Fig. 1: The K113-conjugated Ub subunit employs its classical L8-I44-H68-V70 hydrophobic patch to interact with the hydrophobic groove of BAK.

a Chemical shift perturbations (CSPs) of BAK upon Ub conjugation. Left, superimposed ¹H-¹⁵N HSQC spectra of ¹⁵N-labeled BAK^K113C (black) and Ub^G76C ~ BAK^K113C mimetic (red). Right, the ¹H-¹⁵N CSPs of BAK upon K113-Ub conjugation are plotted against BAK residue number. The secondary elements of BAK are labeled at the top of the CSPs plot. Residues harboring large ¹H-¹⁵N CSPs are highlighted using blue arrows in the ¹H-¹⁵N HSQC spectrum (>0.04 ppm) and mapped on the BAK structure (2IMT.PDB⁴²) in red (>0.02 ppm). The α3-α5 helices constituting the hydrophobic groove of BAK are indicated on its structure. b CSPs of Ub conjugated to BAK. Left, superimposed ¹H-¹⁵N HSQC spectra of ¹⁵N-labeled Ub^G76C (black) and Ub^G76C ~ BAK^K113C mimetic (red). Right, The ¹H-¹⁵N CSPs of Ub upon linking to BAK are plotted against Ub residue number. The secondary elements of Ub are labeled at the top of the CSPs plot. Residues harboring large ¹H-¹⁵N CSPs are highlighted using blue arrows (>0.04 ppm) and mapped on the Ub structure (1UBQ.PDB⁷⁶) in red (>0.02 ppm). Residues of the L8-I44-H68-V70 hydrophobic patch are shown in stick representation. The ¹H-¹⁵N CSP is calculated as (0.5 × ΔH² + 0.1 × ΔN²)^0.5, where ΔH and ΔN stand for the changed chemical shift values in the proton and nitrogen dimensions, respectively. The gray dashed line indicates a CSP of 0.02 ppm.