Evolution of cell division: from shear mechanics to complex molecular machineries.

Cell division depends on sophisticated molecular machinery. However, wall-less forms of bacteria use a much simpler mechanism that mimics spontaneous division of synthetic lipid vesicles. Mercier et al. (2013) show that this "mechanical" division can be activated by increased lipid synthesis. Conceivably, the first cells divided via this route.

Six Functions of Respiration: Isn't It Time to Take Control over ROS Production in Mitochondria, and Aging Along with It?

Cellular respiration is associated with at least six distinct but intertwined biological functions. (1) biosynthesis of ATP from ADP and inorganic phosphate, (2) consumption of respiratory substrates, (3) support of membrane transport, (4) conversion of respiratory energy to heat, (5) removal of oxygen to prevent oxidative damage, and (6) generation of reactive oxygen species (ROS) as signaling molecules. Here we focus on function #6, which helps the organism control its mitochondria. The ROS bursts typically occur when the mitochondrial membrane potential (MMP) becomes too high, e.g., due to mitochondrial malfunction, leading to cardiolipin (CL) oxidation. Depending on the intensity of CL damage, specific programs for the elimination of damaged mitochondria (mitophagy), whole cells (apoptosis), or organisms (phenoptosis) can be activated. In particular, we consider those mechanisms that suppress ROS generation by enabling ATP synthesis at low MMP levels. We discuss evidence that the mild depolarization mechanism of direct ATP/ADP exchange across mammalian inner and outer mitochondrial membranes weakens with age. We review recent data showing that by protecting CL from oxidation, mitochondria-targeted antioxidants decrease lethality in response to many potentially deadly shock insults. Thus, targeting ROS- and CL-dependent pathways may prevent acute mortality and, hopefully, slow aging.

Innate Immunity as an Executor of the Programmed Death of Individual Organisms for the Benefit of the Entire Population.

In humans, over-activation of innate immunity in response to viral or bacterial infections often causes severe illness and death. Furthermore, similar mechanisms related to innate immunity can cause pathogenesis and death in sepsis, massive trauma (including surgery and burns), ischemia/reperfusion, some toxic lesions, and viral infections including COVID-19. Based on the reviewed observations, we suggest that such severe outcomes may be manifestations of a controlled suicidal strategy protecting the entire population from the spread of pathogens and from dangerous pathologies rather than an aberrant hyperstimulation of defense responses. We argue that innate immunity may be involved in the implementation of an altruistic programmed death of an organism aimed at increasing the well-being of the whole community. We discuss possible ways to suppress this atavistic program by interfering with innate immunity and suggest that combating this program should be a major goal of future medicine.

Lipid Dynamics in Diisobutylene-Maleic Acid (DIBMA) Lipid Particles in Presence of Sensory Rhodopsin II.

Amphiphilic diisobutylene/maleic acid (DIBMA) copolymers extract lipid-encased membrane proteins from lipid bilayers in a detergent-free manner, yielding nanosized, discoidal DIBMA lipid particles (DIBMALPs). Depending on the DIBMA/lipid ratio, the size of DIBMALPs can be broadly varied which makes them suitable for the incorporation of proteins of different sizes. Here, we examine the influence of the DIBMALP sizes and the presence of protein on the dynamics of encased lipids. As shown by a set of biophysical methods, the stability of DIBMALPs remains unaffected at different DIBMA/lipid ratios. Coarse-grained molecular dynamics simulations confirm the formation of viable DIBMALPs with an overall size of up to 35 nm. Electron paramagnetic resonance spectroscopy of nitroxides located at the 5th, 12th or 16th carbon atom positions in phosphatidylcholine-based spin labels reveals that the dynamics of enclosed lipids are not altered by the DIBMALP size. The presence of the membrane protein sensory rhodopsin II from Natronomonas pharaonis (NpSRII) results in a slight increase in the lipid dynamics compared to empty DIBMALPs. The light-induced photocycle shows full functionality of DIBMALPs-embedded NpSRII and a significant effect of the protein-to-lipid ratio during preparation on the NpSRII dynamics. This study indicates a possible expansion of the applicability of the DIBMALP technology on studies of membrane protein-protein interaction and oligomerization in a constraining environment.

ATP Analogues for Structural Investigations: Case Studies of a DnaB Helicase and an ABC Transporter.

Nucleoside triphosphates (NTPs) are used as chemical energy source in a variety of cell systems. Structural snapshots along the NTP hydrolysis reaction coordinate are typically obtained by adding stable, nonhydrolyzable adenosine triphosphate (ATP) -analogues to the proteins, with the goal to arrest a state that mimics as closely as possible a physiologically relevant state, e.g., the pre-hydrolytic, transition and post-hydrolytic states. We here present the lessons learned on two distinct ATPases on the best use and unexpected pitfalls observed for different analogues. The proteins investigated are the bacterial DnaB helicase from Helicobacter pylori and the multidrug ATP binding cassette (ABC) transporter BmrA from Bacillus subtilis, both belonging to the same division of P-loop fold NTPases. We review the magnetic-resonance strategies which can be of use to probe the binding of the ATP-mimics, and present carbon-13, phosphorus-31, and vanadium-51 solid-state nuclear magnetic resonance (NMR) spectra of the proteins or the bound molecules to unravel conformational and dynamic changes upon binding of the ATP-mimics. Electron paramagnetic resonance (EPR), and in particular W-band electron-electron double resonance (ELDOR)-detected NMR, is of complementary use to assess binding of vanadate. We discuss which analogues best mimic the different hydrolysis states for the DnaB helicase and the ABC transporter BmrA. These might be relevant also to structural and functional studies of other NTPases.

The microtubule skeleton and the evolution of neuronal complexity in vertebrates.

The evolution of a highly developed nervous system is mirrored by the ability of individual neurons to develop increased morphological complexity. As microtubules (MTs) are crucially involved in neuronal development, we tested the hypothesis that the evolution of complexity is driven by an increasing capacity of the MT system for regulated molecular interactions as it may be implemented by a higher number of molecular players and a greater ability of the individual molecules to interact. We performed bioinformatics analysis on different classes of components of the vertebrate neuronal MT cytoskeleton. We show that the number of orthologs of tubulin structure proteins, MT-binding proteins and tubulin-sequestering proteins expanded during vertebrate evolution. We observed that protein diversity of MT-binding and tubulin-sequestering proteins increased by alternative splicing. In addition, we found that regions of the MT-binding protein tau and MAP6 displayed a clear increase in disorder extent during evolution. The data provide evidence that vertebrate evolution is paralleled by gene expansions, changes in alternative splicing and evolution of coding sequences of components of the MT system. The results suggest that in particular evolutionary changes in tubulin-structure proteins, MT-binding proteins and tubulin-sequestering proteins were prominent drivers for the development of increased neuronal complexity.

Styrene/Maleic Acid Copolymers Form SMALPs by Pulling Lipid Patches out of the Lipid Bilayer.

Amphiphilic copolymers composed of styrene and maleic acid (SMA) monomers caused a major methodical breakthrough in the study of membrane proteins. They were found to directly release phospholipids and membrane proteins both from artificial and natural lipid bilayers, yielding stable water-soluble discoidal SMA/lipid particles (SMALPs) of uniform size. Although many empirical studies indicate the great potency of SMALPs for membrane protein research, the mechanisms of their formation remain obscure. It is unknown which factors account for the very assembly of SMALPs and govern their uniform size. We have developed a coarse-grained (CG) molecular model of SMA copolymers based on the MARTINI CG force field and used it to probe the behavior of SMA copolymers with varying composition/charge/concentration in solution as well as their interaction with lipid membranes. First, we found that SMA copolymers tend to aggregate in solution into clusters, which could account for the uniform size of SMALPs. Next, molecular dynamics (MD) simulations showed that periodic SMA copolymers with styrene/maleic acid ratios of 2:1 ([SSM] n) and 3:1 ([SSSM] n) differently interacted with lipid bilayers. While clusters of 2:1 SMA copolymers induced membrane poration, the clusters of 3:1 SMA copolymers extracted lipid patches from the membrane yielding SMALP-like structures. Extraction of lipid patches was also observed when we simulated the behavior of 3:1 copolymers with varying lengths and statistical distribution of styrene and MA units. Analysis of MD simulation trajectories and comparison with experimental data indicate that the formation of SMALPs requires copolymer molecules with a sufficient number of units made of more than two sequential styrene monomers.

Emergence of cytochrome bc complexes in the context of photosynthesis.

The cytochrome bc (cyt bc) complexes are involved in Q-cycling; they oxidize membrane quinols by high-potential electron acceptors, such as cytochromes or plastocyanin, and generate transmembrane proton gradient. In several prokaryotic lineages, and also in plant chloroplasts, the catalytic core of the cyt bc complexes is built of a four-helical cytochrome b (cyt b) that contains three hemes, a three-helical subunit IV, and an iron-sulfur Rieske protein (cytochrome b6 f-type complexes). In other prokaryotic lineages, and also in mitochondria, the cyt b subunit is fused with subunit IV, yielding a seven- or eight-helical cyt b with only two hemes (cyt bc1 -type complexes). Here we present an updated phylogenomic analysis of the cyt b subunits of cyt bc complexes. This analysis provides further support to our earlier suggestion that (1) the ancestral version of cyt bc complex contained a small four-helical cyt b with three hemes similar to the plant cytochrome b6 and (2) independent fusion events led to the formation of large cyts b in several lineages. In the search for a primordial function for the ancestral cyt bc complex, we address the intimate connection between the cyt bc complexes and photosynthesis. Indeed, the Q-cycle turnover in the cyt bc complexes demands high-potential electron acceptors. Before the Great Oxygenation Event, the biosphere had been highly reduced, so high-potential electron acceptors could only be generated upon light-driven charge separation. It appears that an ancestral cyt bc complex capable of Q-cycling has emerged in conjunction with the (bacterio)chlorophyll-based photosynthetic systems that continuously generated electron vacancies at the oxidized (bacterio)chlorophyll molecules.

Signaling and Adaptation Modulate the Dynamics of the Photosensoric Complex of Natronomonas pharaonis.

Motile bacteria and archaea respond to chemical and physical stimuli seeking optimal conditions for survival. To this end transmembrane chemo- and photoreceptors organized in large arrays initiate signaling cascades and ultimately regulate the rotation of flagellar motors. To unravel the molecular mechanism of signaling in an archaeal phototaxis complex we performed coarse-grained molecular dynamics simulations of a trimer of receptor/transducer dimers, namely NpSRII/NpHtrII from Natronomonas pharaonis. Signaling is regulated by a reversible methylation mechanism called adaptation, which also influences the level of basal receptor activation. Mimicking two extreme methylation states in our simulations we found conformational changes for the transmembrane region of NpSRII/NpHtrII which resemble experimentally observed light-induced changes. Further downstream in the cytoplasmic domain of the transducer the signal propagates via distinct changes in the dynamics of HAMP1, HAMP2, the adaptation domain and the binding region for the kinase CheA, where conformational rearrangements were found to be subtle. Overall these observations suggest a signaling mechanism based on dynamic allostery resembling models previously proposed for E. coli chemoreceptors, indicating similar properties of signal transduction for archaeal photoreceptors and bacterial chemoreceptors.

Origin of first cells at terrestrial, anoxic geothermal fields.

All cells contain much more potassium, phosphate, and transition metals than modern (or reconstructed primeval) oceans, lakes, or rivers. Cells maintain ion gradients by using sophisticated, energy-dependent membrane enzymes (membrane pumps) that are embedded in elaborate ion-tight membranes. The first cells could possess neither ion-tight membranes nor membrane pumps, so the concentrations of small inorganic molecules and ions within protocells and in their environment would equilibrate. Hence, the ion composition of modern cells might reflect the inorganic ion composition of the habitats of protocells. We attempted to reconstruct the "hatcheries" of the first cells by combining geochemical analysis with phylogenomic scrutiny of the inorganic ion requirements of universal components of modern cells. These ubiquitous, and by inference primordial, proteins and functional systems show affinity to and functional requirement for K(+), Zn(2+), Mn(2+), and phosphate. Thus, protocells must have evolved in habitats with a high K(+)/Na(+) ratio and relatively high concentrations of Zn, Mn, and phosphorous compounds. Geochemical reconstruction shows that the ionic composition conducive to the origin of cells could not have existed in marine settings but is compatible with emissions of vapor-dominated zones of inland geothermal systems. Under the anoxic, CO(2)-dominated primordial atmosphere, the chemistry of basins at geothermal fields would resemble the internal milieu of modern cells. The precellular stages of evolution might have transpired in shallow ponds of condensed and cooled geothermal vapor that were lined with porous silicate minerals mixed with metal sulfides and enriched in K(+), Zn(2+), and phosphorous compounds.

Evolution of cytochrome bc complexes: from membrane-anchored dehydrogenases of ancient bacteria to triggers of apoptosis in vertebrates.

This review traces the evolution of the cytochrome bc complexes from their early spread among prokaryotic lineages and up to the mitochondrial cytochrome bc1 complex (complex III) and its role in apoptosis. The results of phylogenomic analysis suggest that the bacterial cytochrome b6f-type complexes with short cytochromes b were the ancient form that preceded in evolution the cytochrome bc1-type complexes with long cytochromes b. The common ancestor of the b6f-type and the bc1-type complexes probably resembled the b6f-type complexes found in Heliobacteriaceae and in some Planctomycetes. Lateral transfers of cytochrome bc operons could account for the several instances of acquisition of different types of bacterial cytochrome bc complexes by archaea. The gradual oxygenation of the atmosphere could be the key evolutionary factor that has driven further divergence and spread of the cytochrome bc complexes. On the one hand, oxygen could be used as a very efficient terminal electron acceptor. On the other hand, auto-oxidation of the components of the bc complex results in the generation of reactive oxygen species (ROS), which necessitated diverse adaptations of the b6f-type and bc1-type complexes, as well as other, functionally coupled proteins. A detailed scenario of the gradual involvement of the cardiolipin-containing mitochondrial cytochrome bc1 complex into the intrinsic apoptotic pathway is proposed, where the functioning of the complex as an apoptotic trigger is viewed as a way to accelerate the elimination of the cells with irreparably damaged, ROS-producing mitochondria. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes.

Phylogenomic reconstruction of archaeal fatty acid metabolism.

While certain archaea appear to synthesize and/or metabolize fatty acids, the respective pathways still remain obscure. By analysing the genomic distribution of the key lipid-related enzymes, we were able to identify the likely components of the archaeal pathway of fatty acid metabolism, namely, a combination of the enzymes of bacterial-type ß-oxidation of fatty acids [acyl-coenzyme A (CoA) dehydrogenase, enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase] with paralogs of the archaeal acetyl-CoA C-acetyltransferase, an enzyme of the mevalonate biosynthesis pathway. These three ß-oxidation enzymes working in the reverse direction could potentially catalyse biosynthesis of fatty acids, with paralogs of acetyl-CoA C-acetyltransferase performing addition of C2 fragments. The presence in archaea of the genes for energy-transducing membrane enzyme complexes, such as cytochrome bc complex, cytochrome c oxidase and diverse rhodopsins, was found to correlate with the presence of the proposed system of fatty acid biosynthesis. We speculate that because these membrane complexes functionally depend on fatty acid chains, their genes could have been acquired via lateral gene transfer from bacteria only by those archaea that already possessed a system of fatty acid biosynthesis. The proposed pathway of archaeal fatty acid metabolism operates in extreme conditions and therefore might be of interest in the context of biofuel production and other industrial applications.

Co-evolution of primordial membranes and membrane proteins.

Studies of the past several decades have provided major insights into the structural organization of biological membranes and mechanisms of many membrane molecular machines. However, the origin(s) of the membrane(s) and membrane proteins remains enigmatic. We discuss different concepts of the origin and early evolution of membranes with a focus on the evolution of the (im)permeability to charged molecules such as proteins, nucleic acids and small ions. Reconstruction of the evolution of F-type and A/V-type membrane ATPases (ATP synthases), which are either proton- or sodium-dependent, might help us to understand not only the origin of membrane bioenergetics but also of membranes themselves. We argue that evolution of biological membranes occurred as a process of co-evolution of lipid bilayers, membrane proteins and membrane bioenergetics.

Dynamics of bioenergetic microcompartments.

The vast majority of life on earth is dependent on harvesting electrochemical potentials over membranes for the synthesis of ATP. Generation of membrane potential often relies on electron transport through membrane protein complexes, which vary among the bioenergetic membranes found in living organisms. In order to maximize the efficient harvesting of the electrochemical potential, energy loss must be minimized, and this is achieved partly by restricting certain events to specific microcompartments, on bioenergetic membranes. In this review we will describe the characteristics of the energy-converting supramolecular structures involved in oxidative phosphorylation in mitochondria and bacteria, and photophosphorylation. Efficient function of electron transfer pathways requires regulation of electron flow, and we will also discuss how this is partly achieved through dynamic re-compartmentation of the membrane complexes into different supercomplexes. In addition to supercomplexes, the supramolecular structure of the membrane, and in particular the role of water layers on the surface of the membrane in the prevention of wasteful proton escape (and therefore energy loss), is discussed in detail. In summary, the restriction of energetic processes to specific microcompartments on bioenergetic membranes minimizes energy loss, and dynamic rearrangement of these structures allows for regulation.

The role of energy in the emergence of biology from chemistry.

Any scenario of the transition from chemistry to biology should include an "energy module" because life can exist only when supported by energy flow(s). We addressed the problem of primordial energetics by combining physico-chemical considerations with phylogenomic analysis. We propose that the first replicators could use abiotically formed, exceptionally photostable activated cyclic nucleotides both as building blocks and as the main energy source. Nucleoside triphosphates could replace cyclic nucleotides as the principal energy-rich compounds at the stage of the first cells, presumably because the metal chelates of nucleoside triphosphates penetrated membranes much better than the respective metal complexes of nucleoside monophosphates. The ability to exploit natural energy flows for biogenic production of energy-rich molecules could evolve only gradually, after the emergence of sophisticated enzymes and ion-tight membranes. We argue that, in the course of evolution, sodium-dependent membrane energetics preceded the proton-based energetics which evolved independently in bacteria and archaea.

Open questions on the origin of life at anoxic geothermal fields.

We have recently reconstructed the 'hatcheries' of the first cells by combining geochemical analysis with phylogenomic scrutiny of the inorganic ion requirements of universal components of modern cells (Mulkidjanian et al. Proc Natl Acad Sci U S A 109:E821-830, 2012). These ubiquitous, and by inference primordial, proteins and functional systems show affinity to and functional requirement for K⁺, Zn²âº, Mn²âº, and phosphate. Thus, protocells must have evolved in habitats with a high K⁺/Na⁺ ratio and relatively high concentrations of Zn, Mn and phosphorous compounds. Geochemical reconstruction shows that the ionic composition conducive to the origin of cells could not have existed in marine settings but is compatible with emissions of vapor-dominated zones of inland geothermal systems. Under an anoxic, CO2-dominated atmosphere, the ionic composition of pools of cool, condensed vapor at anoxic geothermal fields would resemble the internal milieu of modern cells. Such pools would be lined with porous silicate minerals mixed with metal sulfides and enriched in K⁺ ions and phosphorous compounds. Here we address some questions that have appeared in print after the publication of our anoxic geothermal field scenario. We argue that anoxic geothermal fields, which were identified as likely cradles of life by using a top-down approach and phylogenomics analysis, could provide geochemical conditions similar to those which were suggested as most conducive for the emergence of life by the chemists who pursuit the complementary bottom-up strategy.

Common Patterns of Hydrolysis Initiation in P-loop Fold Nucleoside Triphosphatases.

The P-loop fold nucleoside triphosphate (NTP) hydrolases (also known as Walker NTPases) function as ATPases, GTPases, and ATP synthases, are often of medical importance, and represent one of the largest and evolutionarily oldest families of enzymes. There is still no consensus on their catalytic mechanism. To clarify this, we performed the first comparative structural analysis of more than 3100 structures of P-loop NTPases that contain bound substrate Mg-NTPs or their analogues. We proceeded on the assumption that structural features common to these P-loop NTPases may be essential for catalysis. Our results are presented in two articles. Here, in the first, we consider the structural elements that stimulate hydrolysis. Upon interaction of P-loop NTPases with their cognate activating partners (RNA/DNA/protein domains), specific stimulatory moieties, usually Arg or Lys residues, are inserted into the catalytic site and initiate the cleavage of gamma phosphate. By analyzing a plethora of structures, we found that the only shared feature was the mechanistic interaction of stimulators with the oxygen atoms of gamma-phosphate group, capable of causing its rotation. One of the oxygen atoms of gamma phosphate coordinates the cofactor Mg ion. The rotation must pull this oxygen atom away from the Mg ion. This rearrangement should affect the properties of the other Mg ligands and may initiate hydrolysis according to the mechanism elaborated in the second article.

Common Mechanism of Activated Catalysis in P-loop Fold Nucleoside Triphosphatases-United in Diversity.

To clarify the obscure hydrolysis mechanism of ubiquitous P-loop-fold nucleoside triphosphatases (Walker NTPases), we analysed the structures of 3136 catalytic sites with bound Mg-NTP complexes or their analogues. Our results are presented in two articles; here, in the second of them, we elucidated whether the Walker A and Walker B sequence motifs-common to all P-loop NTPases-could be directly involved in catalysis. We found that the hydrogen bonds (H-bonds) between the strictly conserved, Mg-coordinating Ser/Thr of the Walker A motif ([Ser/Thr]WA) and aspartate of the Walker B motif (AspWB) are particularly short (even as short as 2.4 ångströms) in the structures with bound transition state (TS) analogues. Given that a short H-bond implies parity in the pKa values of the H-bond partners, we suggest that, in response to the interactions of a P-loop NTPase with its cognate activating partner, a proton relocates from [Ser/Thr]WA to AspWB. The resulting anionic [Ser/Thr]WA alkoxide withdraws a proton from the catalytic water molecule, and the nascent hydroxyl attacks the gamma phosphate of NTP. When the gamma-phosphate breaks away, the trapped proton at AspWB passes by the Grotthuss relay via [Ser/Thr]WA to beta-phosphate and compensates for its developing negative charge that is thought to be responsible for the activation barrier of hydrolysis.

Mechanisms of Formation, Structure, and Dynamics of Lipoprotein Discs Stabilized by Amphiphilic Copolymers: A Comprehensive Review.

Amphiphilic copolymers consisting of alternating hydrophilic and hydrophobic units account for a major recent methodical breakthrough in the investigations of membrane proteins. Styrene-maleic acid (SMA), diisobutylene-maleic acid (DIBMA), and related copolymers have been shown to extract membrane proteins directly from lipid membranes without the need for classical detergents. Within the particular experimental setup, they form disc-shaped nanoparticles with a narrow size distribution, which serve as a suitable platform for diverse kinds of spectroscopy and other biophysical techniques that require relatively small, homogeneous, water-soluble particles of separate membrane proteins in their native lipid environment. In recent years, copolymer-encased nanolipoparticles have been proven as suitable protein carriers for various structural biology applications, including cryo-electron microscopy (cryo-EM), small-angle scattering, and conventional and single-molecule X-ray diffraction experiments. Here, we review the current understanding of how such nanolipoparticles are formed and organized at the molecular level with an emphasis on their chemical diversity and factors affecting their size and solubilization efficiency.

Activated Q-cycle as a common mechanism for cytochrome bc1 and cytochrome b6f complexes.

Cytochrome bc(1)-complexes of animals and bacteria (hereafter bc(1)), as well as related cytochrome b(6)f complexes of plants and cyanobacteria (hereafter bf) are dimeric quinol:cytochrome c/plastocyanin oxidoreductases capable of translocating protons across energy-converting membranes. The commonly accepted Q-cycle mechanism suggests that these enzymes oxidize two quinol molecules in their catalytic centers P to yield one quinol molecule in another catalytic center N. Earlier, based upon data on flash-induced redox changes of cytochromes b and c(1), voltage generation, and proton transfer in membrane vesicles of Rhodobacter capsulatus, we have put forward a scheme of an "activated Q-cycle" for the bc(1). The scheme suggests that the bc(1) dimers, being "activated" by injection of electrons from the membrane ubiquinol pool via centers N, steadily contain two electrons in their cytochrome b moieties under physiological conditions, most likely, as a bound semiquinone in center N of one monomer and a reduced high-potential heme b in the other monomer. Then the oxidation of each ubiquinol molecule in centers P of an activated bc(1) should result in a complete catalytic cycle leading to the formation of a ubiquinole molecule in the one of enzyme's centers N and to voltage generation. Here it is argued that a similar pre-loading by two electrons can explain the available data on flash-induced reactions in cytochrome b(6)f-complexes of green plants and cyanobacteria.