Bond Christopher J, Marsters James C, Sidhu Sachdev S
Departments of Medicinal Chemistry, 1 DNA Way, South San Francisco, CA 94080, USA.
J Mol Biol. 2003 Sep 19;332(3):643-55. doi: 10.1016/s0022-2836(03)00967-7.
Camelids produce functional antibodies devoid of light chains. Autonomous heavy chain variable (V(H)H) domains in these molecules have adapted to the absence of the light chain in the following ways: bulky hydrophobic residues replace small aliphatic residues in the former light chain interface, and residues from the third complementarity-determining region (CDR3) pack against the framework and stabilize the global V(H)H domain fold. To determine the specific roles of CDR3 residues in framework stabilization, we used nai;ve phage-displayed libraries, combinatorial alanine-scanning mutagenesis and biophysical characterization of purified proteins. Our results indicate that in the most stable scaffolds, the structural residues in CDR3 reside near the boundaries of the loop and pack against the framework to form a small hydrophobic core. These results allow us to differentiate between structural CDR3 residues that should remain fixed, and CDR3 residues that are tolerant to substitution and can therefore be varied to generate functional diversity within phage-displayed libraries. These methods and insights can be applied to the rapid design of heavy chain scaffolds for the identification of novel ligands using synthetic, antibody-phage libraries. In addition, they shed light on the relationships between CDR3 sequence diversity and the structural stability of the V(H)H domain fold.
骆驼科动物产生不含轻链的功能性抗体。这些分子中的自主重链可变区（V(H)H）结构域通过以下方式适应了轻链的缺失：在原轻链界面处，大的疏水残基取代了小的脂肪族残基，并且来自第三互补决定区（CDR3）的残基堆积在框架上并稳定了整个V(H)H结构域的折叠。为了确定CDR3残基在框架稳定中的具体作用，我们使用了天然噬菌体展示文库、组合丙氨酸扫描诱变以及纯化蛋白的生物物理表征。我们的结果表明，在最稳定的支架中，CDR3中的结构残基位于环的边界附近，并堆积在框架上以形成一个小的疏水核心。这些结果使我们能够区分应保持固定的结构CDR3残基和耐受取代的CDR3残基，因此可以对后者进行改变以在噬菌体展示文库中产生功能多样性。这些方法和见解可应用于使用合成抗体噬菌体文库快速设计重链支架，以鉴定新型配体。此外，它们揭示了CDR3序列多样性与V(H)H结构域折叠的结构稳定性之间的关系。
