Abstract
Protein targeting is critical in all living organisms and involves a signal recognition particle (SRP), an SRP receptor, and a translocase. In co-translational targeting, interactions among these proteins are mediated by the ribosome. In chloroplasts, the light-harvesting chlorophyll-binding protein (LHCP) in the thylakoid membrane is targeted post-translationally without a ribosome. A multidomain chloroplast-specific subunit of the SRP, cpSRP43, is proposed to take on the role of coordinating the sequence of targeting events. Here, we demonstrate that cpSRP43 exhibits significant interdomain dynamics that are reduced upon binding its SRP binding partner, cpSRP54. We showed that the affinity of cpSRP43 for the binding motif of LHCP (L18) increases when cpSRP43 is complexed to the binding motif of cpSRP54 (cpSRP54pep). These results support the conclusion that substrate binding to the chloroplast SRP is modulated by protein structural dynamics in which a major role of cpSRP54 is to improve substrate binding efficiency to the cpSRP.
Highlights
Targeting of proteins requires a signal recognition particle (SRP) and multiple protein interactions
The data presented in this study demonstrate that one role of cpSRP54 is to promote light-harvesting chlorophyll-binding protein (LHCP) binding to cpSRP43 by reducing interdomain protein dynamics in cpSRP43
Summary and Conclusions—We have used a combination of smFRET, MD simulations, and ITC to relate the interdomain structural dynamics of cpSRP43 to its function in post-translational targeting of LHCP
Summary
Targeting of proteins requires a signal recognition particle (SRP) and multiple protein interactions. We hypothesize that the order of these interactions will be controlled through changes in cpSRP43 structural dynamics that alter the relative position of these domains in response to association of cpSRP43 with each of its binding partners It is this hypothesis that forms the basis of the present study. Our results show that cpSRP43 alone exhibits wide interdomain conformational dynamic heterogeneity (i.e. high flexibility) but upon binding to cpSRP54 shows reduced regional flexibility We hypothesized that this reduced flexibility favors binding of LHCP targeting substrates, and this was tested using isothermal titration calorimetry (ITC). We found that the affinity of cpSRP43 for L18 increased by ϳ3-fold when cpSRP43 was complexed with a cpSRP54 peptide that corresponds to the cpSRP43 binding site on cpSRP54 Together, these findings support a model in which cpSRP54 modulates the structural dynamics of cpSRP43, which is critical for the promotion of efficient binding and localization of LHCP targeting substrates
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