Journal of Molecular Biology
CommunicationComparison of CH1 Domains in Different Classes of Murine Antibodies
Abstract
The CH1 domains of antibodies belonging to the following five murine immunoglobulin (Ig) classes IgG1, IgG2a, IgG2b, IgG3 and IgA have been compared. The IgG CH1 domain structures are, as would be expected, similar overall, but show local conformational variations. When compared with IgG CH1 domain structures, the IgA CH1 domain displays several significant structural differences, which are a consequence of insertions/ deletions and specific structural constraints. In regions of structural differences in the IgG CH1 domains, the spatial correspondence of residues is not reflected by conventional (Kabat) sequence number. Thus the sequence alignment and numbering for CH1 domains has been revised to be consistent with the three-dimensional alignments.
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Site-Specific Photolabeling of the IgG Fab Fragment Using a Small Protein G Derived Domain
2016, Bioconjugate ChemistryAntibodies are widely used reagents for recognition in both clinic and research laboratories all over the world. For many applications, antibodies are labeled through conjugation to different reporter molecules or therapeutic agents. Traditionally, antibodies are covalently conjugated to reporter molecules via primary amines on lysines or thiols on cysteines. While efficient, such labeling is variable and nonstoichiometric and may affect an antibody’s binding to its target. Moreover, an emerging field for therapeutics is antibody–drug conjugates, where a toxin or drug is conjugated to an antibody in order to increase or incorporate a therapeutic effect. It has been shown that homogeneity and controlled conjugation are crucial in these therapeutic applications. Here we present two novel protein domains developed from an IgG-binding domain of Streptococcal Protein G. These domains show obligate Fab binding and can be used for site-specific and covalent attachment exclusively to the constant part of the Fab fragment of an antibody. The two different domains can covalently label IgG of mouse and human descent. The labeled antibodies were shown to be functional in both an ELISA and in an NK-cell antibody-dependent cellular cytotoxicity assay. These engineered protein domains provide novel tools for controlled labeling of Fab fragments and full-length IgG.
Monoclonal antibody MN423 as a stable mold facilitates structure determination of disordered tau protein
2010, Journal of Structural BiologyFlexibility of intrinsically disordered tau protein is important for performing its functions. It is believed that alteration of the flexibility is instrumental to the assembly of tau protein into paired helical filaments (PHF) in tauopathies. Tau flexibility represents the main obstacle for structure determination of its conformation in physiology and/or pathology. We have alleviated this inherited difficulty by using specific monoclonal antibodies as tau protein surrogate binding partners. In this work we compare two “antibody mold structures”: (1) X-ray structure of the free form of the Alzheimer’s disease PHF core-specific antibody MN423 and (2) previously solved structure of the complex of MN423 with the PHF core C-terminal tau peptide. We found that MN423 combining site is in both structures identical. As a consequence, recombinant tau assumes in the complex a fold determined by the antibody combining site. Obtained results show that MN423 functions as a molecular mold for the PHF core segment, and opens the way for structure determination of other PHF core segments providing that other conformation-specific antibodies are available. Data from in silico docking of tau peptide into antibody mold, obtained in this study, show that biochemical data and computational approaches provide results comparable to X-ray crystallography.
Structure of the Fab fragment of therapeutic antibody Ofatumumab provides insights into the recognition mechanism with CD20
2009, Molecular ImmunologyCD20 is an important drug target for B-cell depletion therapy against certain B-cell lymphomas and autoimmune diseases. The success of anti-CD20 antibody drugs such as Rituximab, Ibritumomab, and Tositumomab has promoted the development of new generation of anti-CD20 antibodies for therapeutic applications. Ofatumumab is a fully human anti-CD20 antibody that is currently in phase III clinical trial for several types of malignancies and autoimmune diseases and is one of the most promising anti-CD20 drugs. Here we report the crystal structure of the Fab fragment of Ofatumumab at 2.2 Å resolution. The antigen combining site is composed of a large, deep pocket formed by six CDR loops. The pocket has a hydrophobic periphery and a positively charged bottom. Structure analysis and comparison with other antibodies suggest that the hydrophobic periphery might interact with the epitope on CD20 that is enriched with hydrophobic residues and very close to cell membrane, and the positively charged bottom might interact with Glu150 of CD20 which is the only negatively charged residue within the epitope. These results provide some insights into the recognition of Ofatumumab with CD20 and explain how the antibody can recognize an epitope so close to the cell membrane.
The Crystal Structure of CD8 in Complex with YTS156.7.7 Fab and Interaction with Other CD8 Antibodies Define the Binding Mode of CD8 αβ to MHC Class I
2008, Journal of Molecular BiologyThe CD8αβ heterodimer interacts with class I pMHC on antigen-presenting cells as a co-receptor for TCR-mediated activation of cytotoxic T cells. To characterize this immunologically important interaction, we used monoclonal antibodies (mAbs) specific to either CD8α or CD8β to probe the mechanism of CD8αβ binding to pMHCI. The YTS156.7 mAb inhibits this interaction and blocks T cell activation. To elucidate the molecular basis for this inhibition, the crystal structure of the CD8αβ immunoglobulin-like ectodomains were determined in complex with mAb YTS156.7 Fab at 2.7 Å resolution. The YTS156.7 epitope on CD8β was identified and implies that residues in the CDR1 and CDR2-equivalent loops of CD8β are occluded upon binding to class I pMHC. To further characterize the pMHCI/CD8αβ interaction, binding of class I tetramers to CD8αβ on the surface of T cells was assessed in the presence of anti-CD8 mAbs. In contrast to YTS156.7, mAb YTS105.18, which is specific for CD8α, does not inhibit binding of CD8αβ to class I tetramers, indicating the YTS105.18 epitope is not occluded in the pMHCI/CD8αβ complex. Together, these data indicate a model for the pMHCI/CD8αβ interaction similar to that observed for CD8αα in the CD8αα/pMHCI complex, but in which CD8α occupies the lower orientation (membrane proximal to the antigen presenting cell), and CD8β occupies the upper position (membrane distal). The implication of this molecular assembly for the function of CD8αβ in T cell activation is discussed.
Crystal Structures of a Quorum-quenching Antibody
2007, Journal of Molecular BiologyA large number of Gram-negative bacteria employ N-acyl homoserine lactones (AHLs) as signaling molecules in quorum sensing, which is a population density-dependent mechanism to coordinate gene expression. Antibody RS2-1G9 was elicited against a lactam mimetic of the N-acyl homoserine lactone and represents the only reported monoclonal antibody that recognizes the naturally-occuring N-acyl homoserine lactone with high affinity. Due to its high cross-reactivity, RS2-1G9 showed remarkable inhibition of quorum sensing signaling in Pseudomonas aeruginosa, a common opportunistic pathogen in humans. The crystal structure of Fab RS2-1G9 in complex with a lactam analog revealed complete encapsulation of the polar lactam moiety in the antibody-combining site. This mode of recognition provides an elegant immunological solution for tight binding to an aliphatic, lipid-like ligand with a small head group lacking typical haptenic features, such as aromaticity or charge, which are often incorporated into hapten design to generate high-affinity antibodies. The ability of RS2-1G9 to discriminate between closely related AHLs is conferred by six hydrogen bonds to the ligand. Conversely, cross-reactivity of RS2-1G9 towards the lactone is likely to originate from conservation of these hydrogen bonds as well as an additional hydrogen bond to the oxygen of the lactone ring. A short, narrow tunnel exiting at the protein surface harbors a portion of the acyl chain and would not allow entry of the head group. The crystal structure of the antibody without its cognate lactam or lactone ligands revealed a considerably altered antibody-combining site with a closed binding pocket. Curiously, a completely buried ethylene glycol molecule mimics the lactam ring and, thus, serves as a surrogate ligand. The detailed structural delineation of this quorum-quenching antibody will aid further development of an antibody-based therapy against bacterial pathogens by interference with quorum sensing.
Structure-based Stability Engineering of the Mouse IgG1 Fab Fragment by Modifying Constant Domains
2006, Journal of Molecular BiologyA semi-rational approach based on structural data was exploited in a search for CH1 and CL domains with improved intrinsic thermodynamic stabilities. Structural and amino acid level comparisons were carried out against known biophysically well-behaving and thermodynamically beneficial scFv and Fab fragments. A number of mutant Fab fragments were constructed by site-directed mutagenesis of regions in the CH1 and CL domains expected to be most sensitive under physical stress conditions. These mutations were located on three sites in the Fab constant domains; a mobile loop in the CH1 domain, residues surrounding the two largest solvated hydrophobic cavities located in the interface of the CH1 and CL domains and the hydrophobic core regions of both CH1 and CL. Expression levels of functional Fab fragments, denaturant-induced unfolding equilibria and circular dichroism spectroscopy were used to evaluate the relative stabilities of the wild-type and the mutant Fab fragments. The highest thermodynamic stability was reached through the mutation strategy, where the hydrophobicity and the packing density of the solvated hydrophobic cavity in the CH1/CL interface was increased by the replacement of the hydrophilic Thr178 in the CL domain by a more hydrophobic residue, valine or isoleucine. The midpoint of the transition curve from native to unfolded states of the protein, measured by fluorescence emission, occurred at concentrations of guanidine hydrochloride of 2.4 M and 2.6 M for the wild-type Fab and the most stable mutants, respectively. Our results illustrate that point mutations targeted to the CH1/CL interface were advantageous for the overall thermodynamic stability of the Fab fragment.
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Corresponding author
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Present address: P. D. Jeffrey, Box 576, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA.