The exact biological role of mitochondrial supercomplexes remains debated, particularly their role in guiding redox proteins across membranes during energy conversion. We integrate multiscale modeling and single particle cryo-electron microscopy (cryo-EM) to examine electron transfer in mitochondrial supercomplexes composed of complexes III and IV (CIII and CIV). Using bioinformatic and entropy-based methods, we generated structural ensembles capturing conformations of CIII's disordered QCR6 hinge within the yeast CIII2CIV2 supercomplex. Molecular and Brownian Dynamics simulations reveal that these negatively charged hinge states electrostatically couple with redox proteins, promoting their binding and directional diffusion across the membrane on millisecond timescales. Rather than hindering transfer, disorder lowers the diffusion barrier. Anionic lipids reinforce this recognition by retaining a membrane pool of redox proteins when hinge length is critical. Cryo-EM models of ΔQCR6 show large rearrangements, yet maintain a robust electrostatic environment enabling surface-mediated transfer despite reduced charge. Overall, electron carriers confined on bioenergetic membranes follow a refolding-guided diffusion mechanism that enhances supercomplex energy conversion efficiency by nearly 30%.

A comprehensive understanding of lipid structure and dynamics is essential to understanding biomembrane functions. Based on the flexible surface model (FSM), we highlight the significance of proteolipid interactions and their out-of-plane couplings, which influence membrane functionality through curvature elastic forces. These forces, underscored by spontaneous curvature and bending rigidity, are paramount to distinguishing the FSM from the earlier fluid-mosaic model. Investigating membrane mimics consisting of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) may contribute to addressing this question [1]. Here we used solid-state deuterium NMR spectroscopy to investigate the structural dynamics of unsaturated phospholipid mixtures with varying PC to PE headgroups [2]. Using POPC-d31 as a probe lipid, we analyzed the structural and mechanical properties of 1,2-dioleoy-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and their binary mixtures. Measurements were performed on liquid-crystalline state multilamellar dispersions as a function of temperature. The order parameter (SCD) profiles indicated that the quadrupolar splitting remains constant across a probe lipid concentration of 1–15 mol% for both PE and PC lipids, while the mol% of POPC-d31 influences the transition from lamellar to hexagonal phase in DOPE lipids. In the liquid-crystalline phase, DOPE lipids exhibited a constrained acyl chain mobility, leading to a more pronounced molecular order than DOPC. Increasing DOPE concentration reduced the area per molecule at the lipid/water interface, affecting acyl chain configurational freedom and increasing order. Additionally, DOPE membranes showed higher bending rigidity than DOPC, highlighting it is driven primarily by increased lipid packing, correlating with the area compressibility modulus. The use of a probe lipid elucidates the intricate properties of complex lipid systems, facilitating a deeper understanding of how membrane mechanics are intertwined with critical biological functions.

The modeling of diffusion processes, particularly in confined or crowded environments, is considered one of the perennial challenges of classical molecular dynamics or MD simulations. By making assumptions of a friction-dominated regime, Brownian dynamics or BD methods offer an elegant computational solution for studying biomolecular diffusion at the milliseconds timescale, still maintaining the pairwise interactions of MD—a tool dubbed atom resolved BD or ARBD. It is nontrivial to conceive such simulations, given requirements to (1) periodically parametrize BD when MD visits novel conformations, (2) reset pH and charge environment, and (3) update visualization and analysis scripts for the redefined Brownian particles.

The mitochondrial permeability transition pore (mPTP) leads to cell death upon its activation. The past several decades have seen many studies regarding the modulation of the mPTP, but little is known about the structural components that contribute to its formation. Evidence now exists, supporting that the ATP synthase c-subunit houses the leak channel for the mPTP. Experimental evidence has shown that ATP Synthase forms voltage-gated and Ca2+-activated channels, consistent to what is known about the opening of the mPTP. Previous studies claim the ATP synthase c-ring is occupied by lipid and/or detergent molecules, claiming that a leak channel cannot be formed. These studies have sparked much controversy regarding the structural identity of the mPTP leak channel. In this work, we discovered the ATP synthase c-subunit forms a leak channel upon introducing voltage. We performed molecular dynamics simulations on the ATP synthase c-subunit under an electric field, ranging from −180 mV to +180 mV. Our results show significant conformational changes of the c-ring, including a dipole on the c-subunit monomer that aids in voltage-sensing, and lipids exiting the c-ring as wetting occurs. We find similar results in high-conductance and low-conductance mutants. Initially, we observed a bottleneck at a key glutamic acid residue that is responsible for proton transport in the c-ring. To overcome this bottleneck, we introduced a series of deprotonations on these glutamic acid residues to promote wetting, resulting in a full leak channel. Additionally, we increased our voltage to 1V once the channel was formed to attract ions into the pore, based off of previous literature. With our results, we seek to answer a long-standing question about the gating mechanism of ATP synthase c-subunit leak channel and the structural identity of the mPTP.

Alzheimer’s disease (AD) is a progressive neurodegenerative disease, resulting in an irreversible deterioration of multiple brain regions associated with cognitive dysfunction. Phosphorylation of the microtubule-associated protein, Tau, is known to occur decades before symptomatic AD. The Src family of tyrosine kinases are known to phosphorylate select tyrosine sites on Tau and promote microtubule disassembly and subsequent neurofibrillary tangle (NFT) formation. Our data show that the proto-oncogene, non-receptor tyrosine kinase Src colocalizes with a range of late (PHF1) to early (MC1) AD-associated phosphorylated Tau epitopes. The strongest co-occurrence is seen with MC1 (probability of MC1 given Src =100%), an early AD-specific conformational dependent epitope. Single-cell RNA sequencing data of 101 subjects show that Src is upregulated in both AD inhibitory and excitatory neurons. The most significantly affected, by orders of magnitude, were excitatory neurons which are the most prone to pathological Tau accumulation. We measured Src phosphorylation by mass spectrometry across a cohort of 48 patient neocortical tissues and found that Src has increased phosphorylation on Ser75, Tyr187, and Tyr440 in AD, showing that Src kinase undergoes distinct phosphorylation alterations in AD. Through Brownian dynamics simulations of Src and Tau, we show that as Tau undergoes the transition into disease-associated paired helical filaments, there is a notable seven-fold increase in Src contact with Tau. These results collectively emphasize Src kinase’s central role in Tau phosphorylation and its close association with Tau epitopes, presenting a promising target for potential therapeutic intervention.### Competing Interest StatementThe authors have declared no competing interest.

Here we address how membrane properties involving head group size, acyl chain unsaturation, and lipid packing affect their structure and dynamics. Strong out-of-plane proteolipid couplings considered by the flexible surface model (FSM) affect protein function through elastic forces described by the spontaneous curvature and the bending rigidity. We hypothesized that the mechanical properties and the molecular packing resulting from different lipid types are captured by introducing a perdeuterated POPC-d31 probe lipid. Di-monounsaturated phosphatidylcholine and phosphatidylethanolamine (DOPC, DOPE), and different ratios of DOPC/DOPE mixtures were studied using MD simulations combined with experimental solid-state 2H NMR spectroscopy. Residual quadrupolar couplings obtained from de-Paked powder-type spectra yielded order parameter (SCD) profiles for the individual acyl segments giving insights into average membrane structural properties such as area per lipid and bilayer thickness [1]. The corresponding spin-lattice relaxation rates (R1Z) for the resolved peaks followed a theoretical square-law dependence on SCD providing the bending rigidity [2]. Furthermore, MD simulations for the lipid systems were carried out and order parameters were calculated and compared against the NMR-observed SCD values. Utilization of the probe lipid POPC-d31 (1−10 mol%)affected the lamellar to hexagonal phase transition temperature of DOPE lipid systems but did not affect the quadrupolar splittings of DOPE and DOPC systems. Higher bending rigidity was observed for DOPE membranes versus DOPC supporting the idea that the membrane bending rigidity is primarily governed by increased lipid packing and is related to the area-compressibility modulus. Use of a probe lipid to capture membrane mechanical properties of complex lipid systems allow investigation of the relationship of membrane mechanics in vital biological functions.

Cellular bioenergetic processes responsible for providing energy to most lifeforms, photosynthesis and respiration, involve cooperative networks of proteins integrated into membrane domains. Exascale computational approaches, hand-in-hand with experiments, are necessary to learn how these processes are achieved. In particular, computations will reveal key organizational principles that underlie the efficient energy harvesting of protein networks. Application of these principles will aid the design of better man-made biohybrid devices in order to harness renewable energy for the world's future energy needs.

Are protein supercomplexes biological redundancies or do they have a functional role? This question has been perplexing biophysicists and bioenergeticists for decades, especially on the significances in crowded environments. Now, cryo-EM has brought forth remarkable insights into the structures of supercomplexes, but functional relevance of their intrinsically disordered regions remains a mystery. First, using a combination of maximum entropy (MaxEnT)-guided molecular simulations with low-resolution cryo-EM data, we have resolved the disordered Qcr6 subunit of respiratory complex III (CIII). Second, porting this whole-CIII model into the crowded yeast CIII2CIV2 supercomplex, long-timescale Brownian Dynamics computations were performed. We found that the negative charge on the disordered region surprisingly cooperates with that of the anionic lipids in the mitochondrial membrane to attract a pool of complementary protein substrates in its vicinity. Third, we discovered how the transient fold of Qcr6 expedites directional diffusion of the substrates from CIII to CIV that simultaneously leverage the supercomplex architecture as well as the electrostatic environment of the membrane. A re-classification of the EM images based on our computational model have brought to light new density features that indeed describe the 2-dimensional dynamic of the substrate in crowded supercomplexes. Finally, using multisequence alignment via Alphafold2, mutations are designed to control the diffusivity of substrates by tuning the disorder of the protein surface, for future biochemical validation.

The mitochondrial permeability transition pore (mPTP) leads to cell death upon its activation. The past several decades have seen many studies regarding the modulation of the mPTP, but little is known about the structure. There has been a growing body of evidence that the ATP synthase c-subunit houses the mPTP leak channel. Experimental evidence has shown that ATP Synthase forms voltage-gated and Ca2+-activated channels, consistent to what is known about mPTP activation. Previous experimental and molecular dynamics simulation studies demonstrate that the ATP synthase c-subunit is occupied by lipid and/or detergent molecules, claiming that a leak channel cannot be formed and that ATP synthase does not play a role in mPTP formation.

Antimicrobial peptides (AMPs) are promising candidates for next generation antimicrobial therapeutics. In mammalian cell membranes cholesterol plays a key regulatory function in antibiotic drug resistance and the immune response. However, the mechanism of action and the selectivity of AMPs towards bacterial membranes are not yet well understood. We hypothesized that differences in biophysical properties of mammalian versus bacterial membranes give rise to differences in peptide-membrane interactions which underlie the AMP mechanism and selectivity. Solid-state 2H NMR spectroscopy was employed to study di-monounsaturated phosphatidylcholine and phosphatidylethanolaminelipids (DOPC versus DOPE) bilayers in the presence of the antimicrobial peptide LL37 (2−4 mol%) and cholesterol (20−30% mol) as model systems mimicking mammalian and bacterial membranes. The lipid systems were probed with POPC-d31 (10 mol%) where the residual quadrupolar couplings yielded segmental order parameters (SCD) for the individual acyl segments. The LL37-peptide decreased the quadrupolar splittings indicating it increased the area per lipid, while cholesterol showed the opposite. The mean-torque model was used to calculate the key bilayer properties of area per lipid and bilayer thickness [2]. The model-free functional dependence of spin-lattice relaxation rates on SCD (square-law plots) indicated greater bending rigidity with cholesterol for both DOPC and DOPE membranes. By contrast LL37 decreased the bending rigidity of the membranes. Additionally all-atom molecular dynamics simulations for bilayers comprising POPE/DMPG/cardiolipin (90:5:5) and DMPC/cholesterol (90:10) were conducted with LL37 peptides on the bilayer surface to capture the molecular-level interactions. We observed partial insertion of the LL37-peptides in the POPE system, while the peptides were repelled from the membrane in the DMPC/cholesterol system. These observations reveal the surprising rivalry between cholesterol and AMPs which underlies AMP mechanism and selectivity.

While N2O2 tetraanionic ligands containing a strong N-amidate σ-donor are generally assumed to stabilise metal high valence states, we herein have shown that, in dianionic Cu(II)-diamido-diphenoxo complexes, H-bonding and electronic effects on the phenolate groups may modulate the electronic structure of their oxidised species from Cu(III) to Cu(II)-phenoxyl radical complexes; and so in the negative potential range. We observe that electron-poor phenolate complexes 22− and 32− oxidise to Cu(III) species, whereas electron rich phenolate complex 12− oxidises to a Cu(II)-phenoxyl radical. Our DFT results suggest that π-electron-rich phenolate rings in 12− are responsible for an increase of the HOMO orbital energy, bringing the HOMO-SOMO gap small enough to favour a ligand-based oxidation process. Further DFT-calculations have also shown that upon changing the o,p-phenol substituent from electron-widthdrawing groups (NO2) to electron-donating ones (OMe), the favoured oxidised state switches from Cu(III) to Cu(II)-radical. These results emphasize the use of the versatile diamido-diphenoxo backbone as a promising way to novel GO-chemical models, as well as molecular switches.

The mechanism of rotatory catalysis in ATP-hydrolyzing molecular motors remains an unresolved puzzle in biological energy transfer. Notwithstanding the wealth of available biochemical and structural information inferred from years of experiments, knowledge on how the coupling between the chemical and mechanical steps within motors enforces directional rotatory movements remains fragmentary. Even more contentious is to pinpoint the rate-limiting step of a multistep rotation process. Here, using vacuolar or V1-type hexameric ATPase as an exemplary rotational motor, we present a model of the complete 4-step conformational cycle involved in rotatory catalysis. First, using X-ray crystallography, a new intermediate or "dwell" is identified, which enables the release of an inorganic phosphate (or Pi) after ATP hydrolysis. Using molecular dynamics simulations, this new dwell is placed in a sequence with three other crystal structures to derive a putative cyclic rotation path. Free-energy simulations are employed to estimate the rate of the hexameric protein transformations and delineate allosteric effects that allow new reactant ATP entry only after hydrolysis product exit. An analysis of transfer entropy brings to light how the side-chain-level interactions transcend into larger-scale reorganizations, highlighting the role of the ubiquitous arginine-finger residues in coupling chemical and mechanical information. An inspection of all known rates encompassing the 4-step rotation mechanism implicates the overcoming of the ADP interactions with V1-ATPase to be the rate-limiting step of motor action.

Antimicrobial peptides (AMPs) exhibit cell selectivity and activity against microorganisms and are promising candidates as pharmaceutical agents. In mammalian cell membranes cholesterol plays a regulatory function in antibiotic drug resistance and the immune response. Our hypothesis is that differences in the peptide-membrane interactions versus cholesterol affect the bilayer properties giving a possible framework for selectivity of AMPs for bacterial membranes. Here we employed solid-state 2H NMR to compare the degree of softening or stiffening by AMPs compared to cholesterol in model di-monounsaturated phosphatidylcholine and phosphatidylethanolamine lipids (DOPC vs.

Cytochrome c2 (cyt. c2) is a major element in electron transfer between redox proteins in bioenergetic membranes. While the interaction between cyt. c2 and anionic lipids abundant in bioenergetic membranes has been reported, their effect on the shuttling activity of cyt. c2 remains elusive. Here, the effect of anionic lipids on the interaction and binding of cyt. c2 to the cytochrome bc1 complex (bc1) is investigated using a combination of molecular dynamics (MD) and Brownian dynamics (BD) simulations. MD is used to generate thermally accessible conformations of cyt. c2 and membrane-embedded bc1, which were subsequently used in multireplica BD simulations of diffusion of cyt. c2 from solution to bc1, in the presence of various lipids. We show that, counterintuitively, anionic lipids facilitate association of cyt. c2 with bc1 by localizing its diffusion to the membrane surface. The observed lipid-mediated bc1 association is further enhanced by the oxidized state of cyt. c2, in line with its physiological function. This lipid-mediated enhancement is salinity-dependent, and anionic lipids can disrupt cyt. c2-bc1 interaction at nonphysiological salt levels. Our data highlight the importance of the redox state of cyt. c2, the lipid composition of the chromatophore membrane, and the salinity of the chromatophore in regulating the efficiency of the electron shuttling process mediated by cyt. c2. The conclusions can be extrapolated to mitochondrial systems and processes, or any bioenergetic membrane, given the structural similarity between cyt. c2 and bc1 and their mitochondrial counterparts.

Adenovirus derived vectors, based on chimpanzee adenovirus Y25 (ChAdOx1) and human adenovirus type 26 are proving critical in combatting the 2019 SARS-CoV-2 pandemic. Following emergency use authorisation, scale up in vaccine administration has inevitably revealed vaccine related adverse effects; too rare to observe even in large Phase-III clinical trials. These include vaccine-induced thrombotic thrombocytopenia (VITT), an ultra-rare adverse event in which patients develop life-threatening blood clots 5-24 days following vaccination. To investigate vector-host interactions of ChAdOx1 underpinning VITT we solved the structure of the ChAdOx1 capsid by CryoEM, and the structure of the primary receptor tropism determining fiber-knob protein by crystallography. These structural insights have enabled us to unravel key protein interactions involved in ChAdOx1 cell entry and a possible means by which it may generate misplaced immunity to platelet factor 4 (PF4), a protein involved in coagulation. We use in vitro cell binding assays to show that the fiber-knob protein uses coxsackie and adenovirus receptor (CAR) as a high affinity binding partner, while it does not form a stable interface with CD46. Computational simulations identified a putative mechanism by which the ChAdOx1 capsid interacts with PF4 by binding in the spaces between hexon proteins, with downstream implications for the causes of VITT. Summary We present the structure of the ChAdOx1 viral vector, derived from chimpanzee adenovirus Y25 at 4.2Å resolution1. ChAdOx1 is in global use in the AstraZeneca vaccine, ChAdOx1 nCoV-19/AZD-1222, to combat the SARS-CoV-2 coronavirus pandemic. Recently observed, rare, adverse events make detailed mechanistic understanding of this vector key to informing proper treatment of affected patients and the development of safer viral vectors. Here, we determine a primary mechanism ChAdOx1 uses to attach to cells is coxsackie and adenovirus receptor (CAR), a protein which is identical in humans and chimpanzees. We demonstrate the vector does not form a stable CD46 interaction, a common species B adenovirus receptor, via its primary attachment protein. Further, we reveal the surface of the ChAdOx1 viral capsid has a strong electronegative potential. Molecular simulations suggest this charge, together with shape complementarity, are a mechanism by which an oppositely charged protein, platelet factor 4 (PF4) may bind the vector surface. PF4 is a key protein involved in the formation of blood clots2, and the target of auto-antibodies in heparin-induced immune thrombotic thrombocytopenia (HITT)3, an adverse reaction to heparin therapy which presents similarly to vaccine-induced immune thrombotic thrombocytopenia (VITT), a rare complication of ChAdOx1 nCoV-19 vaccination4–6. We propose a mechanism in which the ChAdOx1-PF4 complex may stimulate the production of antibodies against PF4, leading to delayed blood clot formation, as observed in VITT.

Antimicrobial peptides (AMPs) are found in the innate immune systems of most living organisms. These peptides exhibit cell selectivity, and activity against a broad spectrum of microorganisms, making them promising candidates as antimicrobial biomaterials. The AMPs are anchored with polymer tethers and biologically synthesized as functionalized biomaterials. We successfully prepared LL-37 conjugated biopolymer materials with antimicrobial activity, which switch to micelles at temperatures between 27-30 oC. Understanding the peptide-membrane interactions represents the basis for AMP's selectivity for bacterial cell membranes. We hypothesized that peptide insertion is assisted by membrane curvature. Models of a gram-negative bacterial outer membrane comprising POPE/DMPG/CL (90:5:5) and a membrane with the composition DMPC/DMPG/CL (90:5:5) were investigated using molecular dynamics simulations. The antimicrobial peptide LL-37 was arranged on the surface of the membranes as "carpets" of ordered peptides. We also performed all-atom molecular dynamics simulations using NAMD 2.13 to visualize the carpet-to-barrel or toroidal-pore transition. To determine the energetically favorable model, the non-bonded interactions between the carpet and pore models were compared using the NAMD energy. From our in-silico observations, the pore model is more favorable than the carpet model for the same peptide/lipid ratio. Critical values of the peptide/lipid ratio required for cell penetration were investigated to determine the rate of peptide insertion into the membrane with different LL-37 concentrations. From the simulation timescale (in microseconds), as the peptide/lipid ratio increased, partial insertion and membrane curvature were observed in the bacterial mimic model. Further, the C-terminal helix of LL-37 was observed to unfold when interacting with phosphate head groups of the lipids. The propensity for AMPs to insert into the lipid bilayer via the N-terminus or C-terminus was similar. These observations from molecular dynamics simulations provide a basis for designing more advanced functionalized antimicrobial-biomaterials.

Two independent structures of the proton-pumping, respiratory cytochrome bo3 ubiquinol oxidase (cyt bo3 ) have been determined by cryogenic electron microscopy (cryo-EM) in styrene-maleic acid (SMA) copolymer nanodiscs and in membrane scaffold protein (MSP) nanodiscs to 2.55- and 2.19-Å resolution, respectively. The structures include the metal redox centers (heme b, heme o3 , and CuB), the redox-active cross-linked histidine-tyrosine cofactor, and the internal water molecules in the proton-conducting D channel. Each structure also contains one equivalent of ubiquinone-8 (UQ8) in the substrate binding site as well as several phospholipid molecules. The isoprene side chain of UQ8 is clamped within a hydrophobic groove in subunit I by transmembrane helix TM0, which is only present in quinol oxidases and not in the closely related cytochrome c oxidases. Both structures show carbonyl O1 of the UQ8 headgroup hydrogen bonded to D75I and R71I In both structures, residue H98I occupies two conformations. In conformation 1, H98I forms a hydrogen bond with carbonyl O4 of the UQ8 headgroup, but in conformation 2, the imidazole side chain of H98I has flipped to form a hydrogen bond with E14I at the N-terminal end of TM0. We propose that H98I dynamics facilitate proton transfer from ubiquinol to the periplasmic aqueous phase during oxidation of the substrate. Computational studies show that TM0 creates a channel, allowing access of water to the ubiquinol headgroup and to H98I.

We observe previously unknown interactions between clinically important adenovirus vector capsids, platelet factor 4, and CAR.

The mitochondrial respiratory chain, formed by five protein complexes, utilizes energy from catabolic processes to synthesize ATP. Complex I, the first and the largest protein complex of the chain, harvests electrons from NADH to reduce quinone, while pumping protons across the mitochondrial membrane. Detailed knowledge of the working principle of such coupled charge-transfer processes remains, however, fragmentary due to bottlenecks in understanding redox-driven conformational transitions and their interplay with the hydrated proton pathways. Complex I from Thermus thermophilus encases 16 subunits with nine iron-sulfur clusters, reduced by electrons from NADH. Here, employing the latest crystal structure of T. thermophilus complex I, we have used microsecond-scale molecular dynamics simulations to study the chemo-mechanical coupling between redox changes of the iron-sulfur clusters and conformational transitions across complex I. First, we identify the redox switches within complex I, which allosterically couple the dynamics of the quinone binding pocket to the site of NADH reduction. Second, our free-energy calculations reveal that the affinity of the quinone, specifically menaquinone, for the binding-site is higher than that of its reduced, menaquinol form-a design essential for menaquinol release. Remarkably, the barriers to diffusive menaquinone dynamics are lesser than that of the more ubiquitous ubiquinone, and the naphthoquinone headgroup of the former furnishes stronger binding interactions with the pocket, favoring menaquinone for charge transport in T. thermophilus. Our computations are consistent with experimentally validated mutations and hierarchize the key residues into three functional classes, identifying new mutation targets. Third, long-range hydrogen-bond networks connecting the quinone-binding site to the transmembrane subunits are found to be responsible for proton pumping. Put together, the simulations reveal the molecular design principles linking redox reactions to quinone turnover to proton translocation in complex I.