Figure 1: NMR analysis of PEX19 farnesylation.
(a) Schematic overview of human PEX19: the unfolded N-terminus of PEX19 interacts with the peroxisomal membrane proteins PEX3 and PEX14, the C-terminus harbours the α-helical cargo binding region and the farnesyl recognition sequence CaaX. Farnesyl transferase catalyses the transfer of the farnesyl moiety from farnesyl pyrophosphate to the cysteine of the CaaX box. (b) Overlay of 1H,15N HSQC spectra of PEX19 CTD with (red) and without (black) farnesylation. Amide resonances that are strongly affected by farnesylation are annotated. (c) Chemical shift perturbation (Δδ) of amide protons induced by farnesylation (top), {1H}-15N heteronuclear NOE (middle) and solvent paramagnetic relaxation enhancement (sPRE) rates for amide protons (bottom) are shown for PEX19 CTD with (red) and without (black) farnesylation. Error bars for heteronuclear NOE data based on s.d. of noise in the individual spectra were calculated using error propagation. Error bars for sPRE data represent fitting error of proton R1 relaxation rates recorded from different concentrations of Gd(DTPA-BMA). The large changes in the C-terminal region that harbours the CaaX box are highlighted by a red box. The amino acid sequence and secondary structure elements of the PEX19 CTD are indicated on top, with aliphatic residues involved in farnesyl binding highlighted in yellow. Point mutations of residues that contact the farnesyl group or involved in PMP recognition are indicated as green and purple letters, respectively.
