1H, 13C and 15N resonance assignments for the microtubule-binding domain of the kinetoplastid kinetochore protein KKT4 from Trypanosoma brucei

Abstract

KKT4 is a kinetoplastid-specific microtubule-binding kinetochore protein that lacks significant similarity to any known kinetochore or microtubule-binding proteins. Here we present the ¹H, ¹³C and ¹⁵N resonance assignments for several fragments from the microtubule-binding domain of KKT4 (KKT4^115-343) from Trypanosoma brucei. These assignments provide the starting point for detailed investigations of the structure, dynamics and interactions of the microtubule-binding region of KKT4.

Keywords: KKT4; Kinetochore; Kinetoplastid; NMR resonance assignments; Trypanosomes.

Fig. 1:

750 MHz ¹H–¹⁵N BEST-TROSY spectra of KKT4^115–343 in 25 mM HEPES, 150 mM NaCl and 0.5 mM TCEP (95% H₂O/5% D₂O), at pH 7.2, 20 °C. a This spectrum is contoured at a low level to highlight the range of peak intensities observed for KKT4^115–343. b The peak assignments for backbone amides of the C-terminal region (residues 231–343) of KKT4^115–343 are annotated. The inset is an expansion of the area outlined with dashed lines. Peaks in the region of 111–114 ppm and upfield of ~ 7.6 ppm are artefacts in the BEST-TROSY arising from incomplete cancellation of signals from the side chain amides of Asn and Gln which have not been assigned. The three circled peaks in a correspond to Y278 (¹⁵N = 120 ppm), T279 (¹⁵N = 117 ppm) and L280 (¹⁵N = 124 ppm) in a minor species involving P281 in a cis peptide bond. The three peaks in squares in a correspond to Y278 (¹⁵N = 123 ppm), T279 (¹⁵N = 118 ppm) and L280 (¹⁵N = 127 ppm) in a minor species involving P277 in a cis peptide bond

Fig. 2:

750 MHz ¹H–¹⁵N BEST-TROSY spectrum of KKT4^115–174 in 25 mM HEPES, 150 mM NaCl and 0.5 mM TCEP (95% H₂O/5% D₂O), at pH 7.2, 20 °C. The peak assignments for backbone amides are annotated. Peaks in the region of 111–114 ppm and upfield of ~ 7.6 ppm are artefacts in the BEST-TROSY arising from incomplete cancellation of signals from the side chain amides of Gln; these NH₂ groups have been assigned in the HSQC spectrum shown in Supplementary Fig. 3A

Fig. 3:

750 MHz ¹H–¹⁵N BEST-TROSY spectrum of KKT4^145–232 in 25 mM HEPES, 150 mM NaCl and 0.5 mM TCEP (95% H₂O/5% D₂O), at pH 7.2, 30 °C. The peak assignments for backbone amides are annotated. Peaks in the region of 111–114 ppm and upfield of ~ 7.6 ppm are artefacts in the BEST-TROSY arising from incomplete cancellation of signals from the side chain amides of Asn and Gln; these NH₂ groups have been assigned in the HSQC spectrum shown in Supplementary Fig. 3B