Bobeck Melissa J, Glick Gary D
Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USA.
Biopolymers. 2007;85(5-6):481-9. doi: 10.1002/bip.20692.
11F8 is a sequence-specific monoclonal anti-ssDNA autoantibody isolated from a lupus prone mouse that forms pathogenic complexes with ssDNA, resulting in kidney damage. Prior studies show that specificity is mediated by a somatic mutation from serine at (31)V(H) to arginine. Reversion back to serine in 11F8 resulted in >30-fold decrease in affinity and altered thermodynamic and kinetic parameters for sequence-specific recognition of its cognate ssDNA ligand. Mutagenesis and structural studies suggest that (R31)V(H) contacts ssDNA via a salt bridge and a bidentate hydrogen bond and may further contribute to specificity by altering binding-site conformation. Fluorescence resonance energy transfer experiments were conducted to assess the kinetics of conformational change during 11F8ssDNA association. The extent of rearrangement between the six complementary determining regions in the 11F8ssDNA complex with germline serine or somatically mutated arginine at residue 31 of the heavy chain was examined. Our studies show that greater conformational change occurs in five of six complementarity determining regions after the heavy chain germline J558 sequence undergoes mutation to arginine at (31)V(H).
11F8是一种从狼疮易感小鼠中分离出的序列特异性单克隆抗单链DNA自身抗体，它与单链DNA形成致病复合物，导致肾脏损伤。先前的研究表明，其特异性由重链可变区（V(H)）第31位的丝氨酸经体细胞突变变为精氨酸介导。11F8中该位点回复为丝氨酸后，其亲和力下降超过30倍，并且其识别同源单链DNA配体的热力学和动力学参数也发生了改变。诱变和结构研究表明，重链可变区第31位的精氨酸（R31）V(H)通过盐桥和双齿氢键与单链DNA接触，并且可能通过改变结合位点构象进一步影响特异性。进行了荧光共振能量转移实验以评估11F8与单链DNA结合过程中构象变化的动力学。研究了重链第31位为胚系丝氨酸或体细胞突变精氨酸的11F8 - 单链DNA复合物中六个互补决定区之间的重排程度。我们的研究表明，重链胚系J558序列在V(H)第31位突变为精氨酸后，六个互补决定区中的五个发生了更大程度的构象变化。
