Changing hydration level in an internal cavity modulates the proton affinity of a key glutamate in cytochrome c oxidase

Cytochrome c oxidase contributes to the transmembrane proton gradient by removing two protons from the high-pH side of the membrane each time the binuclear center active site is reduced. One proton goes to the binuclear center, whereas the other is pumped to the low-pH periplasmic space. Glutamate 286 (Glu286) has been proposed to serve as a transiently deprotonated proton donor. Using unrestrained atomistic molecular dynamics simulations, we show that the size of and water distribution in the hydrophobic cavity that holds Glu286 is controlled by the protonation state of the propionic acid of heme a₃, a group on the proton outlet pathway. Protonation of the propionate disrupts hydrogen bonding to two side chains, allowing a loop to swing open. Continuum electrostatics and atomistic free-energy perturbation calculations show that the resultant changes in hydration and electrostatic interactions lower the Glu proton affinity by at least 5 kcal/mol. These changes in the internal hydration level occur in the absence of major conformational transitions and serve to stabilize needed transient intermediates in proton transport. The trigger is not the protonation of the Glu of interest, but rather the protonation of a residue ∼10 Å away. Thus, unlike local water penetration to stabilize a new charge, this finding represents a specific role for water molecules in the protein interior, mediating proton transfers and facilitating ion transport.Water is essential to the structure, dynamics, and function of biomolecules (1, 2), and its role in protein folding, association (3), and dynamics (4, 5) has been well documented. The highly polar and polarizable water molecules play diverse roles in protein interiors. Water can aid catalysis in enzyme active sites (6–8). Water or water chains are often observed in proteins that are (9, 10) proton or ion transporters or pumps (11–14). Internal cavities holding functional water molecules are believed to have a fairly constant level of hydration throughout the protein reaction cycle, unless significant conformational changes occur (15). Water penetration in response to the ionization or reduction of internal groups has been extensively discussed (16, 17), although it is usually described as part of protein''s local dielectric response.Cytochrome c oxidase (CcO) adds to the transmembrane proton gradient through proton transport coupled to electron transfer reactions (12, 18, 19). In the overall reaction, electrons from four cytochromes c are transferred to oxygen to make two water molecules at the binuclear center (BNC). The four protons needed for chemistry are bound only from the high-pH, N side of the membrane. Coupled to the process, four more protons are transferred across the membrane from the high- to low-pH (P) side of the membrane. Thus, eight charges are transferred across the membrane as each O₂ is reduced.Glu286 is a required, conserved residue that is expected to transfer protons from the D channel either to the BNC or the proton-loading site (PLS) each time CcO is reduced (Fig. 1). Experiments assign a functional pK_a to Glu286 near 9.4 (20). Thus, at higher pH, proton binding to the Glu becomes rate-limiting for steady-state turnover. The current understanding of the reaction cycle shows that protons are pumped in each of the four distinct BNC redox states (12, 18, 19). The reaction mechanism needs Glu286 to be deprotonated twice to pass a proton to the PLS and to the BNC in each CcO reduction step. Previous continuum electrostatics (21–24) and semimacroscopic (25, 26) calculations obtained pK_a values for Glu286 near 9–10. However, recent microscopic calculations have found significantly higher pK_a values of more than 12 (17, 27), making it unclear how a proton could be lost from this site, whereas others do not address the proton affinity of the essential Glu (28, 29). The discrepancy between experiment and simulations may result from technical issues such as the use of static protein structures and limited sampling of protonation states of titratable groups, or it may arise from changes in the protein that have been missed. Thus, a key question remaining is how the proton affinity of this essential Glu is modulated so it can donate a proton to the PLS and the BNC through the reaction cycle.Open in a separate windowFig. 1.Illustration of key residues near the hydrophobic cavity in CcO and general proton pathways to and from Glu286.In this work, computational studies show the hydration level of an internal cavity near Glu286 changes substantially without needing global conformational changes. Rather, the structure of an internal loop is controlled or anchored by the protonation state of the D-propionic acid of heme a₃. This potentially important motion has not been noted in previous computational studies in which part of the protein structure was constrained (21, 27, 28). Both continuum electrostatics and quantum mechanical/classical mechanical (QM/MM) free-energy simulations show that the resultant changes in Glu286 hydration level and electrostatic interactions significantly affect its pK_a (proton affinity). These findings point to a molecular mechanism to modulate the timing of proton transfers in the CcO proton pumping cycle by modifying the proton affinity of this key acid. More generally, the results show that changes in protein internal hydration may occur with only small, distal conformational changes, and these can serve as an important regulatory mechanism in ion transport, thus going beyond being part of generic dielectric response of proteins.     

Detection of genomic variations and DNA polymorphisms and impact on analysis of meiotic recombination and genetic mapping

DNA polymorphisms are important markers in genetic analyses and are increasingly detected by using genome resequencing. However, the presence of repetitive sequences and structural variants can lead to false positives in the identification of polymorphic alleles. Here, we describe an analysis strategy that minimizes false positives in allelic detection and present analyses of recently published resequencing data from Arabidopsis meiotic products and individual humans. Our analysis enables the accurate detection of sequencing errors, small insertions and deletions (indels), and structural variants, including large reciprocal indels and copy number variants, from comparisons between the resequenced and reference genomes. We offer an alternative interpretation of the sequencing data of meiotic products, including the number and type of recombination events, to illustrate the potential for mistakes in single-nucleotide polymorphism calling. Using these examples, we propose that the detection of DNA polymorphisms using resequencing data needs to account for nonallelic homologous sequences.DNA polymorphisms are ubiquitous genetic variations among individuals and include single nucleotide polymorphisms (SNPs), insertions and deletions (indels), and other larger rearrangements (1–3) (Fig. 1 A and B). They can have phenotypic consequences and also serve as molecular markers for genetic analyses, facilitating linkage and association studies of genetic diseases, and other traits in humans (4–6), animals, plants, (7–10) and other organisms. Using DNA polymorphisms for modern genetic applications requires low-error, high-throughput analytical strategies. Here, we illustrate the use of short-read next-generation sequencing (NGS) data to detect DNA polymorphisms in the context of whole-genome analysis of meiotic products.Open in a separate windowFig. 1.(A) SNPs and small indels between two ecotype genomes. (B) Possible types of SVs. Col genotypes are marked in blue and Ler in red. Arrows indicate DNA segments involved in SVs between the two ecotypes. (C) Meiotic recombination events including a CO and a GC (NCO). Centromeres are denoted by yellow dots.There are many methods for detecting SNPs (11–14) and structural variants (SVs) (15–25), including NGS, which can capture nearly all DNA polymorphisms (26–28). This approach has been widely used to analyze markers in crop species such as rice (29), genes associated with diseases (6, 26), and meiotic recombination in yeast and plants (30, 31). However, accurate identification of DNA polymorphisms can be challenging, in part because short-read sequencing data have limited information for inferring chromosomal context.Genomes usually contain repetitive sequences that can differ in copy number between individuals (26–28, 31); therefore, resequencing analyses must account for chromosomal context to avoid mistaking highly similar paralogous sequences for polymorphisms. Here, we use recently published datasets to describe several DNA sequence features that can be mistaken as allelic (32, 33) and describe a strategy for differentiating between repetitive sequences and polymorphic alleles. We illustrate the effectiveness of these analyses by examining the reported polymorphisms from the published datasets.Meiotic recombination is initiated by DNA double-strand breaks (DSBs) catalyzed by the topoisomerase-like SPORULATION 11 (SPO11). DSBs are repaired as either crossovers (COs) between chromosomes (Fig. 1C), or noncrossovers (NCOs). Both COs and NCOs can be accompanied by gene conversion (GC) events, which are the nonreciprocal transfer of sequence information due to the repair of heteroduplex DNA during meiotic recombination. Understanding the control of frequency and distribution of CO and NCO (including GC) events has important implications for human health (including cancer and aneuploidy), crop breeding, and the potential for use in genome engineering. COs can be detected relatively easily by using polymorphic markers in the flanking sequences, but NCO products can only be detected if they are accompanied by a GC event. Because GCs associated with NCO result in allelic changes at polymorphic sites without exchange of flanking sequences, they are more difficult to detect. Recent advances in DNA sequencing have made the analysis of meiotic NCOs more feasible (30–32, 34); however, SVs present a challenge in these analyses. We recommend a set of guidelines for detection of DNA polymorphisms by using genomic resequencing short-read datasets. These measures improve the accuracy of a wide range of analyses by using genomic resequencing, including estimation of COs, NCOs, and GCs.     

Functional heterogeneity of the four voltage sensors of a human L-type calcium channel

Excitation-evoked Ca²⁺ influx is the fastest and most ubiquitous chemical trigger for cellular processes, including neurotransmitter release, muscle contraction, and gene expression. The voltage dependence and timing of Ca²⁺ entry are thought to be functions of voltage-gated calcium (Ca_V) channels composed of a central pore regulated by four nonidentical voltage-sensing domains (VSDs I–IV). Currently, the individual voltage dependence and the contribution to pore opening of each VSD remain largely unknown. Using an optical approach (voltage-clamp fluorometry) to track the movement of the individual voltage sensors, we discovered that the four VSDs of Ca_V1.2 channels undergo voltage-evoked conformational rearrangements, each exhibiting distinct voltage- and time-dependent properties over a wide range of potentials and kinetics. The voltage dependence and fast kinetic components in the activation of VSDs II and III were compatible with the ionic current properties, suggesting that these voltage sensors are involved in Ca_V1.2 activation. This view is supported by an obligatory model, in which activation of VSDs II and III is necessary to open the pore. When these data were interpreted in view of an allosteric model, where pore opening is intrinsically independent but biased by VSD activation, VSDs II and III were each found to supply ∼50 meV (∼2 kT), amounting to ∼85% of the total energy, toward stabilizing the open state, with a smaller contribution from VSD I (∼16 meV). VSD IV did not appear to participate in channel opening.Voltage-gated Ca²⁺ (Ca_V) channels respond to membrane depolarization by catalyzing Ca²⁺ influx. Ca_V-mediated elevation of intracellular [Ca²⁺] regulates such critical physiological functions as neurotransmitter and hormone release, axonal outgrowth, muscle contraction, and gene expression (1). Their relevance to human physiology is evident from the broad phenotypic consequences of Ca_V channelopathies (2). The voltage dependence of Ca_V-driven Ca²⁺ entry relies on the modular organization of the channel-forming α₁ subunit (Fig. 1), which consists of four repeated motifs (I–IV), each comprising six membrane-spanning helical segments (S1–S6) (Fig. 1A). Segments S1–S4 form a voltage-sensing domain (VSD), whereas segments S5 and S6 contribute to the Ca²⁺-conductive pore (1). The VSDs surround the central pore (Fig. 1B). VSDs are structurally and functionally conserved modules (3–5) capable of transducing a change in the cell membrane electrical potential into a change of ion-specific permeability or enzyme activity. VSDs sense depolarization by virtue of a signature motif of positively charged Arg or Lys at every third position of helix S4 (Fig. 1D), which rearranges in response to depolarization (4, 6–10). In contrast to voltage-gated K⁺ (K_V) channels but similar to pseudotetrameric voltage-gated Na⁺ (Na_V) channels, the amino acid sequences encoding each VSD have evolved independently (Fig. 1D). In addition to their distinct primary structure, the four Ca_V VSDs may also gain distinct functional properties from the asymmetrical association of auxiliary subunits, such as β, α₂δ, and calmodulin (1, 11–16) (Fig. 1C). The structural divergence among VSD-driven channels was foreseen by the classical Hodgkin–Huxley model (17), in which four independent “gating particles” control the opening of homotetrameric K_V channels and only three seem sufficient to open Na_V channels. An early study by Kostyuk et al. (18) suggested that only two gating particles are coupled to Ca_V channel opening. We recognize today that gating particles correspond to VSDs, and in Na_V channels, VSDs I–III control Na⁺ influx, whereas VSD IV is associated with fast inactivation (19–21).Open in a separate windowFig. 1.Ca_V membrane topology, putative structure, and S4 helix homology. (A) Ca_V channel-forming α₁ subunits consist of four concatenated repeats, each encompassing one voltage sensor domain (VSD) and a quarter of the central pore domain (PD) (1). Stars indicate the positions of fluorophore labeling. (B) The atomic structure of an Na_V channel (Protein Data Bank ID code 4EKW; top view) (56) shown as a structural representation for the Ca_V α₁ subunit. (C) The α₁ subunit asymmetrically associates with auxiliary β, α₂δ, and calmodulin (CaM) subunits (11–16). (D) Sequence alignment of VSD helix S4 from each of four Ca_V1.2 repeats and the archetypal homotetrameric Shaker K⁺ channel. Conserved, positively charged Arg or Lys is in blue. Residues substituted by Cys for fluorescent labeling are marked: F231 (VSD I), L614 (VSD II), V994 (VSD II), and S1324 (VSD IV).In this study, we used fluorometry to probe the properties of four individual VSDs in a human L-type calcium channel Ca_V1.2, which is a widely expressed regulator of physiological processes, such as cardiac and smooth muscle contractility (22). Although the collective transition of the Ca_V VSDs and the pore has been investigated in studies measuring total charge displacement (gating currents) (23, 24), the activation properties and functional roles of each VSD are unknown. Evidence for the role of each VSD in L-type Ca_V channel operation has been presented from charge neutralization studies, but a clear picture has yet to emerge. Work on a chimeric L-type channel suggests that VSDs I and III drive channel opening (25), whereas other studies on Ca_V1.2 favored the involvement of VSD II over VSD I, with the roles of VSDs III and IV remaining unclear (26, 27).The individual optical reports of four Ca_V1.2 VSDs revealed that each operates with distinct biophysical parameters. We found that VSDs II and III exhibit voltage- and time-dependent characteristics compatible with channel opening and that they can be considered rate-limiting for activation. We compared the voltage and time dependence of the fluorescent signals and ionic currents with the predictions of thermodynamic models relevant to Ca_V domain organization. We found that Ca_V1.2 activation is compatible with a model of allosteric VSD–pore coupling, where VSDs II and III are the primary drivers of channel opening with a smaller contribution by VSD I. We discuss the mechanism of Ca_V1.2 voltage sensitivity, which exhibits similarities to but also clear differences from the related pseudotetrameric Na_V1.4 channels.     

Fe–N2/CO complexes that model a possible role for the interstitial C atom of FeMo-cofactor (FeMoco)

We report here a series of four- and five-coordinate Fe model complexes that feature an axial tri(silyl)methyl ligand positioned trans to a substrate-binding site. This arrangement is used to crudely model a single-belt Fe site of the FeMo-cofactor that might bind N₂ at a position trans to the interstitial C atom. Reduction of a trigonal pyramidal Fe(I) complex leads to uptake of N₂ and subsequent functionalization furnishes an open-shell Fe–diazenido complex. A related series of five-coordinate Fe–CO complexes stable across three redox states is also described. Spectroscopic, crystallographic, and Density Functional Theory (DFT) studies of these complexes suggest that a decrease in the covalency of the Fe–C_alkyl interaction occurs upon reduction and substrate binding. This leads to unusually long Fe–C_alkyl bond distances that reflect an ionic Fe–C bond. The data presented are contextualized in support of a hypothesis wherein modulation of a belt Fe–C interaction in the FeMo-cofactor facilitates substrate binding and reduction.MoFe-nitrogenase catalyzes the fascinating but poorly understood conversion of nitrogen to ammonia at its iron-molybdenum cofactor (FeMoco) (1, 2). The core of the FeMoco was originally thought to be vacant (3). Later work on Azotobacter vinelandii indicated the presence of a light interstitial atom coordinated to six central, so-called “belt Fe atoms” (4). Crystallographic and spectroscopic studies (5, 6), in addition to studies mapping the biosynthetic pathway of C-atom incorporation (7, 8), establish that carbon is the interstitial atom, as shown in Fig. 1.Open in a separate windowFig. 1.A hypothetical N₂-binding event at a belt iron in FeMoco illustrating a proposed Fe–C elongation. The degree and positions of protonation are unknown under electron loading, but the inorganic sulfides are plausible candidate positions.Although the site(s) of N₂ reduction remain(s) uncertain, a body of evidence that includes biochemical, spectroscopic, and computational studies on FeMoco point to a belt Fe center as a plausible candidate (2, 9–13). In a scenario in which N₂ binds terminally to one of the belt Fe centers, the N₂ ligand would initially be coordinated trans to the interstitial C atom (Fig. 1) (10). This hypothesis calls for model complexes that depict such an arrangement to explore factors that might govern substrate coordination and subsequent reduction. Structurally faithful models of the FeMoco that include an Fe₆C unit stabilized by sulfide or other sulfur-based ligands present a formidable synthetic challenge (14). Moreover, N₂ coordination to synthetic iron–sulfur clusters has yet to be established (15). Model complexes featuring a single Fe site with a C-atom anchor positioned trans to an N₂ binding site are unknown, but would provide a useful tool to evaluate how an Fe–C interaction might respond to N₂ binding and the Fe redox state. Model compounds of this type may facilitate the evaluation of theoretical (10) and spectroscopic studies (16) on FeMoco that suggest a single, flexible Fe–C interaction is observed under turnover conditions.It is in this context that we have pursued mononuclear Fe complexes supported by tripodal, tetradentate ligands featuring three phosphine donor arms tethered to a tertiary alkyl anchor. Although a number of such ligands featuring a central C atom has been described (17–19) we reasoned that an alkyl ligand featuring only electropositive substituents adjacent to the C-atom anchor would provide a crude model of the interstitial carbide of the FeMoco and permit a high degree of ionic bonding to a single Fe–N₂ binding site. To achieve this goal, the C-atom anchor of the auxiliary ligand described is surrounded by three electropositive Si centers, in addition to the Fe site. This model system successfully coordinates N₂ trans to the C-atom anchor and shows how the local Fe geometry and the Fe–C interaction respond as a function of such binding. Using CO instead of N₂, this Fe system also presents an opportunity to study the Fe–C interaction as a function of the Fe redox state. As described below, unusually long Fe–C distances can be accessed that are consistent with a much higher degree of ionic character at the Fe–C interaction than would be anticipated for a prototypical Fe alkyl.     

Interacting cytoplasmic loops of subunits a and c of Escherichia coli F1F0 ATP synthase gate H+ transport to the cytoplasm

H⁺-transporting F₁F₀ ATP synthase catalyzes the synthesis of ATP via coupled rotary motors within F₀ and F₁. H⁺ transport at the subunit a–c interface in transmembranous F₀ drives rotation of a cylindrical c₁₀ oligomer within the membrane, which is coupled to rotation of subunit γ within the α₃β₃ sector of F₁ to mechanically drive ATP synthesis. F₁F₀ functions in a reversible manner, with ATP hydrolysis driving H⁺ transport. ATP-driven H⁺ transport in a select group of cysteine mutants in subunits a and c is inhibited after chelation of Ag⁺ and/or Cd⁺² with the substituted sulfhydryl groups. The H⁺ transport pathway mapped via these Ag⁺(Cd⁺²)-sensitive Cys extends from the transmembrane helices (TMHs) of subunits a and c into cytoplasmic loops connecting the TMHs, suggesting these loop regions could be involved in gating H⁺ release to the cytoplasm. Here, using select loop-region Cys from the single cytoplasmic loop of subunit c and multiple cytoplasmic loops of subunit a, we show that Cd⁺² directly inhibits passive H⁺ transport mediated by F₀ reconstituted in liposomes. Further, in extensions of previous studies, we show that the regions mediating passive H⁺ transport can be cross-linked to each other. We conclude that the loop-regions in subunits a and c that are implicated in H⁺ transport likely interact in a single structural domain, which then functions in gating H⁺ release to the cytoplasm.The F₁F₀-ATP synthase of oxidative phosphorylation uses the energy of a transmembrane electrochemical gradient of H⁺ or Na⁺ to mechanically drive the synthesis of ATP via two coupled rotary motors in the F₁ and F₀ sectors of the enzyme (1). H⁺ transport through the transmembrane F₀ sector is coupled to ATP synthesis or hydrolysis in the F₁ sector at the surface of the membrane. Homologous ATP synthases are found in mitochondria, chloroplasts, and many bacteria. In Escherichia coli and other eubacteria, F₁ consists of five subunits in an α₃β₃γδε stoichiometry. F₀ is composed of three subunits in a likely ratio of a₁b₂c₁₀ in E. coli and Bacillus PS3 (2, 3) or a₁b₂c₁₁ in the Na⁺ translocating Ilyobacter tartaricus ATP synthase (1, 4) and may contain as many as 15 c subunits in other bacterial species (5). Subunit c spans the membrane as a hairpin of two α-helices, with the first transmembrane helix (TMH) on the inside and the second TMH on the outside of the c ring (1, 4). The binding of Na⁺ or H⁺ occurs at an essential, membrane-embedded Glu or Asp on cTMH2. High-resolution X-ray structures of both Na⁺- and H⁺-binding c-rings have revealed the details and variations in the cation binding sites (4–8). In the H⁺-translocating E. coli enzyme, Asp-61 at the center of cTMH2 is thought to undergo protonation and deprotonation, as each subunit of the c ring moves past the stationary subunit a. In the functioning enzyme, the rotation of the c ring is thought to be driven by H⁺ transport at the subunit a/c interface. Subunit γ physically binds to the cytoplasmic surface of the c-ring, which results in the coupling of c-ring rotation with rotation of subunit γ within the α₃β₃ hexamer of F₁ to mechanically drive ATP synthesis (1).E. coli subunit a folds in the membrane with five TMHs and is thought to provide aqueous access channels to the H⁺-binding cAsp-61 residue (9, 10). Interaction of the conserved Arg-210 residue in aTMH4 with cTMH2 is thought to be critical during the deprotonation–protonation cycle of cAsp-61 (1, 11, 12). At this time, very limited biophysical or crystallographic information is available on the 3D arrangement of the TMHs in subunit a. TMHs 2–5 of subunit a pack in a four-helix bundle, which was initially defined by cross-linking (13), but now, such a bundle, packing at the periphery of the c-ring, has been viewed directly by high-resolution cryoelectron microscopy in the I. tartaricus enzyme (14). Previously published cross-linking experiments support the identification of aTMH4 and aTMH5 packing at the periphery of the c-ring and the identification of aTHM2 and aTMH3 as the other components of the four-helix bundle seen in these images (13, 15, 16). More recently, published cross-linking experiments identify the N-terminal α-helices of two b subunits, one of which packs at one surface of aTMH2 with close enough proximity to the c-ring to permit cross-linking (17). The other subunit b N-terminal helix packs on the opposite peripheral surface of aTMH2 in a position where it can also be cross-linked to aTMH3 (17). The last helix density shown in Hakulinen and colleagues (14) packs at the periphery of the c-ring next to aTMH5 and is very likely to be aTMH1.The aqueous accessibility of Cys residues introduced into the five TMHs of subunit a has been probed on the basis of their reactivity with and inhibitory effects of Ag⁺ and other thiolate-reactive agents (18–20). Two regions of aqueous access were found with distinctly different properties. One region in TMH4, extending from Asn-214 and Arg-210 at the center of the membrane to the cytoplasmic surface, contains Cys substitutions that are sensitive to inhibition by both N-ethylmaleimide (NEM) and Ag⁺ (18–20; Fig. 1). These NEM- and Ag⁺-sensitive residues in TMH4 pack at or near the peripheral face and cytoplasmic side of the modeled four-helix bundle (11, 13). A second set of Ag⁺-sensitive substitutions in subunit a mapped to the opposite face and periplasmic side of aTMH4 (18, 19), and Ag⁺-sensitive substitutions were also found in TMHs 2, 3, and 5, where they extend from the center of the membrane to the periplasmic surface (19, 20). The Ag⁺-sensitive substitutions on the periplasmic side of TMHs 2–5 cluster at the interior of the four-helix bundle predicted by cross-linking and could interact to form a continuous aqueous pathway extending from the periplasmic surface to the central region of the lipid bilayer (11, 13, 19, 20). We have proposed that the movement of H⁺ from the periplasmic half-channel and binding to the single ionized Asp61 in the c-ring is mediated by a swiveling of TMHs at the a–c subunit interface (16, 21–24). This gating is thought to be coupled with ionization of a protonated cAsp61 in the adjacent subunit of the c-ring and with release of the H⁺ into the cytoplasmic half-channel at the subunit a–c interface. The route of aqueous access to the cytoplasmic side of the c subunit packing at the a–c interface has also been mapped by the chemical probing of Cys substitutions and, more recently, by molecular dynamics simulations (22, 25, 26).Open in a separate windowFig. 1.The predicted topology of subunit a in the E. coli inner membrane. The location of the most Ag⁺-sensitive Cys substitutions are highlighted in red (>85% inhibition) or orange (66–85% inhibition). The five proposed TMHs are shown in boxes, each with a span of 21 amino acids, which is the minimum length required to span the hydrophobic core of a lipid bilayer. The α-helical segments shown in loops 1–2 and 3–4 are consistent with the predictions of TALOS, based on backbone chemical shifts seen by NMR (29). Others have also predicted extensive α-helical regions in these loops (12, 30), but the possible positions remain largely speculative. aArg210 is highlighted in green. Figure is modified from those shown previously (21, 23, 24, 27).We have also reported Ag⁺-sensitive Cys substitutions in two cytoplasmic loops of subunit a (27) and, more recently, in the cytoplasmic loop of subunit c (28). The mechanism by which Ag⁺ inhibited F₁F₀-mediated H⁺ transport was uncertain. Several of these substitutions were also sensitive to inhibition by Cd⁺², and these substitutions provided a means of testing whether Cd⁺² directly inhibits passive H⁺ transport through F₀ (28). In the case of two subunit c loop substitutions, Cd⁺² was shown to directly inhibit passive F₀-mediated transport activity. In this study, we have extended the survey to Cd⁺²-sensitive Cys substitutions in cytoplasmic loops of subunit a. We report four loop substitutions in which Cd⁺² inhibits passive F₀-mediated H⁺ transport. Further, in two cases, we show cross-linking between pairs of Cys substitutions, which lie in subunits a and c, respectively, and which individually mediate passive H⁺ transport activity. These results suggest that the a and c loops, which gate H⁺ release to the cytoplasm, fold into a single domain at the surface of F₀.     

Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics

Mechanisms that regulate the nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions, and is activated by calmodulin binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two electron transfer domains in a FRET dye-labeled neuronal NOS reductase domain, and to understand how calmodulin affects the dynamics to regulate catalysis. We found that calmodulin alters NOS conformational behaviors in several ways: It changes the distance distribution between the NOS domains, shortens the lifetimes of the individual conformational states, and instills conformational discipline by greatly narrowing the distributions of the conformational states and fluctuation rates. This information was specifically obtainable only by single-molecule spectroscopic measurements, and reveals how calmodulin promotes catalysis by shaping the physical and temporal conformational behaviors of NOS.Although proteins adopt structures determined by their amino acid sequences, they are not static objects and fluctuate among ensembles of conformations (1). Transitions between these states can occur on a variety of length scales (Å to nm) and time scales (ps to s) and have been linked to functionally relevant phenomena such as allosteric signaling, enzyme catalysis, and protein–protein interactions (2–4). Indeed, protein conformational fluctuations and dynamics, often associated with static and dynamic inhomogeneity, are thought to play a crucial role in biomolecular functions (5–11). It is difficult to characterize such spatially and temporally inhomogeneous dynamics in bulk solution by an ensemble-averaged measurement, especially in proteins that undergo multiple-conformation transformations. In such cases, single-molecule spectroscopy is a powerful approach to analyze protein conformational states and dynamics under physiological conditions, and can provide a molecular-level perspective on how a protein’s structural dynamics link to its functional mechanisms (12–21).A case in point is the nitric oxide synthase (NOS) enzymes (22–24), whose nitric oxide (NO) biosynthesis involves electron transfer reactions that are associated with relatively large-scale movement (tens of Å) of the enzyme domains (Fig. 1A). During catalysis, NADPH-derived electrons first transfer into an FAD domain and an FMN domain in NOS that together comprise the NOS reductase domain (NOSr), and then transfer from the FMN domain to a heme group that is bound in a separate attached “oxygenase” domain, which then enables NO synthesis to begin (22, 25–27). The electron transfers into and out of the FMN domain are the key steps for catalysis, and they appear to rely on the FMN domain cycling between electron-accepting and electron-donating conformational states (28, 29) (Fig. 1B). In this model, the FMN domain is suggested to be highly dynamic and flexible due to a connecting hinge that allows it to alternate between its electron-accepting (FAD→FMN) or closed conformation and electron-donating (FMN→heme) or open conformation (Fig. 1 A and B) (28, 30–36). In the electron-accepting closed conformation, the FMN domain interacts with the NADPH/FAD domain (FNR domain) to receive electrons, whereas in the electron donating open conformation the FMN domain has moved away to expose the bound FMN cofactor so that it may transfer electrons to a protein acceptor like the NOS oxygenase domain, or to a generic protein acceptor like cytochrome c. In this way, the reductase domain structure cycles between closed and open conformations to deliver electrons, according to a conformational equilibrium that determines the movements and thus the electron flux capacity of the FMN domain (25, 28, 32, 34, 35, 37). A similar conformational switching mechanism is thought to enable electron transfer through the FMN domain in the related flavoproteins NADPH-cytochrome P450 reductase and methionine synthase reductase (38–42).Open in a separate windowFig. 1.(A) The nNOSr ribbon structure (from PDB: 1TLL) showing bound FAD (yellow) in FNR domain (green), FMN (orange) in FMN domain (yellow), connecting hinge (blue), and the Cy3–Cy5 label positions (pink) and distance (42 Å, dashed line). (B) Cartoon of an equilibrium between the FMN-closed and FMN-open states, with Cy dye label positions indicated. (C) Cytochrome c reductase activity of nNOSr proteins in their CaM-bound and CaM-free states. Color scheme of bar graphs: Black, WT nNOSr unlabeled; Red, Cys-lite (CL) nNOSr unlabeled; Blue, E827C/Q1268C CL nNOSr unlabeled; and Dark cyan, E827C/Q1268C CL nNOSr labeled.NOS enzymes also contain a calmodulin (CaM) binding domain that is located just before the N terminus of the FMN domain (Fig. 1B), and this provides an important layer of regulation (25, 27). CaM binding to NOS enzymes increases electron transfer from NADPH through the reductase domain and also triggers electron transfer from the FMN domain to the NOS heme as is required for NO synthesis (31, 32). The ability of CaM, or similar signaling proteins, to regulate electron transfer reactions in enzymes is unusual, and the mechanism is a topic of interest and intensive study. It has long been known that CaM binding alters NOSr structure such that, on average, it populates a more open conformation (43, 44). Recent equilibrium studies have detected a buildup of between two to four discreet conformational populations in NOS enzymes and in related flavoproteins, and in some cases, have also estimated the distances between the bound FAD and FMN cofactors in the different species (26, 36, 37, 39, 40), and furthermore, have confirmed that CaM shifts the NOS population distribution toward more open conformations (34, 36, 45). Although valuable, such ensemble-averaged results about conformational states cannot explain how electrons transfer through these enzymes, or how CaM increases the electron flux in NOS, because answering these questions requires a coordinate understanding of the dynamics of the conformational fluctuations. Indeed, computer modeling has indicated that a shift toward more open conformations as is induced by CaM binding to nNOS should, on its own, actually diminish electron flux through nNOS and through certain related flavoproteins (38). Despite its importance, measuring enzyme conformational fluctuation dynamics is highly challenging, and as far as we know, there have been no direct measures on the NOS enzymes or on related flavoproteins, nor studies on how CaM binding might influence the conformational fluctuation dynamics in NOS.To address this gap, we used single-molecule fluorescence energy resonance transfer (FRET) spectroscopy to characterize individual molecules of nNOSr that had been labeled at two specific positions with Cyanine 3 (Cy3) donor and Cyanine 5 (Cy5) acceptor dye molecules, regarding their conformational states distribution and the associated conformational fluctuation dynamics, which in turn enabled us to determine how CaM binding impacts both parameters. This work provides a unique perspective and a novel study of the NOS enzymes and within the broader flavoprotein family, which includes the mammalian enzymes methionine synthase reductase (MSR) and cytochrome P450 reductase (CPR), and reveals how CaM’s control of the conformational behaviors may regulate the electron transfer reactions of NOS catalysis.     

Aggregation propensities of superoxide dismutase G93 hotspot mutants mirror ALS clinical phenotypes

Protein framework alterations in heritable Cu, Zn superoxide dismutase (SOD) mutants cause misassembly and aggregation in cells affected by the motor neuron disease ALS. However, the mechanistic relationship between superoxide dismutase 1 (SOD1) mutations and human disease is controversial, with many hypotheses postulated for the propensity of specific SOD mutants to cause ALS. Here, we experimentally identify distinguishing attributes of ALS mutant SOD proteins that correlate with clinical severity by applying solution biophysical techniques to six ALS mutants at human SOD hotspot glycine 93. A small-angle X-ray scattering (SAXS) assay and other structural methods assessed aggregation propensity by defining the size and shape of fibrillar SOD aggregates after mild biochemical perturbations. Inductively coupled plasma MS quantified metal ion binding stoichiometry, and pulsed dipolar ESR spectroscopy evaluated the Cu²⁺ binding site and defined cross-dimer copper–copper distance distributions. Importantly, we find that copper deficiency in these mutants promotes aggregation in a manner strikingly consistent with their clinical severities. G93 mutants seem to properly incorporate metal ions under physiological conditions when assisted by the copper chaperone but release copper under destabilizing conditions more readily than the WT enzyme. Altered intradimer flexibility in ALS mutants may cause differential metal retention and promote distinct aggregation trends observed for mutant proteins in vitro and in ALS patients. Combined biophysical and structural results test and link copper retention to the framework destabilization hypothesis as a unifying general mechanism for both SOD aggregation and ALS disease progression, with implications for disease severity and therapeutic intervention strategies.ALS is a lethal degenerative disease of the human motor system (1). Opportunities for improved understanding and clinical intervention arose from the discovery that up to 23.5% of familial ALS cases and 7% of spontaneous cases are caused by mutations in the superoxide dismutase 1 (SOD1) gene encoding human Cu, Zn SOD (2–4). SOD is a highly conserved (5), dimeric, antioxidant metalloenzyme that detoxifies superoxide radicals (6, 7), but overexpression of SOD1 ALS mutants is sufficient to cause disease in mice (8). Misfolded and/or aggregated SOD species are deposited within mouse neuronal and glial inclusions (9, 10), even before symptoms appear (11, 12). Although human familial ALS has a symptomatic phenotype indistinguishable from sporadic cases (13), individual SOD1 mutations can result in highly variable disease progression and penetrance (14, 15).Many nongeneral mechanisms, including loss of activity or gain of function, were postulated to explain the roles of SOD mutants in ALS (3, 16–19). Recently, however, an initial hypothesis proposing that SOD manifests disease symptoms by framework destabilization (protein instability caused by structural defects) and consequent protein misassembly and aggregation has gained renewed support (2, 10, 14, 20–23). Ironically, WT SOD is an unusually stable protein (7, 24–26), and precisely how SOD mutations cause disease remains unclear. For instance, human SOD free cysteine residues C6 and C111 have been implicated in protein aggregation by promoting cross-linking (27, 28) and/or stability changes associated with oxidative modifications (29–33). Mutation of the chemically reactive thiols significantly decreases the irreversible denaturation rate for human and bovine SOD (24, 34). However, ALS mutants in a C6A/C111S SOD (AS-SOD) background (35, 36) maintain the native C57–C146 disulfide bond but can still undergo aggregation, and mutations of the free cysteines can cause ALS (37, 38). These results imply that free cysteines are not strictly required but rather, may alter aggregation kinetics (20). SOD also contains two metal ion cofactors in each subunit: a catalytic copper ion (6) and a structurally stabilizing zinc ion (34, 39, 40) (Fig. 1A). In higher eukaryotes, a copper chaperone for SOD (CCS) plays an important role in catalyzing both the copper incorporation and native disulfide bond formation (41). Structural analyses of apo WT SOD point to greater flexibility or increased solvent accessibility of C6 otherwise buried in the stable dimer interface (42, 43), and molecular dynamics simulations also suggest a critical role for metal ions in protein structure, because SOD’s β-sheet propensity decreases in the absence of metals (44). As a result, apo SOD readily forms protein aggregates (45, 46), but the molecular structures of SOD aggregates are likely polymorphic and represent a controversial topic (23, 47–51). The intertwined effects of the aggregation-enhancing free cysteines, dimer-stabilizing metal ions, and CCS maturation of SOD complicate the study of the ALS-causing SOD mutations themselves, and therefore, a clear cause-and-effect relationship remains obscure and requires deconvolution.Open in a separate windowFig. 1.Comparison of crystallographic and solution structures of WT and G93A SOD. (A) Overall architecture of the WT SOD dimer is displayed in 90° rotated views. G93 (small red spheres) resides on a surface-exposed interstrand loop between the fifth and sixth sequential β-strands of SOD and is expected to be innocuous in facilitating protein stability; however, this site harbors the most substitutions observed to result in ALS. G93 is also distant from both (Upper) the dimer interface and (Lower Left) the SOD active site (gold and silver spheres), which are generally implicated as the major determinants for SOD stability. Small blue spheres denote free cysteines. (Lower Right) The close-up view of the mutation site (boxed region in Lower Left tilted forward) shows high similarity between WT (purple) and G93A (red) SOD crystal structures [Protein Data Bank ID codes 1PU0 (WT) and 2ZKY (G93A)]. Hydrogen bonds characteristic of a β-bulge motif are indicated, whereby G93 (or A93) represents position 1. The main chain carbonyl group of β-barrel cork residue L38 is adjacent to the G93 site. (B) SAXS-derived electron pair P(r) distributions from WT (purple) and G93A (red) SOD samples in solution are compared with the theoretical curve for 1PU0. P(r) plots are normalized to peak height. Ab initio models of WT SOD derived from P(r) data are depicted in purple, with crystal structure docked into mesh envelope. Contributions to major and minor peaks from subunit and dimer dimensions are indicated.To better understand the structural effects of ALS mutations on SOD architecture, we coupled the wealth of crystallographic knowledge on SOD structure (7, 52, 53) with small-angle X-ray scattering (SAXS) experiments to characterize misassembly aggregates of ALS mutant SODs in solution. Over 20 y ago, we solved the first atomic structure of the human WT SOD protein (Fig. 1A) (20, 34) and proposed the framework destabilization hypothesis to explain how diverse mutations located throughout the 153-residue β-barrel enzyme might produce a similar disease phenotype (2), albeit with distinctions in the progression trajectory. Since that time, a staggering number of ALS mutations has been documented in patients [178 (mostly missense) (54)], with a similar phenotype in dogs (55, 56). Solution-based techniques are increasingly being applied to connect structure to biological outcome, for instance, through examination of intermolecular interactions within stress-activated pathways, for instance (57, 58). SAXS, which can probe structures for a wide size range of species, also provides higher resolution insights (59), for instance, over visible light-scattering techniques, readily distinguishing unfolded from folded proteins (60).Here, we monitor the initial events of protein aggregation in a subset of ALS mutants localized to a mutational hotspot site at glycine 93. Specifically, we wished to test a possible structural basis for how G93 mutations (to A, C, D, R, S, or V) modulate age of onset and clinical severity in ALS patients (14, 15). The G93 substitution occurs in a β-bulge region (61) between sequential β-strands of the protein (Fig. 1A) on a protruding loop roughly ∼20 Å from T54, the nearest residue of the opposing subunit, and the metal-containing active site (Fig. S1). A priori, mutation of this outer loop position would not be expected to interfere with active site chemistry or buried molecular interfaces. However, we discovered correlations of aggregation nucleation kinetics of SOD proteins with ALS mutations at this site, the stabilizing effects of metal ion retention, and available data for clinical phenotypes in patients with the same mutation. Furthermore, by measuring and exploiting the dimer geometry to observe intrinsic SOD conformers, we show that G93 mutant proteins natively reveal increased intradimer conformational flexibility in the absence of aggregation, which may reflect an increased tendency for ALS mutants to become metal-deficient and misfolding-prone and further explain the correlation to disease severity. Collective results on G93 mutants, thus, support and extend the framework destabilization hypothesis.     

Tuning a riboswitch response through structural extension of a pseudoknot

Structural and dynamic features of RNA folding landscapes represent critical aspects of RNA function in the cell and are particularly central to riboswitch-mediated control of gene expression. Here, using single-molecule fluorescence energy transfer imaging, we explore the folding dynamics of the preQ₁ class II riboswitch, an upstream mRNA element that regulates downstream encoded modification enzymes of queuosine biosynthesis. For reasons that are not presently understood, the classical pseudoknot fold of this system harbors an extra stem–loop structure within its 3′-terminal region immediately upstream of the Shine–Dalgarno sequence that contributes to formation of the ligand-bound state. By imaging ligand-dependent preQ₁ riboswitch folding from multiple structural perspectives, we reveal that the extra stem–loop strongly influences pseudoknot dynamics in a manner that decreases its propensity to spontaneously fold and increases its responsiveness to ligand binding. We conclude that the extra stem–loop sensitizes this RNA to broaden the dynamic range of the ON/OFF regulatory switch.A variety of small metabolites have been found to regulate gene expression in bacteria, fungi, and plants via direct interactions with distinct mRNA folds (1–4). In this form of regulation, the target mRNA typically undergoes a structural change in response to metabolite binding (5–9). These mRNA elements have thus been termed “riboswitches” and generally include both a metabolite-sensitive aptamer subdomain and an expression platform. For riboswitches that regulate the process of translation, the expression platform minimally consists of a ribosomal recognition site [Shine–Dalgarno (SD)]. In the simplest form, the SD sequence overlaps with the metabolite-sensitive aptamer domain at its downstream end. Representative examples include the S-adenosylmethionine class II (SAM-II) (10) and the S-adenosylhomocysteine (SAH) riboswitches (11, 12), as well as prequeuosine class I (preQ₁-I) and II (preQ₁-II) riboswitches (13, 14). The secondary structures of these four short RNA families contain a pseudoknot fold that is central to their gene regulation capacity. Although the SAM-II and preQ₁-I riboswitches fold into classical pseudoknots (15, 16), the conformations of the SAH (17) and preQ₁-II counterparts are more complex and include a structural extension that contributes to the pseudoknot architecture (14). Importantly, the impact and evolutionary significance of these “extra” stem–loop elements on the function of the SAH and preQ₁-II riboswitches remain unclear.PreQ₁ riboswitches interact with the bacterial metabolite 7-aminomethyl-7-deazaguanine (preQ₁), a precursor molecule in the biosynthetic pathway of queuosine, a modified base encountered at the wobble position of some transfer RNAs (14). The general biological significance of studying the preQ₁-II system stems from the fact that this gene-regulatory element is found almost exclusively in the Streptococcaceae bacterial family. Moreover, the preQ₁ metabolite is not generated in humans and has to be acquired from the environment (14). Correspondingly, the preQ₁-II riboswitch represents a putative target for antibiotic intervention. Although preQ₁ class I (preQ₁-I) riboswitches have been extensively investigated (18–28), preQ₁ class II (preQ₁-II) riboswitches have been largely overlooked despite the fact that a different mode of ligand binding has been postulated (14).The consensus sequence and the secondary structure model for the preQ₁-II motif (COG4708 RNA) (Fig. 1A) comprise ∼80–100 nt (14). The minimal Streptococcus pneumoniae R6 aptamer domain sequence binds preQ₁ with submicromolar affinity and consists of an RNA segment forming two stem–loops, P2 and P4, and a pseudoknot P3 (Fig. 1B). In-line probing studies suggest that the putative SD box (AGGAGA; Fig. 1) is sequestered by pseudoknot formation, which results in translational-dependent gene regulation of the downstream gene (14).Open in a separate windowFig. 1.PreQ₁ class II riboswitch. (A) Chemical structure of 7-aminomethyl-7-deazaguanosine (preQ₁); consensus sequence and secondary structure model for the COG4708 RNA motif (adapted from reference 14). Nucleoside presence and identity as indicated. (B) S. pneumoniae R6 preQ₁-II RNA aptamer investigated in this study. (C) Schematics of an H-type pseudoknot with generally used nomenclature for comparison.Here, we investigated folding and ligand recognition of the S. pneumoniae R6 preQ₁-II riboswitch, using complementary chemical, biochemical, and biophysical methods including selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE), mutational analysis experiments, 2-aminopurine fluorescence, and single-molecule fluorescence resonance energy transfer (smFRET) imaging. In so doing, we explored the structural and functional impact of the additional stem–loop element in the context of its otherwise “classical” H-type pseudoknot fold (29–32) (Fig. 1C). Our results reveal that the unique 3′-stem–loop element in the preQ₁-II riboswitch contributes to the process of SD sequestration, and thus the regulation of gene expression, by modulating both its intrinsic dynamics and its responsiveness to ligand binding.     

Extreme ecosystem instability suppressed tropical dinosaur dominance for 30 million years

A major unresolved aspect of the rise of dinosaurs is why early dinosaurs and their relatives were rare and species-poor at low paleolatitudes throughout the Late Triassic Period, a pattern persisting 30 million years after their origin and 10–15 million years after they became abundant and speciose at higher latitudes. New palynological, wildfire, organic carbon isotope, and atmospheric pCO₂ data from early dinosaur-bearing strata of low paleolatitudes in western North America show that large, high-frequency, tightly correlated variations in δ¹³C_org and palynomorph ecotypes occurred within a context of elevated and increasing pCO₂ and pervasive wildfires. Whereas pseudosuchian archosaur-dominated communities were able to persist in these same regions under rapidly fluctuating extreme climatic conditions until the end-Triassic, large-bodied, fast-growing tachymetabolic dinosaurian herbivores requiring greater resources were unable to adapt to unstable high CO₂ environmental conditions of the Late Triassic.One of the major predictions of models of elevated atmospheric CO₂ is the increased frequency and magnitude of events comprising very high temperatures, an enhanced hydrological cycle, and increased precipitation extremes (1, 2). Because such environmental extremes act as limitations on organisms, past time intervals of elevated CO₂ and associated climate extremes might be expected to profoundly influence biogeographic patterns, especially on land, which is relatively unbuffered climatically compared with the oceans. One such time of elevated CO₂ was the Triassic Period, during which both dinosaurs and mammals first appeared. In particular, it has remained an open question why the global ecological dominance of dinosaurs was delayed in the tropics for at least 30 million years after their first appearance and diversification into the three major clades Sauropodomorpha, Theropoda, and Ornithischia (3, 4). Hypotheses proposed to explain this lag have focused largely on competition (or lack thereof) with nondinosaurian archosaurs, principally those on the line to crocodylians (pseudosuchians), but none provide a clear explanation for this unusual and persistent biogeographic pattern.The rise of dinosaurs to ecological dominance was a diachronous evolutionary event (5–8). Small carnivorous early theropod dinosaurs were widespread at low paleolatitudes, whereas evidence for Triassic herbivorous dinosaurs (i.e., sauropodomorphs and ornithischians) in the tropics is completely absent (6, 7, 9, 10) (Fig. 1). In addition, tropical North American theropod dinosaurs were rare and species-poor (5, 7, 10) compared with higher-latitude assemblages. These patterns have been hypothesized to track largely zonal climatic conditions across Pangaea (6, 8, 11, 12) (Fig. 1), but detailed paleoclimatic data and mechanistic explanations have been lacking. Here, we argue these biogeographic patterns are a result of extreme environmental fluctuations in the tropics enhanced by high atmospheric CO₂, which suppressed large-bodied herbivorous dinosaurs until after the end-Triassic mass extinction.Open in a separate windowFig. 1.Late Triassic Pangean map showing latitudinal climate zones (11, 12) and the distribution of major dinosaur clades. See SI Appendix for occurrence data. Question marks indicate geochronologic uncertainty for the Thailand sauropodomorph and Argentine Laguna Colorada heterodontosaurid occurrences (i.e., they may be Early Jurassic in age). Each dinosaur symbol in most cases represents a region with multiple fossiliferous localities containing the illustrated clades.We present, to our knowledge, the first high-resolution paleoenvironmental multiproxy record from the same sedimentary sequences that produce abundant early dinosaur and other vertebrate fossils (6, 8, 10). Specifically, we sampled fluvial and overbank sediments of the Upper Triassic Chinle Formation of the Chama Basin in north central New Mexico (13, 14). This nonmarine succession from low-paleolatitude Pangaea moved from ∼10°N to 14°N during the late Norian and Rhaetian (15), suggesting that this area would have experienced a semiarid climate through the entire sequence (11) (Fig. 1). The formation in this region contains exceptionally diverse and abundant vertebrate assemblages, which allows the early evolution of dinosaurs, their contemporaneous flora, and their paleoenvironment to be examined through time. Furthermore, tight age control is provided by a recent U–Pb radioisotopic age of 211.9 ± 0.7 Ma from the Hayden Quarry (HQ) in the lower portion of the Petrified Forest Member of the Chama Basin (7), and magnetostratigraphic data (16) that are consistent with a late Norian to Rhaetian age for the sequence.     

Ion-binding properties of a K+ channel selectivity filter in different conformations

K⁺ channels are membrane proteins that selectively conduct K⁺ ions across lipid bilayers. Many voltage-gated K⁺ (K_V) channels contain two gates, one at the bundle crossing on the intracellular side of the membrane and another in the selectivity filter. The gate at the bundle crossing is responsible for channel opening in response to a voltage stimulus, whereas the gate at the selectivity filter is responsible for C-type inactivation. Together, these regions determine when the channel conducts ions. The K⁺ channel from Streptomyces lividians (KcsA) undergoes an inactivation process that is functionally similar to K_V channels, which has led to its use as a practical system to study inactivation. Crystal structures of KcsA channels with an open intracellular gate revealed a selectivity filter in a constricted conformation similar to the structure observed in closed KcsA containing only Na⁺ or low [K⁺]. However, recent work using a semisynthetic channel that is unable to adopt a constricted filter but inactivates like WT channels challenges this idea. In this study, we measured the equilibrium ion-binding properties of channels with conductive, inactivated, and constricted filters using isothermal titration calorimetry (ITC). EPR spectroscopy was used to determine the state of the intracellular gate of the channel, which we found can depend on the presence or absence of a lipid bilayer. Overall, we discovered that K⁺ ion binding to channels with an inactivated or conductive selectivity filter is different from K⁺ ion binding to channels with a constricted filter, suggesting that the structures of these channels are different.K⁺ channels are found in all three domains of life, where they selectively conduct K⁺ ions across cell membranes. Specific stimuli trigger the activation of K⁺ channels, which results in a hinged movement of the inner helix bundle (1–7). This opening on the intracellular side of the membrane initiates ion conduction across the membrane by allowing ions to enter into the channel. After a period, many channels spontaneously inactivate to attenuate the response (8–17). The inactivation process is a timer that terminates the flow of ions in the presence of an activator to help shape the response of the system. Two dominant types of inactivation have been characterized in voltage-dependent channels: N-type and C-type (18). N-type inactivation is fast and involves an N-terminal positively charged “ball” physically plugging the pore of the channel when the membrane is depolarized. C-type inactivation, on the other hand, is a slower process involving a conformational change in the selectivity filter that is initiated by a functional link between the intracellular gate and the selectivity filter (10, 19).Several experimental observations indicate a role for the selectivity filter in C-type inactivation. First, mutations in and around the selectivity filter can alter the kinetics of inactivation (20–23). Second, increasing concentrations of extracellular K⁺ ions decrease the rate of inactivation, as if the ions are stabilizing the conductive conformation of the channel to prevent a conformational change in the selectivity filter (14, 16, 17, 22). Finally, a loss of selectivity of K⁺ over Na⁺ has been observed during the inactivation process in Shaker channels, suggesting a role for the selectivity filter (24, 25). Together, these data indicate that channels in their inactivated and conductive conformations interact with K⁺ ions differently, and suggest that C-type inactivation involves a conformational change in the selectivity filter. Although several structures of K⁺ channels in their conductive state have been solved using X-ray crystallography, there is at present no universally accepted model for the C-type inactivated channel (1, 3–5, 9, 19, 26–28) (Fig. 1B).Open in a separate windowFig. 1.Macroscopic recordings and structural models of KcsA K⁺ channel. (A) Macroscopic currents of WT KcsA obtained by a pH jump from pH 8 to pH 4 reveal channel inactivation. Two models representing the conformation of the channel are shown below. (B) Conductive [Left, Protein Data Bank (PDB) ID code 1K4C] and constricted (Right, PDB ID code 1K4D) conformations of the selectivity filter are shown as sticks, and the ion-binding sites are indicated with green spheres. The thermodynamic properties of the conductive, constricted, and inactivated (Middle) conformations are the subject of this study.Inactivation in the K⁺ channel from Streptomyces lividians (KcsA) has many of the same functional properties of C-type inactivation, which has made it a model to understand its structural features (20). KcsA channels transition from their closed to open gate upon changing the intracellular pH from high to low (Fig. 1A). The rapid flux of ions through the channel is then attenuated by channel inactivation, where most open WT channels are not conducting, suggesting that crystal structures of open KcsA channels would reveal the inactivated channel. In some crystal structures of truncated WT KcsA solved with an open gate, the selectivity filter appears in the constricted conformation, similar to the conformation observed in structures of the KcsA channel determined in the presence of only Na⁺ ions or low concentrations of K⁺ ions (3, 10, 29, 30) (Fig. 1B). Solid-state and solution NMR also indicate that the selectivity filter of the KcsA channel is in the constricted conformation when the cytoplasmic gate is open (31–33).However, a recently published study shows that even when the constricted conformation of KcsA’s selectivity filter is prevented by a nonnatural amino acid substitution, the channel inactivates like WT channels, suggesting the constricted filter does not correspond to the functionally observed inactivation in KcsA (28). In this study, we use isothermal titration calorimetry (ITC) to quantify the ion-binding properties of WT and mutant KcsA K⁺ channels with their selectivity filters in different conformations and EPR spectroscopy to determine the conformation of the channels’ intracellular gates. A comparison of these ion-binding properties leads us to conclude that the conductive and inactivated filters are energetically more similar to each other than the constricted and inactivated filters.     

Nitrogenase-mimic iron-containing chalcogels for photochemical reduction of dinitrogen to ammonia

A nitrogenase-inspired biomimetic chalcogel system comprising double-cubane [Mo₂Fe₆S₈(SPh)₃] and single-cubane (Fe₄S₄) biomimetic clusters demonstrates photocatalytic N₂ fixation and conversion to NH₃ in ambient temperature and pressure conditions. Replacing the Fe₄S₄ clusters in this system with other inert ions such as Sb³⁺, Sn⁴⁺, Zn²⁺ also gave chalcogels that were photocatalytically active. Finally, molybdenum-free chalcogels containing only Fe₄S₄ clusters are also capable of accomplishing the N₂ fixation reaction with even higher efficiency than their Mo₂Fe₆S₈(SPh)₃-containing counterparts. Our results suggest that redox-active iron-sulfide–containing materials can activate the N₂ molecule upon visible light excitation, which can be reduced all of the way to NH₃ using protons and sacrificial electrons in aqueous solution. Evidently, whereas the Mo₂Fe₆S₈(SPh)₃ is capable of N₂ fixation, Mo itself is not necessary to carry out this process. The initial binding of N₂ with chalcogels under illumination was observed with in situ diffuse-reflectance Fourier transform infrared spectroscopy (DRIFTS). ¹⁵N₂ isotope experiments confirm that the generated NH₃ derives from N₂. Density functional theory (DFT) electronic structure calculations suggest that the N₂ binding is thermodynamically favorable only with the highly reduced active clusters. The results reported herein contribute to ongoing efforts of mimicking nitrogenase in fixing nitrogen and point to a promising path in developing catalysts for the reduction of N₂ under ambient conditions.The reduction of atmospheric nitrogen to ammonia is one of the most essential processes for sustaining life. Currently, roughly half of the fixed nitrogen is supplied biologically by nitrogenase, while nearly the other half is from the industrial Haber–Bosch process, which operates under high temperature (400–500 °C) and high pressure (200–250 bar) in the presence of a metallic iron catalyst (1). Nitrogenase, a two-component protein system comprising a MoFe protein and an associated Fe protein, carries out this “fixation” in nature under ambient temperature and pressure (2–4). N₂ substrate binding and activation take place at the iron–molybdenum–sulfur cofactor (FeMoco), and in some cases, Mo-free iron–sulfur cofactor FeFeco and iron–vanadium–sulfur cofactor FeVco cofactors. Electron transfer during this catalytic process is believed to proceed from a [4Fe:4S] cluster located in the Fe protein to another Fe/S cluster (the P cluster) buried in the MoFe protein and finally to the FeMoco (Fig. 1A) (2, 5, 6). Whereas the role of Mo in the reactivity of nitrogenase has been the subject of long debate, iron is now well recognized as the only transition metal essential to all nitrogenases, and recent biochemical and spectroscopic data point to iron as the site of N₂ binding in the FeMoco (7–9). Naturally, understanding and mimicking how the nitrogenase enzyme accomplishes the difficult task of N₂ reduction under ambient conditions is one of the grand challenges in chemistry. To this end, inspired by the molecular structure and function of FeMoco, a number of groups have synthesized transition metal–dinitrogen complexes and examined stoichiometric transformations of their coordinated N₂ into NH₃ and N₂H₄ (9–19). However, the operation of homogeneous transition metal–dinitrogen complexes usually requires organic solvents, strong reducing agents, and often extremely low operation temperatures (5, 10, 12, 14). The prospect of using solar light energy to convert N₂ to ammonia is highly attractive but it represents a great challenge and is a less-investigated line of inquiry. Hamers and co-workers reported that solvated electrons emitted from illuminated diamond can accomplish N₂ reduction (20, 21). Other semiconductor systems such as Fe₂Ti₂O₇ (22), Au/Nb–SrTiO₃/Ru (23), and BiOBr_ov (24) were also reported to perform light-induced N₂ fixation. These systems are not biomimetic and usually exhibit very low conversion efficiency (SI Appendix, Table S1).Open in a separate windowFig. 1.Nitrogenase-inspired biomimetic chalcogels. (A) The two-component proteins of molybdenum nitrogenase: MoFe protein and Fe protein; space filling and stick model structures of the FeMo cofactor and the P cluster. (B) The reaction routes leading to the assembly of FeMoS–SnS, FeMoS–FeS–SnS, and FeMoS–M–SnS (M=Sb³⁺, Sn⁴⁺, Zn²⁺) chalcogel, respectively.Our group has recently developed a new class of porous chalcogenide aerogels by the metathesis reaction, dubbed “chalcogels,” which can be functionalized with biomimetic functionalities (25–27). These materials can easily incorporate Mo₂Fe₆S₈(SPh)₃ or Fe₄S₄ clusters in their structure and have been shown to reduce protons both electrocatalytically and photocatalytically to hydrogen (28, 29). The Mo₂Fe₆S₈(SPh)₃ cluster-based chalcogel was recently demonstrated to be capable of photocatalytically reducing N₂ to NH₃ (30). Inspired by the structure and function of the MoFe protein of nitrogenase which contains both iron–molybdenum–sulfur and iron–sulfur clusters (the P cluster), we prepared a chalcogel that also incorporated two types of clusters: the FeMoco-like Mo₂Fe₆S₈(SPh)₃ and P-cluster–like Fe₄S₄ linked together with units of [Sn₂S₆]^4-, in a 3D superstructure (Fig. 1B) (2, 4, 30). This chalcogel is dubbed “FeMoS–FeS-SnS.” We also prepared two more chalcogels, one with Mo₂Fe₆S₈(SPh)₃ clusters and inert metals such as Sb³⁺, Sn⁴⁺, Zn²⁺ (dubbed “FeMoS–M-SnS”) and a molybdenum-free one, the Fe₄S₄ chalcogel (FeS–SnS) (31, 32). The purpose of using the FeMoS–M–SnS chalcogels was to see if placing the FeMoS clusters farther apart in space would have any effect on the catalytic reaction, whereas that of FeS–SnS was to probe the necessity of Mo. These three chalcogels achieve photocatalytic N₂ reduction but more importantly, and to our surprise, the iron-only FeS–SnS chalcogel is in fact not only capable of N₂ reduction but also with higher rate. Diffuse-reflectance Fourier transform infrared spectroscopy (DRIFTS) experiments performed under light illumination show a clear signature of the N₂ binding process and its subsequent reduction. The results reported here show that the photochemical activation of N₂ using visible light is possible with Mo₂Fe₆S₈(SPh)₃ as well as Fe₄S₄-based materials at room temperature, ambient pressure, and aqueous conditions. Despite the complex progression multielectron/-proton reactions required, we clearly have an unexpectedly viable and robust process that leads to ammonia. Our results also demonstrate that iron rather than molybdenum is the element necessary for photoreduction of N₂ to NH₃.     

Selective molecular transport through the protein shell of a bacterial microcompartment organelle

Bacterial microcompartments are widespread prokaryotic organelles that have important and diverse roles ranging from carbon fixation to enteric pathogenesis. Current models for microcompartment function propose that their outer protein shell is selectively permeable to small molecules, but whether a protein shell can mediate selective permeability and how this occurs are unresolved questions. Here, biochemical and physiological studies of structure-guided mutants are used to show that the hexameric PduA shell protein of the 1,2-propanediol utilization (Pdu) microcompartment forms a selectively permeable pore tailored for the influx of 1,2-propanediol (the substrate of the Pdu microcompartment) while restricting the efflux of propionaldehyde, a toxic intermediate of 1,2-propanediol catabolism. Crystal structures of various PduA mutants provide a foundation for interpreting the observed biochemical and phenotypic data in terms of molecular diffusion across the shell. Overall, these studies provide a basis for understanding a class of selectively permeable channels formed by nonmembrane proteins.The complex behavior of biological systems depends fundamentally on the controlled movement of molecules between cellular compartments. Such processes occur in a wide range of biological contexts through the movement of ions and small molecules across lipid bilayers via proteins—channels and pumps—embedded in the bilayer. Achievements in understanding molecular transport in transmembrane systems have contributed to scientific disciplines from cell biology and physiology to membrane biophysics (1, 2). Interestingly, there exists a second type of system for molecular transport through proteins that is fundamentally different and much less understood. Hundreds of species of bacteria produce large subcellular organelles known as microcompartments (MCPs), which consist of metabolic enzymes encapsulated within proteinaceous shells reminiscent of viral capsids (reviewed in ref. 3). For MCPs to function, substrates and products must move across their outer protein shell, which lacks any lipid-based membrane. In the last several years, 3D structures of the proteins that comprise MCP shells have revealed narrow pores through their centers that have been hypothesized to be the routes by which substrates enter (and products escape from) MCPs (4; reviewed in ref. 5). However, experimental evidence to support this key hypothesis and the molecular principles involved is lacking.The overarching function of MCPs is to optimize metabolic pathways that have toxic or volatile intermediates. MCPs are present across at least 11 different bacterial phyla, where they carry out diverse metabolic processes (6–12). The carboxysome MCP is used to enhance CO₂ fixation in nearly all bacteria that use the Calvin cycle, and it has been estimated that 25% of the carbon fixation on Earth occurs within this proteinaceous bacterial organelle (9). The 1,2-propanediol utilization (Pdu) and ethanolamine utilization (Eut) MCPs are used to optimize 1,2-propanediol (1,2-PD) and ethanolamine catabolism, respectively (13–15), and the degradation of these compounds is thought to promote enteric pathogenesis (12, 16, 17). Although the Pdu and Eut MCPs, the carboxysome, and other metabolically diverse MCPs of unknown function encapsulate distinct sets of enzymes, all have shells built from homologous proteins suggesting they operate by conserved functional principles. Most models of MCP function propose that the protein shell acts as a diffusion barrier that allows passage of substrates (and products) while limiting the escape of a toxic or volatile metabolic intermediate such as CO₂ or toxic aldehyde (9, 18), but selective permeability by MCP shells has not been established experimentally.The shells of MCPs are assembled primarily from a family of small proteins that have so-called bacterial microcompartment (BMC) domains (5). Many BMC domain proteins form flat, hexagonally shaped oligomers that tile into extended sheets that form the basis of the MCP shell (4, 19, 20) (Fig. 1). In most cases, MCP shells are composed of four to eight different types of functionally diversified BMC domain proteins, some of which have pores proposed to mediate the selective movement of metabolites across the shell (4, 7, 8, 18). For example, the PduA shell protein from the Pdu MCP has a small central pore (∼6 Å) that is lined with numerous hydrogen-bond donors and acceptors, leading to a suggested role in the preferential movement of 1,2-PD over the less polar propionaldehyde (a toxic intermediate) (21). In addition, a subgroup of BMC proteins have been crystallized in two distinct conformations where the central pore is either fully closed or opened widely (12–15 Å), suggesting that a gating mechanism might control the movement of larger molecules (such as enzymatic cofactors) across the MCP shell (22, 23). However, no physiological or biochemical studies demonstrating transport or selective movement specifically through any MCP pore have been reported. As a result, the idea that the MCP protein shell is capable of mediating selective diffusion has lacked a clear experimental basis. Furthermore, a recent alternative model for MCP function proposes that enzymes embedded in or tightly associated with the shell could move metabolites into MCPs by vectorial catalysis, in which case functional pores might not be required for metabolite movement (24).Open in a separate windowFig. 1.Structure and function of the propanediol utilization (Pdu) bacterial microcompartment. A few thousand shell proteins (mostly of the BMC family) encapsulate a series of enzymes for metabolizing 1,2-PD. The protein shell of the Pdu MCP has been hypothesized to be selectively permeable allowing substrates such as 1,2-PD to enter through small pores in the center of hexameric shell proteins while restricting the efflux of propionaldehyde, which is toxic to the cell. For clarity, the reaction scheme has been simplified by omitting some of the steps involved in coenzyme B₁₂ recycling.Here, we use the known structure of PduA—a canonical, hexameric BMC-type shell protein in the Pdu MCP—to design a series of mutant shell proteins having central pores with altered sizes and physicochemical properties. A combination of physiological studies on mutant bacteria, biochemical studies on isolated mutant MCPs, and crystal structure studies on the mutant shell proteins, show that the PduA pore serves as a key route for entry of the metabolic substrate (1,2-PD), and that the chemical properties of the PduA pore are tuned to limit the escape of the toxic propionaldehyde intermediate.