Denzin L K, Whitlow M, Voss E W
Department of Microbiology, University of Illinois, Urbana 61801.
J Biol Chem. 1991 Jul 25;266(21):14095-103.
Previous crystallographic studies of high affinity anti-fluorescein monoclonal antibody 4-4-20 (Ka = 1.7 x 10(10) M-1) complexed with fluorescyl ligand resolved active site contact residues involved in binding. For better definition of the relative roles of three light chain antigen contact residues (L27dhis, L32tyr and L34arg), four site-specific mutations (L27dhis to L27lys, L32tyr to L32phe, and L34arg to L34lys and L34his) were generated and expressed in single-chain antigen binding derivatives of monoclonal antibody 4-4-20 containing two different polypeptide linkers (SCA 4-4-20/205c, 25 amino acids and SCA 4-4-20/212, 14 amino acids). Results showed that L27dhis and L32tyr were necessary for wild type binding affinities, however, were not required for near-wild type Qmax values (where Qmax is the maximum fluoroscein fluorescence quenching expressed as percent). Tyrosine L32 which hydrogen bonds with ligand was also characterized at the haptenic level through the use of 9-hydroxyphenylfluoron which lacks the carboxyl group to which L32 tyrosine forms a hydrogen bond. Results demonstrated that wild type SCA and mutant L32phe possessed similar HPF binding characteristics. Active site contact residue L34arg was important for fluorescein quenching maxima and binding affinity (L34his mutant), however, substitution of lysine for arginine at L34 did not have a significant effect on observed Qmax value. In addition, substitutions had no effect on structural and topological characteristics, since all mutants retained similar idiotypic and metatypic properties. Finally, two linkers were comparatively examined to determine relative contributions to mutant binding properties and stability. No linker effects were observed. Collectively, these results verified the importance of these light chain fluorescein contact residues in the binding pocket of monoclonal antibody 4-4-20.
先前对与荧光素配体复合的高亲和力抗荧光素单克隆抗体4-4-20（Ka = 1.7×10¹⁰ M⁻¹）进行的晶体学研究解析了参与结合的活性位点接触残基。为了更好地定义三个轻链抗原接触残基（L27dhis、L32tyr和L34arg）的相对作用，产生了四个位点特异性突变（L27dhis突变为L27lys、L32tyr突变为L32phe，以及L34arg突变为L34lys和L34his），并在含有两种不同多肽接头（SCA 4-4-20/205c，25个氨基酸；SCA 4-4-20/212，14个氨基酸）的单链抗原结合衍生物中表达。结果表明，L27dhis和L32tyr对于野生型结合亲和力是必需的，然而，对于接近野生型的Qmax值（其中Qmax是以百分比表示的最大荧光素荧光猝灭）并非必需。与配体形成氢键的酪氨酸L32也通过使用9-羟基苯基荧光素在半抗原水平进行了表征，9-羟基苯基荧光素缺乏L32酪氨酸与之形成氢键的羧基。结果表明野生型SCA和突变体L32phe具有相似的HPF结合特性。活性位点接触残基L34arg对于荧光素猝灭最大值和结合亲和力很重要（L34his突变体），然而，L34处用赖氨酸取代精氨酸对观察到的Qmax值没有显著影响。此外，这些取代对结构和拓扑特征没有影响，因为所有突变体都保留了相似的独特型和变构型特性。最后，对两种接头进行了比较研究，以确定它们对突变体结合特性和稳定性的相对贡献。未观察到接头效应。总的来说，这些结果证实了这些轻链荧光素接触残基在单克隆抗体4-4-20结合口袋中的重要性。
