Conformational Change in an Anti-integrin Antibody: Structure of OPG2 Fab Bound to a β3 Peptide

Kodandapani, R.; Veerapandian, L.; Ni, Chao-Zhou; Chiou, Chu-Kuan; Whittal, Randy M.; Kunicki, Thomas J.; Ely, Kathryn R.

doi:10.1006/bbrc.1998.9380

Cited by 10 publications

(6 citation statements)

References 31 publications

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“…Again, additional packing interactions between CDR H3 and hapten 3 are introduced as a result of this rearrangement. CDR H3 has also been shown to undergo significant conformational changes upon the binding of antigen in a number of affinity-matured antibodies ( − ). In general, the movement of CDR H3 is not so large (<2 Å) in the affinity-matured antibodies as in the germline antibodies discussed here.…”

Section: Resultsmentioning

confidence: 99%

A Comparative Analysis of the Immunological Evolution of Antibody 28B4

et al. 2001

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In an effort to gain greater insight into the evolution of the redox active, catalytic antibody 28B4, the germline genes used by the mouse to generate this antibody were cloned and expressed, and the X-ray crystal structures of the unliganded and hapten-bound germline Fab of antibody 28B4 were determined. Comparison with the previously determined structures of the unliganded and hapten-bound affinity-matured Fab [Hsieh-Wilson, L. C., Schultz, P. G., and Stevens, R. C. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 5363] shows that the germline antibody binds the p-nitrophenyl ring of hapten 3 in an orientation significantly different from that seen in the affinity-matured antibody, whereas the phosphonate moiety is bound in a similar mode by both antibodies. The affinity-matured antibody 28B4 has more electrostatic and hydrophobic interactions with hapten 3 than the germline antibody and binds the hapten in a lock-and-key fashion. In contrast, significant conformational changes occur in the loops of CDR H3 and CDR L1 upon hapten binding to the germline antibody, consistent with the notion of structural plasticity in the germline antibody-combining site [Wedemayer, G. J., Patten, P. A., Wang, L. H., Schultz, P. G., and Stevens, R. C. (1997) Science 276, 1665]. The structural differences are reflected in the differential binding affinities of the germline Fab (K d ) 25 µM) and 28B4 Fab (K d ) 37 nM) to hapten 3. Nine replacement mutations were found to accumulate in the affinity-matured antibody 28B4 compared to its germline precursor. The effects of each mutation on the binding affinity of the antibody to hapten 3 were characterized in detail in the contexts of both the germline and the affinity-matured antibodies. One of the mutations, Asp95 H Trp, leads to a change in the orientation of the bound hapten, and its presence is a prerequisite for other somatic mutations to enhance the binding affinity of the germline antibody for hapten 3. Thus, the germline antibody of 28B4 acquired functionally important mutations in a stepwise manner, which fits into a multicycle mutation, affinity selection, and clonal expansion model for germline antibody evolution. Two other antibodies, 20-1 and NZA6, with very different antigen specificities were found to be highly homologous to the germline antibody of 28B4, consistent with the notion that certain germline variable-region gene combinations can give rise to polyspecific hapten binding sites [Romesberg, F. E., Spiller, B., Schultz, P. G., and Stevens, R. C. ( ) Science 279, 1929. The ultimate specificity of the polyspecific germline antibody appears to be defined by CDR H3 variability and subsequent somatic mutation. Insights into the evolution of antibody-combining sites provided by this and other structural studies are discussed.The evolution of binding affinity in the immune system involves combinatorial and mutational processes, which in many aspects parallel those that occur in the natural evolution of enzymes. Consequently, the characterization of the affinity mat...

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Section: Resultsmentioning

confidence: 99%

A Comparative Analysis of the Immunological Evolution of Antibody 28B4

et al. 2001

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“…Legitimacy of the antigen-induced models is debated. Supporting experiments include that: i) the circular polarization of fluorescence suggests that the disulfide bonds at the hinge region of the antibody are required to translate the conformational change from the F ab to the F c domain29; ii) X-ray crystallography reports a 15° conformation change of the F ab domains of OPG2 antibody by an antigen, β3 peptide, through carboxyl fragments of F ab domains30. Unfortunately, the small and diverse changes generated by the binding of antigens in most observed cases by crystallography cannot be generalized or identified with signaling3132333435.…”

Section: Discussionmentioning

confidence: 99%

Peptide-Conjugation Induced Conformational Changes in Human IgG1 Observed by Optimized Negative-Staining and Individual-Particle Electron Tomography

Tong

Zhang

Kaspar

et al. 2013

Sci Rep

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Peptides show much promise as potent and selective drug candidates. Fusing peptides to a scaffold monoclonal antibody produces a conjugated antibody which has the advantages of peptide activity yet also has the pharmacokinetics determined by the scaffold antibody. However, the conjugated antibody often has poor binding affinity to antigens that may be related to unknown structural changes. The study of the conformational change is difficult by conventional techniques because structural fluctuation under equilibrium results in multiple structures co-existing. Here, we employed our two recently developed electron microscopy (EM) techniques: optimized negative-staining (OpNS) EM and individual-particle electron tomography (IPET). Two-dimensional (2D) image analyses and three-dimensional (3D) maps have shown that the domains of antibodies present an elongated peptide-conjugated conformational change, suggesting that our EM techniques may be novel tools to monitor the structural conformation changes in heterogeneous and dynamic macromolecules, such as drug delivery vehicles after pharmacological synthesis and development.

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“…Consequently, one could argue that we never deal with the binding of a single charged ligand to a protein, but that simultaneous, interacting equilibria are the rule when proteins and charged ligands are considered. Indeed, recent structural studies on Fab fragments directed against a variety of charged antigens have revealed that significant conformation changes can occur within the Fab as a consequence of antigen binding ( − ). Similarly, other studies using intact monoclonal antibodies directed against fluorescein and lysozyme have revealed that secondary interactions external to the antibody active site can affect ligand binding efficiency, protein dynamics, and variable domain conformation ( − ).…”

Section: Discussionmentioning

confidence: 99%

Allosteric Binding Properties of a Monoclonal Antibody and Its Fab Fragment

et al. 2002

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Detailed equilibrium binding studies were conducted on a monoclonal antibody directed against Pb(II) complexed with a protein conjugate of diethylenetriaminepentaacetic acid (DTPA). Binding curves obtained with DTPA and a cyclohexyl derivative of DTPA in the presence and absence of metal ions were consistent with the anticipated one-site homogeneous binding model. Binding curves obtained with aminobenzyl-DTPA or its complexes with Ca(II), Sr(II), and Ba(II) were highly sigmoidal, characterized by Hill coefficients of 2.3-6.5. Binding curves obtained with the Pb(II) and In(III) complexes of aminobenzyl-DTPA were hyperbolic, but in each case the apparent affinity of the antibody for the chelator-metal complex was higher in the presence of excess chelator than it was in the presence of excess metal ion. In the presence of excess chelator, the equilibrium dissociation constant for the binding of aminobenzyl-DTPA-Pb(II) to the antibody was 9.5 x 10(-)(10) M. Binding curves obtained with the Hg(II) and Cd(II) complexes of aminobenzyl-DTPA were biphasic, indicative of negative cooperativity. Further binding studies demonstrated that aminobenzyl-DTPA-Hg(II) opposed the binding of additional chelator-metal complexes to the antibody more strongly than did aminobenzyl-DTPA-Cd(II). The Fab fragment differed from the intact antibody only in that the apparent affinity of the Fab was generally lower for a given chelator-metal complex. These data are interpreted in terms of a model in which (i) aminobenzyl-DTPA and its complexes bind both to the antigen binding site and to multiple charged sites on the surface of the compact immunoglobulin; and (ii) the bound, highly charged ligands interact in a complicated fashion through the apolar core of the folded antibody.

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Conformational Change in an Anti-integrin Antibody: Structure of OPG2 Fab Bound to a β3 Peptide

Cited by 10 publications

References 31 publications

A Comparative Analysis of the Immunological Evolution of Antibody 28B4

A Comparative Analysis of the Immunological Evolution of Antibody 28B4

Peptide-Conjugation Induced Conformational Changes in Human IgG1 Observed by Optimized Negative-Staining and Individual-Particle Electron Tomography

Allosteric Binding Properties of a Monoclonal Antibody and Its Fab Fragment

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