1g7h: Difference between revisions

Revision as of 13:47, 21 February 2008

CRYSTAL STRUCTURE OF HEN EGG WHITE LYSOZYME (HEL) COMPLEXED WITH THE MUTANT ANTI-HEL MONOCLONAL ANTIBODY D1.3(VLW92A)

OverviewOverview

Antigen-antibody complexes provide useful models for analyzing the thermodynamics of protein-protein association reactions. We have employed site-directed mutagenesis, X-ray crystallography, and isothermal titration calorimetry to investigate the role of hydrophobic interactions in stabilizing the complex between the Fv fragment of the anti-hen egg white lysozyme (HEL) antibody D1.3 and HEL. Crystal structures of six FvD1.3-HEL mutant complexes in which an interface tryptophan residue (V(L)W92) has been replaced by residues with smaller side chains (alanine, serine, valine, aspartate, histidine, and phenylalanine) were determined to resolutions between 1.75 and 2.00 A. In the wild-type complex, V(L)W92 occupies a large hydrophobic pocket on the surface of HEL and constitutes an energetic "hot spot" for antigen binding. The losses in apolar buried surface area in the mutant complexes, relative to wild-type, range from 25 (V(L)F92) to 115 A(2) (V(L)A92), with no significant shifts in the positions of protein atoms at the mutation site for any of the complexes except V(L)A92, where there is a peptide flip. The affinities of the mutant Fv fragments for HEL are 10-100-fold lower than that of the original antibody. Formation of all six mutant complexes is marked by a decrease in binding enthalpy that exceeds the decrease in binding free energy, such that the loss in enthalpy is partly offset by a compensating gain in entropy. No correlation was observed between decreases in apolar, polar, or aggregate (sum of the apolar and polar) buried surface area in the V(L)92 mutant series and changes in the enthalpy of formation. Conversely, there exist linear correlations between losses of apolar buried surface and decreases in binding free energy (R(2) = 0.937) as well as increases in the solvent portion of the entropy of binding (R(2) = 0.909). The correlation between binding free energy and apolar buried surface area corresponds to 21 cal mol(-1) A(-2) (1 cal = 4.185 J) for the effective hydrophobicity at the V(L)92 mutation site. Furthermore, the slope of the line defined by the correlation between changes in binding free energy and solvent entropy approaches unity, demonstrating that the exclusion of solvent from the binding interface is the predominant energetic factor in the formation of this protein complex. Our estimate of the hydrophobic contribution to binding at site V(L)92 in the D1.3-HEL interface is consistent with values for the hydrophobic effect derived from classical hydrocarbon solubility models. We also show how residue V(L)W92 can contribute significantly less to stabilization when buried in a more polar pocket, illustrating the dependence of the hydrophobic effect on local environment at different sites in a protein-protein interface.

About this StructureAbout this Structure

1G7H is a Protein complex structure of sequences from Gallus gallus and Mus musculus. Active as Lysozyme, with EC number 3.2.1.17 Full crystallographic information is available from OCA.

ReferenceReference

Estimation of the hydrophobic effect in an antigen-antibody protein-protein interface., Sundberg EJ, Urrutia M, Braden BC, Isern J, Tsuchiya D, Fields BA, Malchiodi EL, Tormo J, Schwarz FP, Mariuzza RA, Biochemistry. 2000 Dec 19;39(50):15375-87. PMID:11112523

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 ==Overview==
-Antigen-antibody complexes provide useful models for analyzing the, thermodynamics of protein-protein association reactions. We have employed, site-directed mutagenesis, X-ray crystallography, and isothermal titration, calorimetry to investigate the role of hydrophobic interactions in, stabilizing the complex between the Fv fragment of the anti-hen egg white, lysozyme (HEL) antibody D1.3 and HEL. Crystal structures of six FvD1.3-HEL, mutant complexes in which an interface tryptophan residue (V(L)W92) has, been replaced by residues with smaller side chains (alanine, serine, valine, aspartate, histidine, and phenylalanine) were determined to, resolutions between 1.75 and 2.00 A. In the wild-type complex, V(L)W92, occupies a large hydrophobic pocket on the surface of HEL and constitutes, an energetic "hot spot" for antigen binding. The losses in apolar buried, surface area in the mutant complexes, relative to wild-type, range from 25, (V(L)F92) to 115 A(2) (V(L)A92), with no significant shifts in the, positions of protein atoms at the mutation site for any of the complexes, except V(L)A92, where there is a peptide flip. The affinities of the, mutant Fv fragments for HEL are 10-100-fold lower than that of the, original antibody. Formation of all six mutant complexes is marked by a, decrease in binding enthalpy that exceeds the decrease in binding free, energy, such that the loss in enthalpy is partly offset by a compensating, gain in entropy. No correlation was observed between decreases in apolar, polar, or aggregate (sum of the apolar and polar) buried surface area in, the V(L)92 mutant series and changes in the enthalpy of formation., Conversely, there exist linear correlations between losses of apolar, buried surface and decreases in binding free energy (R(2) = 0.937) as well, as increases in the solvent portion of the entropy of binding (R(2) =, 0.909). The correlation between binding free energy and apolar buried, surface area corresponds to 21 cal mol(-1) A(-2) (1 cal = 4.185 J) for the, effective hydrophobicity at the V(L)92 mutation site. Furthermore, the, slope of the line defined by the correlation between changes in binding, free energy and solvent entropy approaches unity, demonstrating that the, exclusion of solvent from the binding interface is the predominant, energetic factor in the formation of this protein complex. Our estimate of, the hydrophobic contribution to binding at site V(L)92 in the D1.3-HEL, interface is consistent with values for the hydrophobic effect derived, from classical hydrocarbon solubility models. We also show how residue, V(L)W92 can contribute significantly less to stabilization when buried in, a more polar pocket, illustrating the dependence of the hydrophobic effect, on local environment at different sites in a protein-protein interface.
+Antigen-antibody complexes provide useful models for analyzing the thermodynamics of protein-protein association reactions. We have employed site-directed mutagenesis, X-ray crystallography, and isothermal titration calorimetry to investigate the role of hydrophobic interactions in stabilizing the complex between the Fv fragment of the anti-hen egg white lysozyme (HEL) antibody D1.3 and HEL. Crystal structures of six FvD1.3-HEL mutant complexes in which an interface tryptophan residue (V(L)W92) has been replaced by residues with smaller side chains (alanine, serine, valine, aspartate, histidine, and phenylalanine) were determined to resolutions between 1.75 and 2.00 A. In the wild-type complex, V(L)W92 occupies a large hydrophobic pocket on the surface of HEL and constitutes an energetic "hot spot" for antigen binding. The losses in apolar buried surface area in the mutant complexes, relative to wild-type, range from 25 (V(L)F92) to 115 A(2) (V(L)A92), with no significant shifts in the positions of protein atoms at the mutation site for any of the complexes except V(L)A92, where there is a peptide flip. The affinities of the mutant Fv fragments for HEL are 10-100-fold lower than that of the original antibody. Formation of all six mutant complexes is marked by a decrease in binding enthalpy that exceeds the decrease in binding free energy, such that the loss in enthalpy is partly offset by a compensating gain in entropy. No correlation was observed between decreases in apolar, polar, or aggregate (sum of the apolar and polar) buried surface area in the V(L)92 mutant series and changes in the enthalpy of formation. Conversely, there exist linear correlations between losses of apolar buried surface and decreases in binding free energy (R(2) = 0.937) as well as increases in the solvent portion of the entropy of binding (R(2) = 0.909). The correlation between binding free energy and apolar buried surface area corresponds to 21 cal mol(-1) A(-2) (1 cal = 4.185 J) for the effective hydrophobicity at the V(L)92 mutation site. Furthermore, the slope of the line defined by the correlation between changes in binding free energy and solvent entropy approaches unity, demonstrating that the exclusion of solvent from the binding interface is the predominant energetic factor in the formation of this protein complex. Our estimate of the hydrophobic contribution to binding at site V(L)92 in the D1.3-HEL interface is consistent with values for the hydrophobic effect derived from classical hydrocarbon solubility models. We also show how residue V(L)W92 can contribute significantly less to stabilization when buried in a more polar pocket, illustrating the dependence of the hydrophobic effect on local environment at different sites in a protein-protein interface.
 ==About this Structure==
@@ Line 15: / Line 15: @@
 [[Category: Mus musculus]]
 [[Category: Protein complex]]
-[[Category: Braden, B.C.]]
+[[Category: Braden, B C.]]
 [[Category: Isern, J.]]
-[[Category: Mariuzza, R.A.]]
+[[Category: Mariuzza, R A.]]
-[[Category: Sundberg, E.J.]]
+[[Category: Sundberg, E J.]]
 [[Category: Urrutia, M.]]
 [[Category: hydrolase inhibitor/hydrolase]]
-''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Fri Feb 15 15:51:30 2008''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 12:46:57 2008''