科研之友
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摘要
We present the crystal structure and biophysical characterization of a human V-L [variable domain immunoglobulin (Ig) light chain] single-domain intrabody that binds to the huntingtin (Htt) protein and has been engineered for antigen recognition in the absence of its intradomain disulfide bond, otherwise conserved in the Ig fold. Analytical ultracentrifugation demonstrated that the alpha Htt-V-L 12.3 domain is a stable monomer under physiological conditions even at concentrations >20 mu M. Using peptide SPOT arrays, we identified the minimal binding epitope to be EKLMKAFESLKSFQ, comprising the N-terminal residues 5-18 of Htt and including the first residue of the poly-Gin stretch. X-ray structural analysis of alpha Htt-V-L both as apo protein and in the presence of the epitope peptide revealed several interesting insights: first, the role of mutations acquired during the combinatorial selection process of the alpha Htt-V-L 12.3 domain-initially starting from a single-chain Fv fragment-that are responsible for its stability as an individually soluble Ig domain, also lacking the disulfide bridge, and second, a previously unknown mode of antigen recognition, revealing a novel paratope. The Htt epitope peptide adopts a purely alpha-helical structure in the complex with alpha Htt-V-L and is bound at the base of the complementarity-determining regions (CDRs) at the concave beta-sheet that normally gives rise to the interface between the V-L domain and its paired V-H (variable domain Ig heavy chain) domain, while only few interactions with CDR-L1 and CDR-L3 are formed. Notably, this noncanonical mode of antigen binding may occur more widely in the area of in vitro selected antibody fragments, including other Ig-like scaffolds, possibly even if a V-H domain is present.
- 出版日期2011-12-2
- 单位河北医科大学
