Unveiling interatomic distances influencing the reaction coordinates in alanine dipeptide isomerization: An explainable deep learning approach.

The present work shows that the free energy landscape associated with alanine dipeptide isomerization can be effectively represented by specific interatomic distances without explicit reference to dihedral angles. Conventionally, two stable states of alanine dipeptide in vacuum, i.e., C7eq (ß-sheet structure) and C7ax (left handed α-helix structure), have been primarily characterized using the main chain dihedral angles, φ (C-N-Cα-C) and ψ (N-Cα-C-N). However, our recent deep learning combined with the "Explainable AI" (XAI) framework has shown that the transition state can be adequately captured by a free energy landscape using φ and θ (O-C-N-Cα) [Kikutsuji et al., J. Chem. Phys. 156, 154108 (2022)]. In the perspective of extending these insights to other collective variables, a more detailed characterization of the transition state is required. In this work, we employ interatomic distances and bond angles as input variables for deep learning rather than the conventional and more elaborate dihedral angles. Our approach utilizes deep learning to investigate whether changes in the main chain dihedral angle can be expressed in terms of interatomic distances and bond angles. Furthermore, by incorporating XAI into our predictive analysis, we quantified the importance of each input variable and succeeded in clarifying the specific interatomic distance that affects the transition state. The results indicate that constructing a free energy landscape based on the identified interatomic distance can clearly distinguish between the two stable states and provide a comprehensive explanation for the energy barrier crossing.

Improvement of the Green-Red Förster Resonance Energy Transfer-Based Ca2+ Indicator by Using the Green Fluorescent Protein, Gamillus, with a Trans Chromophore as the Donor.

To monitor the Ca2+ dynamics in cells, various genetically encoded Ca2+ indicators (GECIs) based on Förster resonance energy transfer (FRET) between fluorescent proteins are widely used for live imaging. Conventionally, cyan and yellow fluorescent proteins have been often used as FRET pairs. Meanwhile, bathochromically shifted indicators with green and red fluorescent protein pairs have various advantages, such as low toxicity and autofluorescence in cells. However, it remains difficult to develop them with a similar level of dynamic range as cyan and yellow fluorescent protein pairs. To improve this, we used Gamillus, which has a unique trans-configuration chromophore, as a green fluorescent protein. Based on one of the best high-dynamic-range GECIs, Twitch-NR, we developed a GECI with 1.5-times higher dynamic range (253%), Twitch-GmRR, using RRvT as a red fluorescent protein. Twitch-GmRR had high brightness and photostability and was successfully applied for imaging the Ca2+ dynamics in live cells. Our results suggest that Gamillus with trans-type chromophores contributes to improving the dynamic range of GECIs. Therefore, selection of the cis-trans isomer of the chromophore may be a fundamental approach to improve the dynamic range of green-red FRET indicators, unlimited by GECIs.

Accelerated Molecular Dynamics and AlphaFold Uncover a Missing Conformational State of Transporter Protein OxlT.

Transporter proteins change their conformations to carry their substrate across the cell membrane. The conformational dynamics is vital to understanding the transport function. We have studied the oxalate transporter (OxlT), an oxalate:formate antiporter from Oxalobacter formigenes, significant in avoiding kidney stone formation. The atomic structure of OxlT has been recently solved in the outward-open and occluded states. However, the inward-open conformation is still missing, hindering a complete understanding of the transporter. Here, we performed a Gaussian accelerated molecular dynamics simulation to sample the extensive conformational space of OxlT and successfully predicted the inward-open conformation where cytoplasmic substrate formate binding was preferred over oxalate binding. We also identified critical interactions for the inward-open conformation. The results were complemented by an AlphaFold2 structure prediction. Although AlphaFold2 solely predicted OxlT in the outward-open conformation, mutation of the identified critical residues made it partly predict the inward-open conformation, identifying possible state-shifting mutations.

Molecular Design of FRET Probes Based on Domain Rearrangement of Protein Disulfide Isomerase for Monitoring Intracellular Redox Status.

Multidomain proteins can exhibit sophisticated functions based on cooperative interactions and allosteric regulation through spatial rearrangements of the multiple domains. This study explored the potential of using multidomain proteins as a basis for Förster resonance energy transfer (FRET) biosensors, focusing on protein disulfide isomerase (PDI) as a representative example. PDI, a well-studied multidomain protein, undergoes redox-dependent conformational changes, enabling the exposure of a hydrophobic surface extending across the b' and a' domains that serves as the primary binding site for substrates. Taking advantage of the dynamic domain rearrangements of PDI, we developed FRET-based biosensors by fusing the b' and a' domains of thermophilic fungal PDI with fluorescent proteins as the FRET acceptor and donor, respectively. Both experimental and computational approaches were used to characterize FRET efficiency in different redox states. In vitro and in vivo evaluations demonstrated higher FRET efficiency of this biosensor in the oxidized form, reflecting the domain rearrangement and its responsiveness to intracellular redox environments. This novel approach of exploiting redox-dependent domain dynamics in multidomain proteins offers promising opportunities for designing innovative FRET-based biosensors with potential applications in studying cellular redox regulation and beyond.

Characterization of FLOWERING LOCUS C 5 in Brassica rapa L.

Brassica rapa L., which includes Chinese cabbage, turnip, and pak choi, has more complex flowering time regulation than does Arabidopsis thaliana due to the presence of multiple paralogous flowering time genes. FLOWERING LOCUS C (FLC) is one of the key genes regulating the flowering time, and B. rapa has four FLC paralogs. BrFLC5 on the reference genome is deemed a pseudogene because of a mutation (from G to A) in the splice site of the third intron, but there are some accessions with a G nucleotide in the splice site. In this study, we genotyped 310 B. rapa accessions and found that 19 had homozygous and 81 had heterozygous putative functional BrFLC5 alleles. Accessions of turnip showed the highest proportion with a functional BrFLC5 allele. BrFLC5 acts as a floral repressor when overexpressed in A. thaliana. The BrFLC5 expression level varied in pre-vernalized plants, and this transcriptional variation was not associated with the G/A polymorphism in the third intron. Three accessions having a higher BrFLC5 expression in pre-vernalized plants had a 584-bp insertion in the promoter region. Many regions homologous to this 584-bp sequence are present in the B. rapa genome, and this 584-bp inserted region has tandem duplications of an AT-rich sequence in its central region. The possibility that a high expression of a functional BrFLC5 could contribute to producing premature bolting-resistant lines in B. rapa vegetables is discussed. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01405-0.

Structure and mechanism of oxalate transporter OxlT in an oxalate-degrading bacterium in the gut microbiota.

An oxalate-degrading bacterium in the gut microbiota absorbs food-derived oxalate to use this as a carbon and energy source, thereby reducing the risk of kidney stone formation in host animals. The bacterial oxalate transporter OxlT selectively uptakes oxalate from the gut to bacterial cells with a strict discrimination from other nutrient carboxylates. Here, we present crystal structures of oxalate-bound and ligand-free OxlT in two distinct conformations, occluded and outward-facing states. The ligand-binding pocket contains basic residues that form salt bridges with oxalate while preventing the conformational switch to the occluded state without an acidic substrate. The occluded pocket can accommodate oxalate but not larger dicarboxylates, such as metabolic intermediates. The permeation pathways from the pocket are completely blocked by extensive interdomain interactions, which can be opened solely by a flip of a single side chain neighbouring the substrate. This study shows the structural basis underlying metabolic interactions enabling favourable symbiosis.

Molecular mechanism on forcible ejection of ATPase inhibitory factor 1 from mitochondrial ATP synthase.

IF1 is a natural inhibitor protein for mitochondrial FoF1 ATP synthase that blocks catalysis and rotation of the F1 by deeply inserting its N-terminal helices into F1. A unique feature of IF1 is condition-dependent inhibition; although IF1 inhibits ATP hydrolysis by F1, IF1 inhibition is relieved under ATP synthesis conditions. To elucidate this condition-dependent inhibition mechanism, we have performed single-molecule manipulation experiments on IF1-inhibited bovine mitochondrial F1 (bMF1). The results show that IF1-inhibited F1 is efficiently activated only when F1 is rotated in the clockwise (ATP synthesis) direction, but not in the counterclockwise direction. The observed rotational-direction-dependent activation explains the condition-dependent mechanism of IF1 inhibition. Investigation of mutant IF1 with N-terminal truncations shows that the interaction with the γ subunit at the N-terminal regions is crucial for rotational-direction-dependent ejection, and the middle long helix is responsible for the inhibition of F1.

Quantitative trait locus (QTL) analysis and fine-mapping for Fusarium oxysporum disease resistance in Raphanus sativus using GRAS-Di technology.

Fusarium wilt is a significant disease in radish, but the genetic mechanisms controlling yellows resistance (YR) are not well understood. This study aimed to identify YR-QTLs and to fine-map one of them using F2:3 populations developed from resistant and susceptible radish parents. In this study, two high-density genetic maps each containing shared co-dominant markers and either female or male dominant markers that spanned 988.6 and 1127.5 cM with average marker densities of 1.40 and 1.53 cM, respectively, were generated using Genotyping by Random Amplicon Sequencing-Direct (GRAS-Di) technology. We identified two YR-QTLs on chromosome R2 and R7, and designated the latter as ForRs1 as the major QTL. Fine mapping narrowed down the ForRs1 locus to a 195 kb region. Among the 16 predicted genes in the delimited region, 4 genes including two receptor-like protein and -kinase genes (RLP/RLK) were identified as prime candidates for ForRs1 based on the nucleotide sequence comparisons between the parents and their predicted functions. This study is the first to use a GRAS-Di for genetic map construction of cruciferous crops and fine map the YR-QTL on the R7 chromosome of radish. These findings will provide groundbreaking insights into radish YR breeding and understanding the genetics of YR mechanism.

Comparative transcriptome analysis during tuberous stem formation in Kohlrabi (B. oleracea var. gongylodes) at early growth periods (seedling stages).

Tuberous stem of kohlrabi is an important agronomic trait, however, the molecular basis of tuberization is poorly understood. To elucidate the tuberization mechanism, we conducted a comparative transcriptomic analysis between kohlrabi and broccoli at 10 and 20 days after germination (DAG) as tuberous stem initiated between these time points. A total of 5580 and 2866 differentially expressed transcripts (DETs) were identified between genotypes (kohlrabi vs. broccoli) and growth stages (10 DAG vs. 20 DAG), respectively, and most of the DETs were down-regulated in kohlrabi. Gene ontology (GO) and KEGG pathway enrichment analyses showed that the DETs between genotypes are involved in cell wall loosening and expansion, cell cycle and division, carbohydrate metabolism, hormone transport, hormone signal transduction and in several transcription factors. The DETs identified in those categories may directly/indirectly relate to the initiation and development of tuberous stem in kohlrabi. In addition, the expression pattern of the hormone synthesis related DETs coincided with the endogenous IAA, IAAsp, GA, ABA, and tZ profiles in kohlrabi and broccoli seedlings, that were revealed in our phytohormone analysis. This is the first report on comparative transcriptome analysis for tuberous stem formation in kohlrabi at early growth periods. The resulting data could provide significant insights into the molecular mechanism underlying tuberous stem development in kohlrabi as well as in other tuberous organ forming crops.

Widespread and transgenerational retrotransposon activation in inter- and intraspecies recombinant inbred populations of Lotus japonicus.

Transposable elements (TEs) constitute a large proportion of genomes of multicellular eukaryotes, including flowering plants. TEs are normally maintained in a silenced state and their transpositions rarely occur. Hybridization between distant species has been regarded as a 'shock' that stimulates genome reorganization, including TE mobilization. However, whether crosses between genetically close parents that result in viable and fertile offspring can induce TE transpositions has remained unclear. Here, we investigated the activation of long terminal repeat (LTR) retrotransposons in three Lotus japonicus recombinant inbred line (RIL) populations. We found that at least six LTR retrotransposon families were activated and transposed in 78% of the RILs investigated. LORE1a, one of the transposed LTR retrotransposons, showed transgenerational epigenetic activation, indicating the long-term effects of epigenetic instability induced by hybridization. Our study highlights TE activation as an unexpectedly common event in plant reproduction.

Identification of a Male Sterile Candidate Gene in Lilium x formolongi and Transfer of the Gene to Easter Lily (L. longiflorum) via Hybridization.

Pollen-free varieties are advantageous in promoting cut-flower production. In this study, we identified a candidate mutation which is responsible for pollen sterility in a strain of Lilium × formolongi, which was originally identified as a naturally occurred male-sterile plant in a seedling population. The pollen sterility occurred due to the degradation of pollen mother cells (PMCs) before meiotic cell division. Genetic analysis suggested that the male-sterile phenotype is attributed to one recessive locus. Transcriptome comparison between anthers of sterile and fertile plants in a segregated population identified a transcript that was expressed only in pollen-fertile plants, which is homologous to TDF1 (DEFECTIVE in TAPETAL DEVELOPMENT and FUNCTION1) in Arabidopsis, a gene encoding a transcription factor AtMYB35 that is known as a key regulator of pollen development. Since tdf1 mutant shows male sterility, we assumed that the absence transcript of the TDF1-like gene, named as LflTDF1, is the reason for pollen sterility observed in the mutant. A 30 kbp-long nanopore sequence read containing LflTDF1 was obtained from a pollen-fertile accession. PCR analyses using primers designed from the sequence suggested that at least a 30kbp-long region containing LflTDF1 was deleted or replaced by unknown sequence in the pollen-sterile mutant. Since the cross between L. × formolongi and Easter lily (L. longiflorum) is compatible, we successfully introgressed the male-sterile allele, designated as lfltdf1, to Easter lily. To our knowledge, this is the first report of molecular identification of a pollen-sterile candidate gene in lily. The identification and marker development of LflTDF1 gene will assist pollen-free lily breeding of Easter lilies and other lilies.

Explaining reaction coordinates of alanine dipeptide isomerization obtained from deep neural networks using Explainable Artificial Intelligence (XAI).

A method for obtaining appropriate reaction coordinates is required to identify transition states distinguishing the product and reactant in complex molecular systems. Recently, abundant research has been devoted to obtaining reaction coordinates using artificial neural networks from deep learning literature, where many collective variables are typically utilized in the input layer. However, it is difficult to explain the details of which collective variables contribute to the predicted reaction coordinates owing to the complexity of the nonlinear functions in deep neural networks. To overcome this limitation, we used Explainable Artificial Intelligence (XAI) methods of the Local Interpretable Model-agnostic Explanation (LIME) and the game theory-based framework known as Shapley Additive exPlanations (SHAP). We demonstrated that XAI enables us to obtain the degree of contribution of each collective variable to reaction coordinates that is determined by nonlinear regressions with deep learning for the committor of the alanine dipeptide isomerization in vacuum. In particular, both LIME and SHAP provide important features to the predicted reaction coordinates, which are characterized by appropriate dihedral angles consistent with those previously reported from the committor test analysis. The present study offers an AI-aided framework to explain the appropriate reaction coordinates, which acquires considerable significance when the number of degrees of freedom increases.

Development of a New DNA Marker for Fusarium Yellows Resistance in Brassica rapa Vegetables.

In vegetables of Brassica rapa L., Fusarium oxysporum f. sp. rapae (For) or F. oxysporum f. sp. conglutinans (Foc) cause Fusarium yellows. A resistance gene against Foc (FocBr1) has been identified, and deletion of this gene results in susceptibility (focbr1-1). In contrast, a resistance gene against For has not been identified. Inoculation tests showed that lines resistant to Foc were also resistant to For, and lines susceptible to Foc were susceptible to For. However, prediction of disease resistance by a dominant DNA marker on FocBr1 (Bra012688m) was not associated with disease resistance of For in some komatsuna lines using an inoculation test. QTL-seq using four F2 populations derived from For susceptible and resistant lines showed one causative locus on chromosome A03, which covers FocBr1. Comparison of the amino acid sequence of FocBr1 between susceptible and resistant alleles (FocBr1 and FocBo1) showed that six amino acid differences were specific to susceptible lines. The presence and absence of FocBr1 is consistent with For resistance in F2 populations. These results indicate that FocBr1 is essential for For resistance, and changed amino acid sequences result in susceptibility to For. This susceptible allele is termed focbr1-2, and a new DNA marker (focbr1-2m) for detection of the focbr1-2 allele was developed.

Optimizing Go-MARTINI Coarse-Grained Model for F-BAR Protein on Lipid Membrane.

Coarse-grained (CG) molecular dynamics (MD) simulations allow us to access much larger length and time scales than atomistic MD simulations, providing an attractive alternative to the conventional simulations. Based on the well-known MARTINI CG force field, the recently developed Go-MARTINI model for proteins describes large-amplitude structural dynamics, which has not been possible with the commonly used elastic network model. Using the Go-MARTINI model, we conduct MD simulations of the F-BAR Pacsin1 protein on lipid membrane. We observe that structural changes of the non-globular protein are largely dependent on the definition of the native contacts in the Go model. To address this issue, we introduced a simple cutoff scheme and tuned the cutoff distance of the native contacts and the interaction strength of the Lennard-Jones potentials in the Go-MARTINI model. With the optimized Go-MARTINI model, we show that it reproduces structural fluctuations of the Pacsin1 dimer from atomistic simulations. We also show that two Pacsin1 dimers properly assemble through lateral interaction on the lipid membrane. Our work presents a first step towards describing membrane remodeling processes in the Go-MARTINI CG framework by simulating a crucial step of protein assembly on the membrane.

Crystalline chitin hydrolase is a burnt-bridge Brownian motor.

Motor proteins are essential units of life and are well-designed nanomachines working under thermal fluctuations. These proteins control moving direction by consuming chemical energy or by dissipating electrochemical potentials. Chitinase A from bacterium Serratia marcescens (SmChiA) processively moves along crystalline chitin by hydrolysis of a single polymer chain to soluble chitobiose. Recently, we directly observed the stepping motions of SmChiA labeled with a gold nanoparticle by dark-field scattering imaging to investigate the moving mechanism. Time constants analysis revealed that SmChiA moves back and forth along the chain freely, because forward and backward states have a similar free energy level. The similar probabilities of forward-step events (83.5%=69.3%+14.2%) from distributions of step sizes and chain-hydrolysis (86.3%=(1/2.9)/(1/2.9+1/18.3)×100) calculated from the ratios of time constants of hydrolysis and the backward step indicated that SmChiA moves forward as a result of shortening of the chain by a chitobiose unit, which stabilizes the backward state. Furthermore, X-ray crystal structures of sliding intermediate and molecular dynamics simulations showed that SmChiA slides forward and backward under thermal fluctuation without large conformational changes of the protein. Our results demonstrate that SmChiA is a burnt-bridge Brownian ratchet motor.

Learning reaction coordinates via cross-entropy minimization: Application to alanine dipeptide.

We propose a cross-entropy minimization method for finding the reaction coordinate from a large number of collective variables in complex molecular systems. This method is an extension of the likelihood maximization approach describing the committor function with a sigmoid. By design, the reaction coordinate as a function of various collective variables is optimized such that the distribution of the committor pB * values generated from molecular dynamics simulations can be described in a sigmoidal manner. We also introduce the L2-norm regularization used in the machine learning field to prevent overfitting when the number of considered collective variables is large. The current method is applied to study the isomerization of alanine dipeptide in vacuum, where 45 dihedral angles are used as candidate variables. The regularization parameter is determined by cross-validation using training and test datasets. It is demonstrated that the optimal reaction coordinate involves important dihedral angles, which are consistent with the previously reported results. Furthermore, the points with pB *∼0.5 clearly indicate a separatrix distinguishing reactant and product states on the potential of mean force using the extracted dihedral angles.