Hexa-histidin tag position influences disulfide structure but not binding behavior of in vitro folded N-terminal domain of rat corticotropin-releasing factor receptor type 2a

Abstract

The oxidative folding, particularly the arrangement of disulfide bonds of recombinant extracellular N-terminal domains of the corticotropin-releasing factor receptor type 2a bearing five cysteines (C2 to C6), was investigated. Depending on the position of a His-tag, two types of disulfide patterns were found. In the case of an N-terminal His-tag, the disulfide bonds C2-C3 and C4-C6 were found, leaving C5 free, whereas the C-terminal position of the His-tag led to the disulfide pattern C2-C5 and C4-C6, and leaving C3 free. The latter pattern is consistent with the disulfide arrangement of the extracellular N-terminal domain of the corticotropin-releasing factor (CRF) receptor type 1, which has six cysteines (C1 to C6) and in which C1 is paired with C3. However, binding data of the two differently disulfide-bridged domains show no significant differences in binding affinities to selected ligands, indicating the importance of the C-terminal portion of the N-terminal receptor domains, particularly the disulfide bond C4-C6 for ligand binding.

Figure 1.

Alignment of the expressed CRF_2(a) receptor N termini with an N- and C-terminal (His)₆-tag, respectively.

Figure 2.

Disulfide pattern of the expressed, in vitro refolded CRF_2(a) receptor N termini with an N- and C-terminal (His)₆-tag (B,C), respectively, in comparison to the in vivo folded CRF₁ receptor N terminus (A). Unbound cysteines are marked with *. Cysteine 1 in CRF_2(a) receptor N termini is located in the signal sequence and labeled in italics.

Figure 3.

Inhibition of specific [¹²⁵I-Tyr⁰]-urocortin 1 binding to CRF-rNT by the unlabeled CRF-like ligands urocortin 1 (8–40) and astressin by using the SPA technology. All values are given as mean ± SEM. (A) Binding of urocortin 1 (8–40) to the CRF_2(a) receptor N terminus with C-terminal (His)₆-tag (filled circles) and N-terminal (His)₆-tag (open circles). (B) Binding of astressin to the CRF_2(a) receptor N terminus with C-terminal (His)₆-tag (filled circles) and N-terminal (His)₆-tag (open circles).