Cross-reactive binding of cyclic peptides to an anti-TGFalpha antibody Fab fragment: an X-ray structural and thermodynamic analysis.
The monoclonal antibody tAb2 binds the N-terminal sequence of transforming growth factor alpha, VVSHFND. With the help of combinatorial peptide libraries it is possible to find homologous peptides that bind tAb2 with an affinity similar to that of the epitope. The conformational flexibility of short peptides can be constrained by cyclization in order to improve their affinity to the antibody and their stability towards proteolysis. Two cyclic peptides which are cross-reactive binders for tAb2 were selected earlier using combinatorial peptide libraries. One is cyclized by an amide bond between the N-alpha group and the side-chain of the last residue (cyclo-SHFNEYE), and the other by a disulfide bridge (cyclo-CSHFNDYC). The complex structures of tAb2 with the linear epitope peptide VVSHFND and with cyclo-SHFNEYE were determined by X-ray diffraction. Both peptides show a similar conformation and binding pattern in the complex. The linear peptide SHFNEYE does not bind tAb2, but cyclo-SHFNEYE is stabilized in a loop conformation suitable for binding. Hence the cyclization counteracts the exchange of aspartate in the epitope sequence to glutamate. Isothermal titration calorimetry was used to characterize the binding energetics of tAb2 with the two cyclic peptides and the epitope peptide. The binding reactions are enthalpically driven with an unfavorable entropic contribution under all measured conditions. The association reactions are characterized by negative DeltaC(p) changes and by the uptake of one proton per binding site. A putative candidate for proton uptake during binding is the histidine residue in each of the peptides. Hydrogen bonds and the putative formation of an electrostatic pair between the protonated histidine and a carboxy group may contribute markedly to the favorable enthalpy of complex formation. Implications to cyclization of peptides for stabilization are discussed.
Hahn
M
M
Institut für Biochemie, Universitätsklinikum Charité, Monbijoustr. 2, Humboldt-Universität zu Berlin, 10117, Germany.
Winkler
D
D
Welfle
K
K
Misselwitz
R
R
Welfle
H
H
Wessner
H
H
Zahn
G
G
Scholz
C
C
Seifert
M
M
Harkins
R
R
Schneider-Mergener
J
J
Höhne
W
W
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D000915:Antibody Affinity; D001483:Base Sequence; D001666:Binding Sites, Antibody; D002151:Calorimetry; D022801:Complementarity Determining Regions / Q000737:chemistry / Q000276:immunology; D003429:Cross Reactions / Q000276:immunology*; D018360:Crystallography, X-Ray; D019277:Entropy; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D006860:Hydrogen Bonding; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000276:immunology; D007145:Immunoglobulin kappa-Chains / Q000737:chemistry / Q000276:immunology; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010456:Peptides, Cyclic / Q000737:chemistry* / Q000276:immunology*; D011487:Protein Conformation; D055672:Static Electricity; D016211:Transforming Growth Factor alpha / Q000737:chemistry / Q000276:immunology*; D014867:Water / Q000737:chemistry / Q000378:metabolism
D000911:Antibodies, Monoclonal; D022801:Complementarity Determining Regions; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007145:Immunoglobulin kappa-Chains; D010456:Peptides, Cyclic; D016211:Transforming Growth Factor alpha; D014867:Water; D006639:Histidine