Conformational free energy landscape of inward- and outward-facing states of peptidase-containing ATP-binding cassette transporter PCAT1
Membrane transporters rely on large-scale structural changes to actively transport materials across the cell membrane. Structural studies of transporters are often hampered by the conformational flexibility of these proteins. We have recently developed a system-specific, mol. dynamics based, enhanced sampling method to study the conformational free energy landscapes of membrane transporters (Moradi et al., PNAS 110:18916 2013, Moradi et al., JCTC 10:2866 2014, Moradi et al., Nat Commun 6:8393 2015). Here we have employed this novel method to study a peptidase-contg. ATP-binding cassette (ABC) transporter (PCAT). PCATs process and secrete polypeptides and proteins, functioning as a sec-independent protein-translocation machine. All ABC transporters are powered by ATP binding and hydrolysis in a pair of conserved nucleotide binding domains (NBDs) which form a closed dimer upon ATP binding and sep. upon hydrolysis. NBD dimerization (dissocn.) results in the stabilization of the outward-facing (inward-facing) state at the transmembrane domains (TMDs), consistent with the alternating-access mechanism. In addn. to these domains, PCATs have two peptidase domains (PDs) which belong to the cysteine protease superfamily. The first crystal structure of a full-length PCAT, PCAT1 was reported recently in the inward-facing state, along with its structure in an outward-facing (but occluded) state, excluding the PD domains (Lin et al., Nature 523:425 2015). Although these crystal structures are generally consistent with the structures of other ABC transporters, the detailed structural information such as the degree of opening to the intra- or extracellular side in the inward- or outward-facing state remains questionable. We have performed a large set of equil., nonequil., biased, and unbised simulations based on both crystal structure of PCAT1 modeled in explicit membrne and water to characterize its conformational landscape in both nucleotide-free inward-facing and ATP-bound outward-facing states.