Fig. 3: Mutational analyses of the amino-acid residue-binding pockets of the NTD and LarA.
From: Structural basis for the allosteric activation of Lon by the heat shock protein LarA
a In vitro reactions showing the essential role of Arg29 in the NTD of CcLon for LarA-mediated degradation of SciP. b SEC-MALS analysis showing the key role of Arg29 of the NTD in mediating LarA binding. c, In vitro reactions of CcLon demonstrating the important role of Val57 in the hydrophobic binding pocket of LarA for mediating SciP degradation. The gels shown in (a) and (c) are representative of three independent experiments with consistent results. Source data are provided as a Source Data file. d In vitro reactions investigating the role of the hydrophobic residues in the C-terminal degron of SciP for LarA-mediated degradation. Densitometry analysis of SciP remaining was performed for gel data run in triplicate. p values were calculated using an unpaired two-sided Welch’s t-test. In the absence of LarA (left graph): L81D (p = 0.0012 **), L84D (p = 0.0002 ***), and I89D (p = 0.0106 *). In the presence of LarA (right graph): L81D vs. L84D + I89D (p = 0.0002 ***), L81D vs. L81D + L84D + I89D (p = 0.0015 **), L84D vs. L84D + I89D (p = 0.0017 **), L84D vs. L81D + L84D + I89D (p = 0.0098 **), I89D vs. L84D + I89D (p = 0.0012 **), and I89D vs. L81D + L84D + I89D (p = 0.0074 **). The number sign (#) in (c) and (d) denotes the various LarA or SciP constructs, which are either wild-type (wt) or with indicated mutations, used in the reactions. e Fluorescence polarization (FP) assays to detect the binding between different NTD constructs and fluorescein isothiocyanate (FITC) labeled peptides (pep-) derived from the C-terminal degron of LarA. f, g FP assays to detect the interaction of FITC-labeled peptides, derived from the wild-type (wt) C-terminal sequences of SciP (pep-SciP-wt)(f) or LarA (pep-LarA-wt)(g), with indicated proteins or protein complexes. h FP assays showing poor protein binding of the LarA or SciP peptides with all hydrophobic residues replaced by alanine (-mut). Data in (d-h) are presented as mean values ± SD; n = 3 independent measurements. Source data are provided as a Source Data file.
