The catalytic mechanism of the mitochondrial methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFD2)

Methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFD2) is a new drug target that is expressed in cancer cells but not in normal adult cells, which provides an Achilles heel to selectively kill cancer cells. Despite the availability of crystal structures of MTHFD2 in the inhibitor- and cofactor-bound forms, key information is missing due to technical limitations, including (a) the location of absolutely required Mg²⁺ ion, and (b) the substrate-bound form of MTHFD2. Using computational modeling and simulations, we propose that two magnesium ions are present at the active site whereby (i) Arg233, Asp225, and two water molecules coordinate MgA2+, while MgA2+ together with Arg233 stabilize the inorganic phosphate (P_i); (ii) Asp168 and three water molecules coordinate MgB2+, and MgB2+ further stabilizes P_i by forming a hydrogen bond with two oxygens of P_i; (iii) Arg201 directly coordinates the P_i; and (iv) through three water-mediated interactions, Asp168 contributes to the positioning and stabilization of MgA2+, MgB2+ and P_i. Our computational study at the empirical valence bond level allowed us also to elucidate the detailed reaction mechanisms. We found that the dehydrogenase activity features a proton-coupled electron transfer with charge redistribution connected to the reorganization of the surrounding water molecules which further facilitates the subsequent cyclohydrolase activity. The cyclohydrolase activity then drives the hydration of the imidazoline ring and the ring opening in a concerted way. Furthermore, we have uncovered that two key residues, Ser197/Arg233, are important factors in determining the cofactor (NADP⁺/NAD⁺) preference of the dehydrogenase activity. Our work sheds new light on the structural and kinetic framework of MTHFD2, which will be helpful to design small molecule inhibitors that can be used for cancer treatment.     

Viscoelastic Transient of Confined Red Blood Cells

The unique ability of a red blood cell to flow through extremely small microcapillaries depends on the viscoelastic properties of its membrane. Here, we study in vitro the response time upon flow startup exhibited by red blood cells confined into microchannels. We show that the characteristic transient time depends on the imposed flow strength, and that such a dependence gives access to both the effective viscosity and the elastic modulus controlling the temporal response of red cells. A simple theoretical analysis of our experimental data, validated by numerical simulations, further allows us to compute an estimate for the two-dimensional membrane viscosity of red blood cells, ηmem2D ∼ 10⁻⁷ N⋅s⋅m⁻¹. By comparing our results with those from previous studies, we discuss and clarify the origin of the discrepancies found in the literature regarding the determination of ηmem2D, and reconcile seemingly conflicting conclusions from previous works.     

Topography Mediates the Influence of Cover Crops on Soil Nitrate Levels in Row Crop Agricultural Systems

Supplying adequate amounts of soil N for plant growth during the growing season and across large agricultural fields is a challenge for conservational agricultural systems with cover crops. Knowledge about cover crop effects on N comes mostly from small, flat research plots and performance of cover crops across topographically diverse agricultural land is poorly understood. Our objective was to assess effects of both leguminous (red clover) and non-leguminous (winter rye) cover crops on potentially mineralizable N (PMN) and NO3--N levels across a topographically diverse landscape. We studied conventional, low-input, and organic managements in corn-soybean-wheat rotation. The rotations of low-input and organic managements included rye and red clover cover crops. The managements were implemented in twenty large undulating fields in Southwest Michigan starting from 2006. The data collection and analysis were conducted during three growing seasons of 2011, 2012 and 2013. Observational micro-plots with and without cover crops were laid within each field on three contrasting topographical positions of depression, slope and summit. Soil samples were collected 4–5 times during each growing season and analyzed for NO3--N and PMN. The results showed that all three managements were similar in their temporal and spatial distributions of NO₃ ^—N. Red clover cover crop increased NO3--N by 35% on depression, 20% on slope and 32% on summit positions. Rye cover crop had a significant 15% negative effect on NO3--N in topographical depressions but not in slope and summit positions. The magnitude of the cover crop effects on soil mineral nitrogen across topographically diverse fields was associated with the amount of cover crop growth and residue production. The results emphasize the potential environmental and economic benefits that can be generated by implementing site-specific topography-driven cover crop management in row-crop agricultural systems.     

Dominance Genetic Variation Contributes Little to the Missing Heritability for Human Complex Traits

For human complex traits, non-additive genetic variation has been invoked to explain “missing heritability,” but its discovery is often neglected in genome-wide association studies. Here we propose a method of using SNP data to partition and estimate the proportion of phenotypic variance attributed to additive and dominance genetic variation at all SNPs (hSNP2 and δSNP2) in unrelated individuals based on an orthogonal model where the estimate of hSNP2 is independent of that of δSNP2. With this method, we analyzed 79 quantitative traits in 6,715 unrelated European Americans. The estimate of δSNP2 averaged across all the 79 quantitative traits was 0.03, approximately a fifth of that for additive variation (average hSNP2 = 0.15). There were a few traits that showed substantial estimates of δSNP2, none of which were replicated in a larger sample of 11,965 individuals. We further performed genome-wide association analyses of the 79 quantitative traits and detected SNPs with genome-wide significant dominance effects only at the ABO locus for factor VIII and von Willebrand factor. All these results suggest that dominance variation at common SNPs explains only a small fraction of phenotypic variation for human complex traits and contributes little to the missing narrow-sense heritability problem.     

Sexually antagonistic genetic variance for fitness in an ancestral and a novel environment

The intersex genetic correlation for fitness (rwfm), a standardized measure of the degree to which male and female fitness covary genetically, has consequences for important evolutionary processes, but few estimates are available and none have explored how it changes with environment. Using a half-sibling breeding design, we estimated the genetic (co)variance matrix (G) for male and female fitness, and the resulting rwfm, in Drosophila serrata. Our estimates were performed in two environments: the laboratory yeast food to which the population was well adapted and a novel corn food. The major axis of genetic variation for fitness in the two environments, accounting for 51.3 per cent of the total genetic variation, was significant and revealed a strong signal of sexual antagonism, loading negatively in both environments on males but positively on females. Consequently, estimates of rwfm were negative in both environments (−0.34 and −0.73, respectively), indicating that the majority of genetic variance segregating in this population has contrasting effects on male and female fitness. The possible strengthening of the negative rwfm in this novel environment may be a consequence of no history of selection for amelioration of sexual conflict. Additional studies from a diverse range of novel environments will be needed to determine the generality of this finding.     

Dominance of Deleterious Alleles Controls the Response to a Population Bottleneck

Population bottlenecks followed by re-expansions have been common throughout history of many populations. The response of alleles under selection to such demographic perturbations has been a subject of great interest in population genetics. On the basis of theoretical analysis and computer simulations, we suggest that this response qualitatively depends on dominance. The number of dominant or additive deleterious alleles per haploid genome is expected to be slightly increased following the bottleneck and re-expansion. In contrast, the number of completely or partially recessive alleles should be sharply reduced. Changes of population size expose differences between recessive and additive selection, potentially providing insight into the prevalence of dominance in natural populations. Specifically, we use a simple statistic, BR≡∑xipop1/∑xjpop2, where x _i represents the derived allele frequency, to compare the number of mutations in different populations, and detail its functional dependence on the strength of selection and the intensity of the population bottleneck. We also provide empirical evidence showing that gene sets associated with autosomal recessive disease in humans may have a B _R indicative of recessive selection. Together, these theoretical predictions and empirical observations show that complex demographic history may facilitate rather than impede inference of parameters of natural selection.     

Isolation and Structure of Sclerothionine,a Metabolite of Sclerotinia Fungus

A new sulfur-containing imidazole compound, m.p. 218~223°C (decomp.), ${[\alpha ]}_{\text{D}}^{24}+{7.4}^{°}$ in water), C₁₁H₁₉N₃O₃S was isolated from sclerotia of Sclerotinia libertiana and named sclerothionine. The chemical structure of sclerothionine was identified with 2-hydroxyethyl-ergothioneine which was synthesized from ethylene chlorhydrine and ergothioneine.     

Indolicidin Binding Induces Thinning of a Lipid Bilayer

We use all-atom molecular dynamics simulations on a massive scale to compute the standard binding free energy of the 13-residue antimicrobial peptide indolicidin to a lipid bilayer. The analysis of statistical convergence reveals systematic sampling errors that correlate with reorganization of the bilayer on the microsecond timescale and persist throughout a total of 1.4 ms of sampling. Consistent with experimental observations, indolicidin induces membrane thinning, although the simulations significantly overestimate the lipophilicity of the peptide.Antimicrobial peptides are a component of the innate immune system of eukaryotes (1). As such, they must interact with pathogenic membranes, either during translocation or by disrupting their structural integrity (2). Here we examine the binding of the 13-residue cationic antimicrobial peptide indolicidin (3) (ILPWKWPWWPWRR-NH₂) to a lipid membrane as a first step towards elucidating its mechanism of action.Molecular solutes interact with lipid membranes in many cellular processes (4). Computational approaches such as molecular dynamics simulations have been widely used to characterize these interactions (5). However, molecular dynamics simulations can require unfeasibly long times to reach equilibrium (6). Therefore, it is common to compute equilibrium properties of solute insertion into lipid bilayers using umbrella sampling (7) simulations in which the solute is restrained along the bilayer normal using harmonic restraining potentials, or umbrellas, centered at zi0 values distributed between bulk water and the bilayer center.It is often assumed that equilibrium properties rapidly attain convergence in umbrella sampling simulations; accordingly, convergence measures are rarely published (8). However, we have recently shown that umbrella sampling simulations require up to 100 ns per umbrella (3 μs in total) to eliminate systematic sampling errors in the standard free energy of binding, ΔGbind0, of an arginine side-chain analog from bulk water to a lipid bilayer (8). The fact that umbrella sampling has been used to investigate the bilayer insertion of substantially larger solutes (9) motivates a systematic evaluation of statistical sampling convergence of ΔGbind0 for indolicidin in a lipid bilayer.To estimate ΔGbind0 of indolicidin to a lipid bilayer, we conducted 60 sets of umbrella-sampling simulations while systematically varying the initial conformation. In each umbrella sampling simulation, each umbrella was simulated for 1.5 μs, yielding a total simulation time of 1.4 ms and 60 independent free energy or potential of mean force (PMF) profiles from bulk water to the center of a POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine) lipid bilayer.The PMF profiles indicate that indolicidin strongly binds to the bilayer, partitioning inside the lipid headgroups (Fig. 1, A and E). Importantly, the mean estimate of ΔGbind0 decays exponentially with equilibration time t_eq, indicating that systematic sampling errors in individual simulations continued to decrease throughout the 1.5-μs interval as rare events led to more favorable states (Fig. 1 B). The low frequency of transitions to more favorable states exacerbates the requirement for massive sampling using multiple independent simulations.Open in a separate windowFigure 1PMF for indolicidin partitioning into a POPC bilayer. (A) Average PMF from 60 independent umbrella-sampling simulations based on 1 < t ≤ 1.5 μs/umbrella. (B) Average ΔGbind0 from 50-ns time intervals per umbrella (t_eq < t ≤ t_eq+50 ns) as a function of equilibration time, t_eq. (Solid line) Single exponential fit to the mean over 0.5 < t_eq ≤ 1.5 μs. (C) Mean values of ΔGbind0 from the 10-μs/umbrella simulations (crosses) together with the mean values of ΔGbind0 (triangles) and exponential fit from panel B. PMF and ΔGbind0 profiles obtained from each of the 60 independent simulations are shown in Fig. S1 in the Supporting Material. (D–F) Representative conformations after 1.5 μs of simulation at zi0 = (D) 3 nm, (E) 1.2 nm, and (F) 0.0 nm. To see this figure in color, go online.Computational limitations precluded extending all 60 sets of umbrella-sampling simulations to even longer times. Instead, we identified the two simulations at each umbrella that appeared to be most representative of equilibrium and extended each to 10 μs per umbrella (see Methods in the Supporting Material). The resulting estimates of ΔGbind0 continued to decrease until t_eq = 4 μs (68 μs in total), after which they stabilized at the asymptotic limit of the exponential fit of the shorter simulation data (ΔGbind0 = −26 ± 5 kcal/mol; Fig. 1 C).As indolicidin approaches the bilayer, it is drawn closer (Fig. 2 A) as salt bridges form between the peptide and the phospholipid headgroups (Fig. 2 B), inducing their protrusion (Figs. 1 D and ​and22 C). At large separation distances, this state is attained only when the peptide becomes highly extended (Fig. 2, D and E). As indolicidin is inserted more deeply, the surface of the lipid bilayer invaginates (Figs. 1 E and ​and22 C), maintaining peptide-lipid salt bridges (Fig. 2 B) and leading to the formation of a pore when the solute is near the bilayer center (Figs. 1 F and ​and22 C, and see Fig. S2, Fig. S3, Fig. S4, and Fig. S5 in the Supporting Material). These Boltzmann-weighted ensemble averages may not be mechanistically representative of nonequilibrium binding events (8,10).Open in a separate windowFigure 2Slow equilibration of bilayer and peptide. (A–D) Color quantifies conformational reorganization for t_eq < t ≤ t_eq + 100 ns as a function of t_eq and |zi0|. (A) Deviation of insertion depth, z, from zi0, Δz ≡ z − zi0; (B) number of peptide-lipid salt bridges, N_SB; (C) volume change of the bilayer’s proximal leaflet in the radial vicinity of the solute, V_ε; and (D) peptide end-to-end distance (EED). There is no sampling for t > 0.5 μs at |zi0| ≥ 4.5 nm. (E) Representative time-series of a trajectory at zi0 = 3.9 nm. (F) Representative conformation at 10 μs for |zi0| = 1.2 nm. To see this figure in color, go online.The reorganization of the peptide, the bilayer, and the ionic interactions between them became more pronounced with increasing simulation time at peptide insertion depths shallower than the global free energy minimum (|zi0| >1.4 nm; Fig. 1 A and Fig. 2, B–D). These conformational transitions are likely the source of the systematic drift of ΔGbind0. Reorganization of the bilayer also controls the rate of equilibration during membrane insertion of an arginine side chain (8,9) and a cyclic arginine nonamer (11), suggesting that slow reorganization of lipids around cationic solutes presents a general impediment to simulation convergence.Consistent with the perturbation of membrane thickness observed by in situ atomic force microscopy (12), our results suggest that indolicidin insertion induces local thinning of the bilayer (Fig. 1, E and F, and Fig. 2, C and F). The different conformational ensembles sampled by the peptide in water and in the lipid bilayer (Fig. 2 D) are consistent with the observations that indolicidin is disordered in solution (13) and adopts stable conformations in the presence of detergent (14). Although the peptide’s conformation continued to change when it was deeply inserted (Fig. 2 D), the amount of water in the bilayer’s hydrophobic core converged relatively rapidly (see Fig. S2). Indolicidin can induce the formation of hydrated, porelike defects (see Fig. S2, Fig. S3, Fig. S4, and Fig. S5) but does not act as a chloride carrier (see Fig. S6 and Fig. S7). Future studies of the mechanism of indolicidin action will examine the effect of multiple peptide binding.The PMF profile presented in this Letter is strikingly different from that computed by Yeh et al. (15) using different force field parameters for indolicidin partitioning into a DMPC (1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine) bilayer, from which the binding free energy was estimated to be 0 kcal/mol (15). However, that study comprised only 25 ns per umbrella and likely suffers from systematic sampling errors induced by initial conditions (see Fig. S8).Our estimate of the binding affinity is much larger than the values obtained for indolicidin and large unilamellar POPC vesicles using isothermal titration calorimetry, −7.4 kcal/mol (16), and equilibrium dialysis, −8.8 kcal/mol (13). Such a discrepancy suggests that the relative accuracy of binding free energies for amino-acid side-chain analogs (8,9,17) does not necessarily extend to polypeptides. Although more work is needed to elucidate the source of this discrepancy, this study underlines the importance of attaining convergence before evaluating force-field accuracy.Importantly, this work also highlights the extensive sampling required to remove systematic errors induced by initial conditions in atomistic simulations of peptides in membranes. Slow equilibration of the system is due to rare transitions across hidden free energy barriers involving reorganization of the membrane. Two simple recommendations are 1), evaluating the time-dependence of ensemble averages, and 2), conducting multiple simulations with different initial conditions. We have recently shown that by using enhanced sampling techniques it is possible to identify the locations of hidden free energy barriers without a priori knowledge (9). Future research will examine strategies for speeding up the crossing of these barriers, such as optimized order parameters including bilayer reorganization and enhanced sampling techniques including a random walk along the order parameter (9).     

Controlling Electron Transfer between the Two Cofactor Chains of Photosystem I by the Redox State of One of Their Components

Two functional electron transfer (ET) chains, related by a pseudo-C₂ symmetry, are present in the reaction center of photosystem I (PSI). Due to slight differences in the environment around the cofactors of the two branches, there are differences in both the kinetics of ET and the proportion of ET that occurs on the two branches. The strongest evidence that this is indeed the case relied on the observation that the oxidation rates of the reduced phylloquinone (PhQ) cofactor differ by an order of magnitude. Site-directed mutagenesis of residues involved in the respective PhQ-binding sites resulted in a specific alteration of the rates of semiquinone oxidation. Here, we show that the PsaA-F689N mutation results in an ∼100-fold decrease in the observed rate of PhQA− oxidation. This is the largest change of PhQA− oxidation kinetics observed so far for a single-point mutation, resulting in a lifetime that exceeds that of the terminal electron donor, P700+. This situation allows a second photochemical charge separation event to be initiated before PhQA− has decayed, thereby mimicking in PSI a situation that occurs in type II reaction centers. The results indicate that the presence of PhQA− does not impact the overall quantum yield and leads to an almost complete redistribution of the fractional utilization of the two functional ET chains, in favor of the one that does not bear the charged species. The evolutionary implications of these results are also briefly discussed.     

uS5/Rps2 residues at the 40S ribosome entry channel enhance initiation at suboptimal start codons in vivo

The eukaryotic 43S pre-initiation complex (PIC) containing Met-tRNAiMet in a ternary complex (TC) with eIF2-GTP scans the mRNA leader for an AUG codon in favorable “Kozak” context. AUG recognition triggers rearrangement of the PIC from an open conformation to a closed state with more tightly bound Met-tRNAiMet. Yeast ribosomal protein uS5/Rps2 is located at the mRNA entry channel of the 40S subunit in the vicinity of mRNA nucleotides downstream from the AUG codon or rRNA residues that communicate with the decoding center, but its participation in start codon recognition was unknown. We found that nonlethal substitutions of conserved Rps2 residues in the entry channel reduce bulk translation initiation and increase discrimination against poor initiation codons. A subset of these substitutions suppress initiation at near-cognate UUG start codons in a yeast mutant with elevated UUG initiation, and also increase discrimination against AUG codons in suboptimal Kozak context, thus resembling previously described substitutions in uS3/Rps3 at the 40S entry channel or initiation factors eIF1 and eIF1A. In contrast, other Rps2 substitutions selectively discriminate against either near-cognate UUG codons, or poor Kozak context of an AUG or UUG start codon. These findings suggest that different Rps2 residues are involved in distinct mechanisms involved in discriminating against different features of poor initiation sites in vivo.     

Partitioning Heritability of Regulatory and Cell-Type-Specific Variants across 11 Common Diseases

Regulatory and coding variants are known to be enriched with associations identified by genome-wide association studies (GWASs) of complex disease, but their contributions to trait heritability are currently unknown. We applied variance-component methods to imputed genotype data for 11 common diseases to partition the heritability explained by genotyped SNPs (hg2) across functional categories (while accounting for shared variance due to linkage disequilibrium). Extensive simulations showed that in contrast to current estimates from GWAS summary statistics, the variance-component approach partitions heritability accurately under a wide range of complex-disease architectures. Across the 11 diseases DNaseI hypersensitivity sites (DHSs) from 217 cell types spanned 16% of imputed SNPs (and 24% of genotyped SNPs) but explained an average of 79% (SE = 8%) of hg2 from imputed SNPs (5.1× enrichment; p = 3.7 × 10⁻¹⁷) and 38% (SE = 4%) of hg2 from genotyped SNPs (1.6× enrichment, p = 1.0 × 10⁻⁴). Further enrichment was observed at enhancer DHSs and cell-type-specific DHSs. In contrast, coding variants, which span 1% of the genome, explained <10% of hg2 despite having the highest enrichment. We replicated these findings but found no significant contribution from rare coding variants in independent schizophrenia cohorts genotyped on GWAS and exome chips. Our results highlight the value of analyzing components of heritability to unravel the functional architecture of common disease.     

Statistical Mechanics Provides Novel Insights into Microtubule Stability and Mechanism of Shrinkage

Microtubules are nano-machines that grow and shrink stochastically, making use of the coupling between chemical kinetics and mechanics of its constituent protofilaments (PFs). We investigate the stability and shrinkage of microtubules taking into account inter-protofilament interactions and bending interactions of intrinsically curved PFs. Computing the free energy as a function of PF tip position, we show that the competition between curvature energy, inter-PF interaction energy and entropy leads to a rich landscape with a series of minima that repeat over a length-scale determined by the intrinsic curvature. Computing Langevin dynamics of the tip through the landscape and accounting for depolymerization, we calculate the average unzippering and shrinkage velocities of GDP protofilaments and compare them with the experimentally known results. Our analysis predicts that the strength of the inter-PF interaction (Ems) has to be comparable to the strength of the curvature energy (Emb) such that Ems−Emb≈1kBT, and questions the prevalent notion that unzippering results from the domination of bending energy of curved GDP PFs. Our work demonstrates how the shape of the free energy landscape is crucial in explaining the mechanism of MT shrinkage where the unzippered PFs will fluctuate in a set of partially peeled off states and subunit dissociation will reduce the length.     

Physicochemical Properties and Amino Acid Composition of Alkaline Proteinase from Aspergillus sojae

Some physicochemical properties and amino acid composition of alkaline proteinase from Aspergillus sojae were found to be as follows: The isoelectric point was at pH 5.1. The molecular weight was 25,500 using the Sheraga-Mandelkern’s formula, based upon the values of the sedimentation coefficient $(s_{20,w}^{°}=\text{?}2.82\text{?}\text{S})$, the intrinsic viscosity ([η] = 0.027 dl/g), and the partial specific volume $({\displaystyle \overline{V}}\text{?}=\text{?}0.726\text{?}\text{ml}/\text{g})$. The enzyme contains 16.8% of nitrogen and is composed of 250 residues of amino acid; Asp₃₁ Glu₁₉, Gly₂₇, Ala₃₂, Val₁₈, Leu₁₄, Ile₁₄, Ser₂₈, Thr₁₈, ${\text{(}{\displaystyle \stackrel{}{{\displaystyle \stackrel{?}{\text{Cys C}}}}}\text{ys})}_1$, Met₂, Pro₆, Phe₇, Tyr₈, Trp₂, His₅, Lys₁₄, Arg₃, (amide-NH₃)₂₀.     

Thermodynamic examination of 1- to 5-nt purine bulge loops in RNA and DNA constructs

Bulge loops are common features of RNA structures that are involved in the formation of RNA tertiary structures and are often sites for interactions with proteins and ions. Minimal thermodynamic data currently exist on the bulge size and sequence effects. Using thermal denaturation methods, thermodynamic properties of 1- to 5-nt adenine and guanine bulge loop constructs were examined in 10 mM MgCl₂ or 1 M KCl. The ΔG37∘ loop parameters for 1- to 5-nt purine bulge loops in RNA constructs were between 3.07 and 5.31 kcal/mol in 1 M KCl buffer. In 10 mM magnesium ions, the ΔΔG° values relative to 1 M KCl were 0.47–2.06 kcal/mol more favorable for the RNA bulge loops. The ΔG37∘ loop parameters for 1- to 5-nt purine bulge loops in DNA constructs were between 4.54 and 5.89 kcal/mol. Only 4- and 5-nt guanine constructs showed significant change in stability for the DNA constructs in magnesium ions. A linear correlation is seen between the size of the bulge loop and its stability. New prediction models are proposed for 1- to 5-nt purine bulge loops in RNA and DNA in 1 M KCl. We show that a significant stabilization is seen for small bulge loops in RNA in the presence of magnesium ions. A prediction model is also proposed for 1- to 5-nt purine bulge loop RNA constructs in 10 mM magnesium chloride.