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Sandra J. Smith-Gill, Ph.D.
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Contacts in Antibody-Protein Interface |
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The structure of the complex of antibody HyHEL-10 in complex with Hen Egg White Lyzoyme (PDB 1D:3Hfm, Padlan EA, Silverton EW, Sheriff S, Cohen GH, Smith-Gill SJ and Davies DR (1989) Structure of an antibody-antigen complex: crystal structure of the HyHEL-10 Fab-lysozyme complex. Proc Natl Acad Sci U S A 86: 5938-5942.). Heavy chain, light chain, and HEL are shown by purple, yellow, and red, respectively, with side chains of epitope and paratope contact residues. Computational comparisons of the complexes of the HyHEL-10 complex with those of 2 closely related antibodies HyHEL-8 and HyHEL-26 shows that higher electostatic interactions, measured both in number and contributions to binding energies, leads to higher binding specificity but not necessarily affinty. Strong salt bridges, their networking, and electrostatically driven binding limits binding flexibility and cross-reactivity through geometric constraints. Details are found in
Sinha N, Mohan S, Lipschultz CA and Smith-Gill SJ (2002) Differences in electrostatic properties at antibody-antigen binding sites: Implications for specificity and cross-reactivity. Biophys J 83: 2946-2968 Sinha N and Smith-Gill SJ (2002) Electrostatics in protein binding and function. Current Protein & Peptide Science 3: 601-614. |
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Molecular Dynamics Reveal Important Antibody-Protein Interactions |
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The X-ray structure of the antibody HyHEL-63 (cyan) uncomplexed and complexed with Hen Egg White Lysozyme (yellow) has shown that there are small but significant, local conformational changes in the antibody paratope on binding. The structure also reveals that most of the charged epitope residues face the antibody. Details are in
Li YL, Li HM, Smith-Gill SJ and Mariuzza RA (2000) The conformations of the X-ray structure Three-dimensional structures of the free and antigen-bound Fab from monoclonal antilysozyme antibody HyHEL-63. Biochemistry 39: 6296-6309. Salt links and electrostatic interactions provide much of the free energy of binding. Most of the charged residues face in interface in the X-ray structure. The importance of the salt link between Lys97 of HEL and Asp27 of the antibody heavy chain is revealed by molecular dynamics simulations. After 1NSec of MD simulation at 100°C the overall conformation of the complex has changed, but the salt link persists. Details are described in Sinha N and Smith-Gill SJ (2002) Electrostatics in protein binding and function. Current Protein & Peptide Science 3: 601-614. |
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Dynamic View of Interface Interactions |
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Overlays of 10 confomers from a 20ps explicit molecular dynamics simulation reveals significant fluctuations of interface side chains Asp32 from the antibody heavy chain, and Lys96 and Lys97 from HEL, in the HyHEL-63-HEL complex. (Unpublished work of N. Sinha) |
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Alanine Scanning of "Coincident" Epitopes Recognized by Related Antibodies |
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Alanine scanning of residues on HEL recognized by 4 closely related antibodies, showing that the antibodies recognize “coincident� epitopes, with essentially the same “footprints� but differing in the details of their atomic contacts. Figure illustrates qualitative differences in the binding energy contributions, with red representing “hot spot� residues contributing the most binding energy, blue the least, and yellow an intermediate amount.
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Overlay of C-α backbones of the HH26-HEL model and HH26-HEL crystal structure |
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The moderate resolution structure of HH10-HEL was energy minimized and was used to homology model the complex of the closely related HH26-HEL.Our calculations show that the HH26 model described here and the X-ray crystal structure have high degree of similarity, with RMSD of 1.03 Ã….. In this figure, the crystal structure of HH26-HEL (PDB accession code 1NDM) is shown in red, and the model in green. The residues involved in Hydrogen-bond formation are similar and there are identical salt-bridges shared by the X-ray structure and the model. Thus, homology modeling combined with energy minimization of a lower resolution structure (e.g. HH10) can extend the value of existing x-ray crystallography structures by predicting a structure of a closely related protein which compares well to its higher resolution x-ray structure. |
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Diagram of 2-step (3-step) bimolecular association model |
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Panel (a) shows sensorgram overlays of a series of binding curves, determined by surface plasmon resonance on a Biacore instrument, on the same ligand and constant analyte concentration, with the association time (Ta) varying from 2min to 250min. Conventional protocols with short Ta include only a small portion of the binding curves, including at most that encompassing the first 3 curves (2, 5, and 10 min, respectively). The sensorgrams are actually a series of data points, which appear as a continuous curve because of their high density. The four rate constants for the series are determined globally by fitting all the sensorgrams to the model,, and used to generate the fitted total bound and for component’s for each binding curve.. In this model, as illustrated in the yellow inset (b), the concentration of encounter complex, [AB]*, increases first, and k1 predominates the initial observed k-on. As encounter complexes dock, the amount of docked complex, AB, increases, and in high affinity complexes [AB] begins to decrease as it is converted to AB, or, alternatively, reaches an equilibrium level, and the relative rates of complex docking, k2, and dissociation (k-1) become rate limiting to produce a biphasic curve. The time (T50) at which the 2 components curves cross, [AB]* = AB = ½ total bound.; T50 is unique for each complex and set of experimental conditions, and at high (saturating) analyte concentrations approaches a concentration-independent value, T50(MIN), which may be determined empirically by simulation, or by calculating the T½ of k2 in cases where k2 > k-1. period, undocked complexes dissociate first, and k-1 predominates the observed k-off. Eventually, the rate of undocking (k-2) becomes rate limiting to the net observed k-off, resulting in a biphasic dissociation curve. The portions of the curves which are particularly sensitive to each rate constant are indicated in the inset (b). Thus, if there are significant differences between k-1 and k-2, the apparent net off-rate will become slower as Ta is increased.
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This page was last updated on 2/7/2008.