Solution structure of lysine-free (K0) ubiquitin

Abstract

Lysine-free ubiquitin (K0-Ub) is commonly used to study the ubiquitin-signaling pathway, where it is assumed to have the same structure and function as wild-type ubiquitin (wt-Ub). However, the K0-Ub (15) N heteronuclear single quantum correlation NMR spectrum differs significantly from wt-Ub and the melting temperature is depressed by 19°C, raising the question of the structural integrity and equivalence to wt-Ub. The three-dimensional structure of K0-Ub was determined by solution NMR, using chemical shift and residual dipolar coupling data. K0-Ub adopts the same backbone structure as wt-Ub, and all significant chemical shifts can be related to interactions impacted by the K to R mutations.

Keywords: CS-Rosetta; K0-Ub; NMR; ubiquitin.

Figure 1

Spectra comparison between ubiquitin and K0-Ub. (A) ¹⁵N-¹H HSQC spectra overlay of ubiquitin (blue) and K0-Ub (red). (B) CSP between ubiquitin and K0-Ub. Average CSP of ¹H and ¹⁵N chemical shifts is calculated by the formula: CSP = [(Δδ²_NH + Δδ²_N/25)/2]^1/2 (ppm), where Δδ_NH and Δδ_N are the amide proton and nitrogen shift differences for conserved residues between ubiquitin and K0-Ub, respectively. “KR” represents lysine to arginine mutations, “P” represents proline and residues 24 and 53 are missing due to solvent exchange. Hydrogen bonds are connected with gray lines and salt bridges are connected with black lines.

Figure 2

Solution structure of K0-Ub compared with wt-Ub. (A) Plot of rescored Rosetta all-atom energy versus Cα RMSD relative to the lowest-energy model (bold dot on vertical axis). (B) 20 lowest-energy CS-Rosetta models of K0-Ub superimposed. (C) Superposition of best K0-Ub model (green) with ubiquitin (orange; PDB: 1UBQ).