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An insertion mutation that distorts antibody binding site architecture enhances function of a human antibody.

ABSTRACT: The structural and functional significance of somatic insertions and deletions in antibody chains is unclear. Here, we demonstrate that a naturally occurring three-amino-acid insertion within the influenza virus-specific human monoclonal antibody 2D1 heavy-chain variable region reconfigures the antibody-combining site and contributes to its high potency against the 1918 and 2009 pandemic H1N1 influenza viruses. The insertion arose through a series of events, including a somatic point mutation in a predicted hot-spot motif, introduction of a new hot-spot motif, a molecular duplication due to polymerase slippage, a deletion due to misalignment, and additional somatic point mutations. Atomic resolution structures of the wild-type antibody and a variant in which the insertion was removed revealed that the three-amino-acid insertion near the base of heavy-chain complementarity-determining region (CDR) H2 resulted in a bulge in that loop. This enlarged CDR H2 loop impinges on adjacent regions, causing distortion of the CDR H1 architecture and its displacement away from the antigen-combining site. Removal of the insertion restores the canonical structure of CDR H1 and CDR H2, but binding, neutralization activity, and in vivo activity were reduced markedly because of steric conflict of CDR H1 with the hemagglutinin antigen.

SUBMITTER: Krause JC

PROVIDER: S-EPMC3037006 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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An insertion mutation that distorts antibody binding site architecture enhances function of a human antibody.

Krause Jens C JC Ekiert Damian C DC Tumpey Terrence M TM Smith Patricia B PB Wilson Ian A IA Crowe James E JE

mBio 20110208 1

The structural and functional significance of somatic insertions and deletions in antibody chains is unclear. Here, we demonstrate that a naturally occurring three-amino-acid insertion within the influenza virus-specific human monoclonal antibody 2D1 heavy-chain variable region reconfigures the antibody-combining site and contributes to its high potency against the 1918 and 2009 pandemic H1N1 influenza viruses. The insertion arose through a series of events, including a somatic point mutation in ...[more]

PMID: 21304166

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Project description:BackgroundHuman γS-crystallin is an important component of the human eye lens nucleus and cortex. The mutation V42M in the molecule causes severe congenital cataract in children. We compare the structure of the mutant protein with that of the wild type in order to understand how structural changes in the mutant relate to the mechanism of opacification.MethodsBoth proteins were made using conventional cloning and expression procedures. Secondary and tertiary structural features of the proteins were analyzed using spectral methods. Structural stabilities of the proteins were analyzed using chemical and thermal denaturation methods. Self-aggregation was monitored using extrinsic spectral probes. Molecular modeling was used to compare the structural features of the two proteins.ResultsWhile the wild type and mutant have the same secondary structure, molecular modeling and fluorescence analysis suggest the mutant to have a more open tertiary structure, with a larger hydrophobic surface. Experiments using extrinsic probes reveal that the mutant readily self-aggregates, with the suggestion that the aggregates might be similar to amyloidogenic fibrils. Chemical denaturation indicates that while the wild type exhibits the classic two-state transition, V42M goes through an intermediate state, and has a distinctly lower stability than the wild type. The temperature of thermal unfolding of the mutant is also distinctly lower. Further, the mutant readily precipitates and scatters light more easily than the wild type.ConclusionThe replacement of valine in position 42 by the longer and bulkier methionine in human γS-crystallin perturbs the compact β-sheet core packing topology in the N-terminal domain of the molecule, exposes nonpolar residues thereby increasing the surface hydrophobicity and weakens the stability of the protein, thus promoting self-aggregation leading to light scattering particles. This set of changes in the properties of the mutant offers a molecular insight into the mechanism of opacification.

Dataset Information

An insertion mutation that distorts antibody binding site architecture enhances function of a human antibody.

Publications

An insertion mutation that distorts antibody binding site architecture enhances function of a human antibody.

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