Molecular mechanism of cardiolipin-mediated assembly of respiratory chain supercomplexes.

Mitochondria produce most of the ATP consumed by cells through the respiratory chain in their inner membrane. This process involves protein complexes assembled into larger structures, the respiratory supercomplexes (SCs). Cardiolipin (CL), the mitochondrial signature phospholipid, is crucial for the structural and functional integrity of these SCs, but it is as yet unclear by what mechanism it operates. Our data disclose the mechanism for bulk CL in gluing SCs, steering their formation, and suggest how it may stabilize specific interfaces. We describe self-assembly molecular dynamics simulations of 9 cytochrome bc1 (CIII) dimers and 27 cytochrome c oxidase (CIV) monomers from bovine heart mitochondria embedded in a CL-containing model lipid bilayer, aimed at mimicking the crowdedness and complexity of mitochondrial membranes. The simulations reveal a large diversity of interfaces, including those of existing experimental CIII/CIV SC models and an alternative interface with CIV rotated by 180°. SC interfaces enclose 4 to 12 CLs, a ∼10 fold enrichment from the bulk. Half of these CLs glue complexes together using CL binding sites at the surface of both complexes. Free energy calculations demonstrate a larger CL binding strength, compared to other mitochondrial lipids, that is exclusive to these binding sites and results from non-additive electrostatic and van der Waals forces. This study provides a key example of the ability of lipids to selectively mediate protein-protein interactions by altering all ranges of forces, lubricate protein interfaces and act as traffic control agents steering proteins together.

Molecular dynamics simulations reveal that apo-HisJ can sample a closed conformation.

The Escherichia coli histidine binding protein HisJ is a type II periplasmic binding protein (PBP) that preferentially binds histidine and interacts with its cytoplasmic membrane ABC transporter, HisQMP2 , to initiate histidine transport. HisJ is a bilobal protein where the larger Domain 1 is connected to the smaller Domain 2 via two linking strands. Type II PBPs are thought to undergo "Venus flytrap" movements where the protein is able to reversibly transition from an open to a closed conformation. To explore the accessibility of the closed conformation to the apo state of the protein, we performed a set of all-atom molecular dynamics simulations of HisJ starting from four different conformations: apo-open, apo-closed, apo-semiopen, and holo-closed. The simulations reveal that the closed conformation is less dynamic than the open one. HisJ experienced closing motions and explored semiopen conformations that reverted to closed forms resembling those found in the holo-closed state. Essential dynamics analysis of the simulations identified domain closing/opening and twisting as main motions. The formation of specific inter-hinge strand and interdomain polar interactions contributed to the adoption of the closed apo-conformations although they are up to 2.5-fold less prevalent compared with the holo-closed simulations. The overall sampling of the closed form by apo-HisJ provides a rationale for the binding of unliganded PBPs with their cytoplasmic membrane ABC transporters.

Identification of cardiolipin binding sites on cytochrome c oxidase at the entrance of proton channels.

The respiratory chain or oxidative phosphorylation system (OxPhos) generates most of the chemical energy (ATP) used by our cells. The cytochrome c oxidase (CcO) is one of three protein complexes of OxPhos building up a proton gradient across the inner mitochondrial membrane, which is ultimately used by the ATP synthase to produce ATP. We present molecular dynamic simulations of CcO in a mimic of the mitochondrial membrane, and identify precise binding sites of cardiolipin (CL, signature phospholipid of mitochondria) on the protein surface. Two of these CL binding sites reveal pathways linking CLs to the entrance of the D and H proton channels across CcO. CLs being able to carry protons our results strongly support an involvement of CLs in the proton delivery machinery to CcO. The ubiquitous nature of CL interactions with the components of the OxPhos suggests that this delivery mechanism might extend to the other respiratory complexes.

Key issues in the computational simulation of GPCR function: representation of loop domains.

Some key concerns raised by molecular modeling and computational simulation of functional mechanisms for membrane proteins are discussed and illustrated for members of the family of G protein coupled receptors (GPCRs). Of particular importance are issues related to the modeling and computational treatment of loop regions. These are demonstrated here with results from different levels of computational simulations applied to the structures of rhodopsin and a model of the 5-HT2A serotonin receptor, 5-HT2AR. First, comparative Molecular Dynamics (MD) simulations are reported for rhodopsin in vacuum and embedded in an explicit representation of the membrane and water environment. It is shown that in spite of a partial accounting of solvent screening effects by neutralization of charged side chains, vacuum MD simulations can lead to severe distortions of the loop structures. The primary source of the distortion appears to be formation of artifactual H-bonds, as has been repeatedly observed in vacuum simulations. To address such shortcomings, a recently proposed approach that has been developed for calculating the structure of segments that connect elements of secondary structure with known coordinates, is applied to 5-HT2AR to obtain an initial representation of the loops connecting the transmembrane (TM) helices. The approach consists of a simulated annealing combined with biased scaled collective variables Monte Carlo technique, and is applied to loops connecting the TM segments on both the extra-cellular and the cytoplasmic sides of the receptor. Although this initial calculation treats the loops as independent structural entities, the final structure exhibits a number of interloop interactions that may have functional significance. Finally, it is shown here that in the case where a given loop from two different GPCRs (here rhodopsin and 5-HT2AR) has approximately the same length and some degree of sequence identity, the fold adopted by the loops can be similar. Thus, in such special cases homology modeling might be used to obtain initial structures of these loops. Notably, however, all other loops in these two receptors appear to be very different in sequence and structure, so that their conformations can be found reliably only by ab initio, energy based methods and not by homology modeling.