Conformational dynamics of a membrane protein chaperone enables spatially regulated substrate capture and release

Abstract

Membrane protein biogenesis poses enormous challenges to cellular protein homeostasis and requires effective molecular chaperones. Compared with chaperones that promote soluble protein folding, membrane protein chaperones require tight spatiotemporal coordination of their substrate binding and release cycles. Here we define the chaperone cycle for cpSRP43, which protects the largest family of membrane proteins, the light harvesting chlorophyll a/b-binding proteins (LHCPs), during their delivery. Biochemical and NMR analyses demonstrate that cpSRP43 samples three distinct conformations. The stromal factor cpSRP54 drives cpSRP43 to the active state, allowing it to tightly bind substrate in the aqueous compartment. Bidentate interactions with the Alb3 translocase drive cpSRP43 to a partially inactive state, triggering selective release of LHCP's transmembrane domains in a productive unloading complex at the membrane. Our work demonstrates how the intrinsic conformational dynamics of a chaperone enables spatially coordinated substrate capture and release, which may be general to other ATP-independent chaperone systems.membrane protein biogenesis | molecular chaperone | signal recognition particle | protein dynamics | NMR spectroscopy P rotein homeostasis is essential for all cells and requires proper control of the folding, localization, and interactions of proteins. The biogenesis of membrane proteins poses a particular challenge to protein homeostasis. Before arrival at the membrane, newly synthesized membrane proteins need to traverse aqueous cellular compartments where they are highly prone to aggregation. Thus, the posttranslational targeting of membrane proteins relies critically on effective molecular chaperones that maintain nascent membrane proteins in translocation competent states. Many examples illustrate the intimate link between chaperone function and membrane protein biogenesis: SecB, Skp, and SurA protect bacterial outer membrane proteins (1-5), and Hsp70 homologs assist the import of mitochondrial or chloroplast proteins (6).Our understanding of membrane protein chaperones lags far behind that for soluble proteins, such as DnaK and GroEL. All chaperones need to switch between "open" and "closed" conformations to allow substrate release and binding, respectively. For many chaperones that promote the folding of soluble proteins, these switches can be driven either by ATPase cycles, such as Hsp70 (7) and GroEL (8), or by changes in environmental conditions, such as the acid-induced HdeA (9, 10) and oxidationinduced Hsp33 (11). In contrast, membrane protein chaperones must regulate their action spatially: they must effectively capture substrate proteins in the aqueous phase, and then facilely and productively release them at the target membrane. With few exceptions (1, 2), how membrane protein chaperones achieve spatiotemporal coordination of their chaperone cycle is not well understood.The light harvesting chlorophyll a/b-binding proteins (LHCPs) provide an excellent model system to address these quest...

Comparative Analysis of Light-Harvesting Antennae and State Transition in chlorina and cpSRP Mutants

“…Interestingly, levels of LHC proteins were clearly higher in ffc than in chaos, chaos/ffc, and cpftsy (Fig. 1C), indicating that cpSRP43 functions predominantly and independently from cpSRP54 in targeting of LHC proteins to the thylakoid membranes (Tzvetkova-Chevolleau et al, 2007;Liang et al, 2016). In summary, our initial results suggest that malfunction of the cpSRP pathway depresses steady-state levels of both LHCI and LHCII, while strongly reduced Chl b biosynthesis preferentially affects LHCII.…”

Anionic Phospholipids and the Albino3 Translocase Activate Signal Recognition Particle-Receptor Interaction during Light-harvesting Chlorophyll a/b-binding Protein Targeting

“…cpSRP43 con-tains a substrate binding domain that recognizes LHCPs and two chromodomains, CD2 and CD3 (32,33). CD2 binds the C-terminal tail of the cpSRP54 M-domain to form the cpSRP, and CD3 provides an interaction site with the Alb3 translocase (see below) (34,35).…”

A Protean Clamp Guides Membrane Targeting of Tail-Anchored Proteins

“…The dynamic opening of the Get3•TA complex is unexpected, as crystallographic analyses suggested that in open Get3, the hydrophobic groove for TMD binding becomes discontiguous (7,26). Intriguingly, our recent (12) and current measurements showed that Get3•TA complexes exhibit high kinetic stability: Using a membrane protein chaperone, cpSRP43 (27,28), as an inert TA trap, the timescale for spontaneous dissociation of Bos1 from Get3 was measured to be ∼4 h (Fig. S4B).…”

“…Plasmids for recombinant expression of Get3, Get4/5, Get1CD, and a superactive mutant of the cpSRP43 chaperone (intein-cpSRP43) and for in vitro translation of 3xStrep-SUMOnc-Bos1-opsin have been described (10,12,14,28). DNA encoding 2xStrep-Sbh1 was in the pACYCDuet-1 vector.…”

“…Get3, Get4/5, Get1CD, and intein-cpSRP43 were expressed and purified as described (10,12,14,28). E. coli S30 lysate and T7 RNA polymerase were prepared as described (12,37).…”