SUBSTRATE SEQUENCE DETERMINANTS GOVERNING THE SPECIFICITY OF CLPS1 IN PLANT CHLOROPLASTS

AGUILAR LUCERO, DIANELA; CECCARELLI, EDUARDO A.; MOGK, AXEL; ROSANO, GERMÁN L.

Congreso; LVI SAIB Meeting ?XV SAMIGE Meeting; 2020

Proteolysis is not a random process. Components of proteolytic complexes recognize certain features present in proteins thatneed to be eliminated. The ClpS family of adaptors of the Clp system identifies N-terminal destabilizing residues in theirtargets. This phenomenon is known as the bacterial N-end rule and dictates that substrates bearing N-terminal phenylalanine,tyrosine, tryptophan, or leucine are recognized by ClpS and then delivered to the protease. In Arabidopsis thaliana, ClpS1seems to be the cognate N-recognin but its specificity is still not fully elucidated. Its structure has only been defined by insilico modeling, and residues responsible for substrate recognition have been presumed by sequence alignment to bacterialhomologs. In this work, the sequence determinants that regulate the specificity of chloroplastic ClpS1 were analyzed. To gaininsight into the structure of ClpS1 and its ligand binding properties, we undertook a high-resolution solution-state NMRanalysis. A residue-specific analysis showed that ClpS1 contains two distinctive regions, one intrinsically disordered, fromresidue 1 to 36 and a folded region, rich in secondary structure elements that contain residues 39?110. To understand thestructural determinants that modulate the binding of ClpS1 to its substrates, increasing amounts of L-Phe-amide were addedtoClpS1 and the changes were followed in the 1H-15N correlation NMR spectra. Sub-stoichiometric additions of L-Phe-amideled to the disappearance of a group of ClpS1 resonances around some residues and a decrease in intensity for others. At higherL-Phe-amide: ClpS1 ratios, a new set of resonances appeared, whose intensities increased with additions of the substrate.These results indicate that ClpS1 binds to L-Phe-amide and that the interaction is in slow exchange, suggesting that the affinityof the protein for this substrate is high (Kd < 10 µM). The chemical shift perturbation analysis upon L-Phe-amide additionrevealed that the binding site is located between the first and second α-helices and the N-terminal portion of the first β-sheet.NMR titration analysis using L-Trp-amide as a substrate gave similar profiles. NMR titration experiments with L-Trp-amideand L-Phe-amide revealed that the Kd for each interactor lies in the low µM range. For these reasons we used fluorescenceanisotropy, a technique better suited for Kd determination. The fluorescence anisotropy of L-Trp-amide was recorded whilevarying the concentration of ClpS1. ClpS1 bound to L-Trp-amide with a Kd of 2.20 µM. To calculate the Kd for the bindingof ClpS1 to L-Phe-amide, an anisotropy competition assay was set up. ClpS1 bound to L-Phe-amide with a similar but higherstrength than L-Trp-amide. In conclusion, ClpS1 shows an overall architecture and secondary structure elements common tobacterial ClpS. Substrate binding does not elicit major conformational changes but instead provokes local remodeling of keyresidues at or near the proposed substrate-binding cavity. Furthermore, ClpS1 interacts with L-Phe-amide and L-Trp-amidewith high affinity and the binding site is located at the first and second α-helices.