Transient local secondary structure in the intrinsically disordered C-term of the Albino3 insertase

Albino3 (Alb3) is an integral membrane protein fundamental to the targeting and insertion of light-harvesting complex (LHC) proteins into the thylakoid membrane. Alb3 contains a stroma-exposed C-terminus (Alb3-Cterm) that is responsible for binding the LHC-loaded transit complex before LHC membrane insertion. Alb3-Cterm has been reported to be intrinsically disordered, but precise mechanistic details underlying how it recognizes and binds to the transit complex are lacking, and the functional roles of its four different motifs have been debated. Using a novel combination of experimental and computational techniques such as single-molecule fluorescence resonance energy transfer, circular dichroism with deconvolution analysis, site-directed mutagenesis, trypsin digestion assays, and all-atom molecular dynamics simulations in conjunction with enhanced sampling techniques, we show that Alb3-Cterm contains transient secondary structure in motifs I and II. The excellent agreement between the experimental and computational data provides a quantitatively consistent picture and allows us to identify a heterogeneous structural ensemble that highlights the local and transient nature of the secondary structure. This structural ensemble was used to predict both the inter-residue distance distributions of single molecules and the apparent unfolding free energy of the transient secondary structure, which were both in excellent agreement with those determined experimentally. We hypothesize that this transient local secondary structure may play an important role in the recognition of Alb3-Cterm for the LHC-loaded transit complex, and these results should provide a framework to better understand protein targeting by the Alb3-Oxa1-YidC family of insertases.