Fig. 4.

Ltn10 and Ltn40 are functionally distinct. (A) An engineered disulfide (red) locks the CC3 variant into the Ltn10 fold (15), but replacement of Trp⁵⁵ (blue) with Asp (W55D) yields exclusively the Ltn40 species. With the exception of signals adjacent to the amino acid substitution, the HSQC spectrum of W55D is identical to the Ltn40 spectrum obtained with WT Ltn. (B) Ca²⁺ flux response of XCR1 expressing HEK293 cells to WT (black), CC3 (orange), and W55D (blue) Ltn. Dashed line marks the addition of 200 nM chemokine. (C) Elution profiles for WT (black), CC3 (orange), and W55D (blue) Ltn from heparin-Sepharose. (D) Heparin selectively precipitates the Ltn40 conformation. (Left) Before the addition of heparin tetrasaccharide, HSQC signals are observed for both W55D (blue) and CC3 (orange). (Center and Right) Heparin addition results in the broadening (Center) and disappearance of W55D amide peaks (Right). (E) Titration of WT Ltn with semipurified heparin shifts the conformational equilibrium toward Ltn40 based on changes in the Trp⁵⁵ emission wavelength (blue). CC3 emission is unaltered by heparin (orange). Emission maxima for Ltn10 and Ltn40 are indicated. (F) Ca²⁺ flux response of WT (black) and CC3 (orange) after incubation with an equimolar concentration of semipurified heparin. Heparin prevents WT Ltn-XCR1 association, as sequential addition of CC3 elicits XCR1 activation.