Molecular Biology. https://doi.org/l0.l006/jmbi.l997.l508 [6] Saphire EO, Parren PWHI, Pantophlet R, Zwick MB, Morris GM, Rudd PM, Dwek RA, Stanfield RL, Burton DR, Wilson IA (2001) Crystal structure of a neutralizing human IgG against HIV-l: A template for vaccine design. Journal of Biomolecular NMR. https://doi.org/l0.l007/sl0858-020-00332-y [14] Kerr RA, Keire DA, Ye H (2019) The impact of standard accelerated stability conditions on antibody higher order structure as assessed by mass spectrometry. mAbs. https://doi.org/l0.l080/l9420862.20l9.l599632 [15] Karageorgos I, Gallagher ES, Galvin C, Gallagher DT, Hudgens JW (2017) Biophysical characterization and structure of the Fab fragment from the NIST reference antibody, RM 8671.

1. Summary

As monoclonal antibodies have become a vital resource in medicine, knowledge of their complex molecular structures has increased in importance. Thousands of antibody components (Fab and Fc fragments) are described in the Protein Data Bank [1]. Whole antibodies have been imaged by electron microscopy methods [2, 3] and in a few cases, crystallized [4-6]. The central hinge lacks a unique stable conformation and its dynamic properties are important to antibody function. Monte Carlo and molecular dynamics simulations and small-angle scattering methods have been used to analyze the wide range of configurations that are accessible to antibodies in solution [7, 8]. In order to support the development of antibody-based medicines, the National Institute of Standards and Technology (NIST) has released an extensively characterized IgGİK monoclonal antibody (mAb), called the NISTmAb Reference Material 8671 [9-14]. To facilitate modeling of whole antibodies we now report the construction of an all-atom 3-D model of the NISTmAb.

3. Methods

The NISTmAb antibody model was made from five parts as shown in Figure 1, using copies of its previously reported Fab (PDBID 5K8A [15]) and Fc (PDBID 5VGP [16]) fragment crystal structures. All parts of the hinge are derived from the whole-mAb crystal structure 1IGT [4], which uniquely includes the entire 4-Cys core, sequence CPPC, with both disulfides intact. The additional crosslink in 1IGT (its hinge has 3 disulfides) was removed to make NISTmAb, according to the hinge-region alignment in Figure 2 (top).

In order to obtain the correct upper and lower hinge lengths, the two underlined residues of 1IGT in Figure 2 (КС) were removed. The new gap between ... CPPC and PAPN... was spliced together by moving the initial proline of PAPN. onto the location of the deleted lysine. Both heavy chains were spliced simultaneously. Then, appropriate mutations were made, using the rotamer library in PyMOL2 [17] to give the hinge model the correct NISTmAb sequence (FASTA format data file). Figure 2 shows the initial 1IGT hinge and the derived NISTmAb hinge.

The initial model was minimized to remove local strain introduced by splicing and to produce a refined, energy-minimized model with correct local geometry. The model was parameterized using the tLEaP program in Amberl8 [18]. The ffl4SB force field for proteins was used, and the glycan 06j-l force field was used for glycans [19, 20]. Minimization was performed in vacuo for 1000 steps total, using the steepest descent algorithm for the first 500 steps before switching to the conjugate gradient algorithm for 500 steps. Plots of energy and maximum gradient vs. minimization step are shown in Figure 3.

The model includes all 1326 amino acids in four chains. The two light chains are designated as L and M. Both light chains have amino acids 1 to 213. The two heavy chains are designated as H and V chains and have amino acids 1 to 450. As in 5VGP, the G1F/G0F glycans, (i.e. the H-chain glycan has nine sugar groups including one fucose and one terminal galactose, while the V chain has only eight, lacking galactose) attach to Asn300. Thus, the H and V chains have identical amino acid sequence, but slightly different glycans. Each heavy chain has pyroglutamate (PCA) at the N-terminus, a commonly observed (at protein N-termini) conversion from the genetically encoded Glutamine, known to be present in the NISTmAb at near quantitative abundance [21]. In addition, while both chains in 5VGP lacked three Cterminal residues which were disordered in the Fc crystal structure, they are added to the model for the sake of completeness. The model includes all known atoms and has the composition given in Table 1.

To facilitate subsequent comparisons, the model has been oriented with the Fc dyad along the z-axis, placed with the xyz origin at the midpoint between the CA atoms of the two Proline 241 residues, at the top of the Fc. See Figure 4.

4. Impact

As the field of antibody therapeutics continues to grow, it is likely that structural and mechanistic models will serve increasingly to guide molecular design strategies. The reported model is intended to provide an all-atom structure that can be used as a reference for the interpretation of biophysical measurements and a starting point for further modeling and dynamics studies. Molecular dynamics simulation, together with continuing developments in metrology, are expected to lead to improved understanding of hinge properties, enabling better models of biological functions such as bivalent binding and signaling.

Hinge properties potentially involved in antibody function whose study may be facilitated by the present model include the sharp bend near the hinge C-terminus, at the GGP sequence 239-241. Crystal structures 1IGT [4], 1IGY [5], 1HZH [6] and 3AY4 [22] all have a similar bend, and in 3AY4 the bend appears necessary for Fc gamma receptor binding and thus effector signaling [22]. Additionally, the hinge core containing the paired CPPC motif forms a covalent ring of 28 atoms that includes two disulfides, the constrained nature of which could limit the conformational landscape of the Fab and Fc domains. Finally, the radius of gyration of this model, 5.6 nm, agrees closely with available solution state measured values for NISTmAb from scattering methods [7].

Sidebar

Data DOI: https://doi.org/10.18434/mds2-2396

Key words: intact antibody; molecular model; NISTmAb; RM8671.

Accepted: June 30, 2021

Published: July 15, 2021

How to cite this article: Bergonzo C, Gallagher DT (2021) Atomic Model Structure of the NIST Monoclonal Antibody (NISTmAb) Reference Material. J Res Natl Inst Stan 126:126012. https://doi.org/10.6028/jres.126.012

Acknowledgements

We thank John Schiel, John Marino and Joseph Curtis for helpful discussions, overall program direction and testing of models.

About the authors: The authors are Research Chemists in the Biomolecular Measurement Division at NIST.

The National Institute of Standards and Technology is an agency of the U.S. Department of Commerce.

Footnote

1 Extensive information on NISTmAb is available at the website nistmab.ibbr.umd.edu.

2 Certain commercial equipment, instruments, or materials are identified in this paper to foster understanding. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.

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