cpSRP43 is both highly flexible and stable: Structural insights using a combined experimental and computational approach

Abstract

The novel multidomain protein, cpSRP43, is a unique subunit of the post-translational chloroplast signal recognition particle (cpSRP) targeting pathway in higher plants. The cpSRP pathway is responsible for targeting and insertion of light-harvesting chlorophyll a/b binding proteins (LHCPs) to the thylakoid membrane. Nuclear-encoded LHCPs are synthesized in the cytoplasm then imported into the chloroplast. Upon emergence into the stroma, LHCPs form a soluble transit complex with the cpSRP heterodimer, which is composed of cpSRP43 and cpSRP54, a 54 kDa subunit homologous to the universally conserved GTPase in cytosolic SRP pathways. cpSRP43 is irreplaceable as a chaperone to LHCPs in their translocation to the thylakoid membrane and remarkable in its ability to dissolve aggregates of LHCPs without the need for external energy input. In previous studies, cpSRP43 has demonstrated significant flexibility and interdomain dynamics. However, the high flexibility and structural dynamics of cpSRP43 is yet unexplained by current crystal structures of cpSRP43. This is due, in part, to the fact that free full length cpSRP43 is so flexible that it is unable to crystalize. In this study, we explore the structural stability of cpSRP43 under different conditions using various biophysical techniques and find that this protein is concurrently highly stable and flexible. This conclusion is interesting considering that stable proteins typically possess a non-dynamic structure. Molecular dynamics (MD) simulations which correlated with data from biophysical experimentation were used to explain the basis of the extraordinary stability of cpSRP43. This combination of biophysical data and microsecond-level MD simulations allows us to obtain a detailed perspective of the conformational landscape of these proteins.