High-resolution structure of eukaryotic Fibrillarin interacting with Nop56 amino-terminal domain

Experimental verification of the Nop56^1–166–Nop1^83–327 binding interface. (A) Left panel: overlay of size-exclusion chromatograms of Nop56^1–166 with Nop1^83–327 (black), Nop56^1–166 with Nop1^83–321 (green) and Nop56^1–166-Q35A/N39A with Nop1^83–327 (pink). Right panel: isothermal titration calorimetry binding curves of Nop1^83–327 with Nop56^1–166 (black) and Nop1^83–327 with Nop56^1–166-Q35A/N39A (pink). The approximate K_D values determined for each complex are given in the respective color. Only approximate K_D values can be provided, as stirring caused partial precipitation of Nop1^83–327 during the measurements, thus impeding an accurate estimate of the Nop1^83–327 concentration. The K_D of the Nop1^83–321–Nop56^1–166 complex could not be measured due to the poor solubility of Nop1^83–321. (B) Overlay of size-exclusion chromatograms of Nop56^1–166 with Nop1^83–327 (black), Nop56^1–166 with Nop1^83–327-R231E (magenta) and Nop56^1–166-E10K/E11K with Nop1^83–327 (red). (C) Left panel: overlay of the structures of the Sc Nop1^83–327–Nop56^1–166 complex and Pf Nop5–Fib complex showing the potential charge repulsion between Pf Nop5^1–123-R122 and K40 (pink) and Sc Nop1^83–327-K229 (blue) in a putative cross-species complex. Sc Nop56^1–166 is in green cyan and Pf Fib is in green. Middle panel: overlay of the structures of the Sc Nop1^83–327–Nop56^1–166 complex and Pf Nop5–Fib complex showing the steric clash between Pf Fib-R170 (green) and Nop56^1–166-α1 (green cyan) as well as the charge repulsion between Pf Fib-K143 and Nop56^1–166-R129 and R140 in a putative cross-species complex. Right panel: overlay of size-exclusion chromatograms showing that Nop56^1–166-E71K/Y158 and Nop1^83–327-D263R/M263K do not form a complex with wild-type Nop1^83–327 and Nop56^1–166, respectively. Nop56^1–166-E71K/Y158K is also unable to interact with Nop1^83–327-E222K, as expected. All chromatograms in A–C show the absorbance at 280 nm and use the following color code: Nop1^83–327 alone, blue; Nop56^1–166 alone, green cyan; Nop1^83–327 with Nop56^1–166, black; Nop1^83–327 with Nop56^1–166-Q35A/N39A, hot pink; Nop1^83–321 with Nop56^1–166, green; Nop1^83–327 with Nop56^1–166-E10K/E11K, red; Nop1^83–327-R231E with Nop56^1–166, pink; Nop1^83–327 with Nop56^1–166-E71K/Y158K, orange; Nop1^83–327-E222K with Nop56^1–166-E71K/Y158K, olive; Nop1^83–327-D263R/M263K with Nop56^1–166, purple. (D) 15% SDS polyacrylamide gels showing the content of all elution peaks from the chromatograms in A–C. Lanes belonging to a specific chromatogram are labeled in the same color as the corresponding chromatogram and as in the legend. Multiple lanes from the same chromatogram represent multiple fractions with increasing elution volumes. The faint band at ∼ 23 kDa corresponds to amino-terminal His-tagged Nop56^1–166, present because of incomplete cleavage.