Role of Structural Flexibility of cpSRP43 in Binding Substrates during Post-Translational Targeting

The ability of the chloroplast signal recognition particle (cpSRP) to post-translationally target light harvesting complex proteins (LHCs) to the thylakoid membrane relies on a chloroplast-specific subunit, cpSRP43. cpSRP43 is a multidomain protein that forms a heterodimer with the conserved GTPase, cpSRP54, and subsequently interacts with LHCP substrates to form a soluble targeting complex in the chloroplast stroma. Single-molecule Forster Resonance Energy Transfer (smFRET) and molecular dynamics simulations was employed to determine how the inter-domain structural dynamics of cpSRP43 is affected by binding to cpSRP54 to help address how the formation of the cpSRP heterodimer enables LHCPs to bind and adopt an insertion-competent transit complex. Our results reveal significant inter-domain dynamics (i.e. flexibility) in cpSRP43 across the whole protein with 3 major conformations being identified. Upon binding to cpSRP54, there is a reduction in the flexibility of cpSRP43 in certain regions of the protein. Using isothermal titration calorimetry, we found that the affinity of the L18 recognition site of LHCP to cpSRP43 increased when cpSRP43 was complexed with a cpSRP54 peptide, which corresponds to the cpSRP43 binding site on cpSRP54. These results support the model that cpSRP54 promotes transit complex formation by regulating the inter-domain structural dynamics of cpSRP43.