Development of equation of motion deciphering locomotion including omega turns of Caenorhabditis elegans.

Locomotion is a fundamental behavior of Caenorhabditis elegans (C. elegans). Previous works on kinetic simulations of animals helped researchers understand the physical mechanisms of locomotion and the muscle-controlling principles of neuronal circuits as an actuator part. It has yet to be understood how C. elegans utilizes the frictional forces caused by the tension of its muscles to perform sequenced locomotive behaviors. Here, we present a two-dimensional rigid body chain model for the locomotion of C. elegans by developing Newtonian equations of motion for each body segment of C. elegans. Having accounted for friction-coefficients of the surrounding environment, elastic constants of C. elegans, and its kymogram from experiments, our kinetic model (ElegansBot) reproduced various locomotion of C. elegans such as, but not limited to, forward-backward-(omega turn)-forward locomotion constituting escaping behavior and delta-turn navigation. Additionally, ElegansBot precisely quantified the forces acting on each body segment of C. elegans to allow investigation of the force distribution. This model will facilitate our understanding of the detailed mechanism of various locomotive behaviors at any given friction-coefficients of the surrounding environment. Furthermore, as the model ensures the performance of realistic behavior, it can be used to research actuator-controller interaction between muscles and neuronal circuits.

Understanding the molecular mechanism of pathogenic variants of BIR2 domain in XIAP-deficient inflammatory bowel disease.

X-linked inhibitor of apoptosis protein (XIAP) deficiency causes refractory inflammatory bowel disease. The XIAP protein plays a pivotal role in the pro-inflammatory response through the nucleotide-binding oligomerization domain-containing signaling pathway that is important in mucosal homeostasis. We analyzed the molecular mechanism of non-synonymous pathogenic variants (PVs) of XIAP BIR2 domain. We generated N-terminally green fluorescent protein-tagged XIAP constructs of representative non-synonymous PVs. Co-immunoprecipitation and fluorescence cross-correlation spectroscopy showed that wild-type XIAP and RIP2 preferentially interacted in live cells, whereas all non-synonymous PV XIAPs failed to interact properly with RIP2. Structural analysis showed that various structural changes by mutations, such as hydrophobic core collapse, Zn-finger loss, and spatial rearrangement, destabilized the two loop structures (174-182 and 205-215) that critically interact with RIP2. Subsequently, it caused a failure of RIP2 ubiquitination and loss of protein deficiency by the auto-ubiquitination of all XIAP mutants. These findings could enhance our understanding of the role of XIAP mutations in XIAP-deficient inflammatory bowel disease and may benefit future therapeutic strategies.

Development of quantitative and continuous measure for severity degree of Alzheimer's disease evaluated from MRI images of 761 human brains.

BACKGROUND: Alzheimer's disease affects profoundly the quality of human behavior and cognition. The very broad distribution of its severity across various human subjects requires the quantitative diagnose of Alzheimer's disease beyond the conventional tripartite classification of cohorts such as cognitively normal (CN), mild cognitive impairment (MCI), Alzheimer's disease (AD). The unfolding of such broad distributions by the quantitative and continuous degree of AD severity is necessary for the precise diagnose in the cross-sectional study of different stages in AD. RESULTS: We conducted the massive reanalysis on MRI images of 761 human brains based on the accumulated bigdata of Alzheimer's Disease Neuroimaging Initiative. The score matrix of cortical thickness profile at cortex points of subjects was constructed by statistically learning the cortical thickness data of 761 human brains. We also developed a new and simple algebraic predictor which provides the quantitative and continuous degree of AD severity of subjects along the scale from 0 for fully CN to 1 for fully AD state. The mathematical measure of a new predictor for the degree of AD severity is presented based on a covariance correlation matrix of cortical thickness profile between human subjects. One can remove the uncertainty in the determination of different stages in AD by the quantitative degree of AD severity and thus go far beyond the tripartite classification of cohorts. CONCLUSIONS: We unfold the nature of broad distribution of AD severity of subjects even within a given cohort by the scale from 0 for fully CN to 1 for fully AD state. The quantitative and continuous degree of AD severity developed in this study would be a good practical measure for diagnosing the different stages in AD severity.

Computational design of a thermolabile uracil-DNA glycosylase of Escherichia coli.

Polymerase chain reaction (PCR) is a powerful tool to diagnose infectious diseases. Uracil DNA glycosylase (UDG) is broadly used to remove carryover contamination in PCR. However, UDG can contribute to false negative results when not inactivated completely, leading to DNA degradation during the amplification step. In this study, we designed novel thermolabile UDG derivatives by supercomputing molecular dynamic simulations and residual network analysis. Based on enzyme activity analysis, thermolability, thermal stability, and biochemical experiments of Escherichia coli-derived UDG and 22 derivatives, we uncovered that the UDG D43A mutant eliminated the false negative problem, demonstrated high efficiency, and offered great benefit for use in PCR diagnosis. We further obtained structural and thermodynamic insights into the role of the D43A mutation, including perturbed protein structure near D43; weakened pairwise interactions of D43 with K42, N46, and R80; and decreased melting temperature and native fraction of the UDG D43A mutant compared with wild-type UDG.

Impaired formation of high-order gephyrin oligomers underlies gephyrin dysfunction-associated pathologies.

Gephyrin is critical for the structure, function, and plasticity of inhibitory synapses. Gephyrin mutations have been linked to various neurological disorders; however, systematic analyses of the functional consequences of these mutations are lacking. Here, we performed molecular dynamics simulations of gephyrin to predict how six reported point mutations might change the structural stability and/or function of gephyrin. Additional in silico analyses revealed that the A91T and G375D mutations reduce the binding free energy of gephyrin oligomer formation. Gephyrin A91T and G375D displayed altered clustering patterns in COS-7 cells and nullified the inhibitory synapse-promoting effect of gephyrin in cultured neurons. However, only the G375D mutation reduced gephyrin interaction with GABAA receptors and neuroligin-2 in mouse brain; it also failed to normalize deficits in GABAergic synapse maintenance and neuronal hyperactivity observed in hippocampal dentate gyrus-specific gephyrin-deficient mice. Our results provide insights into biochemical, cell-biological, and network-activity effects of the pathogenic G375D mutation.

Interpretation of XIAP Variants of Uncertain Significance in Paediatric Patients with Refractory Crohn's Disease.

BACKGROUND AND AIMS: Mutations in XIAP can lead to the development of treatment-refractory severe paediatric Crohn's disease [CD], for which haematopoietic stem cell transplantation is the primary therapeutic option. The interpretation of variants of uncertain significance [VUSs] in XIAP needs to be scrutinized. METHODS: Targeted next-generation sequencing was performed for 33 male paediatric patients with refractory CD admitted at a tertiary referral hospital. To obtain functional data, biomolecular cell assays and supercomputing molecular dynamics simulations were performed. RESULTS: Nine unrelated male patients harboured hemizygous XIAP variants. Four known pathogenic variants and one novel pathogenic variant [p.Lys168Serfs*12] were identified in five patients, and two novel VUSs [p.Gly205del and p.Pro260Ser] and one known VUS [p.Glu350del] were identified in the remaining four. Among children with VUSs, only the subject with p.Gly205del exhibited defective NOD2 signalling. Using molecular dynamics simulation, we determined that the altered backbone torsional energy of C203 in XIAP of p.G205del was ~2 kcal/mol, suggesting loss of zinc binding in the mutant XIAP protein and poor coordination between the mutant XIAP and RIP2 proteins. Elevated auto-ubiquitination of zinc-depleted p.G205del XIAP protein resulted in XIAP protein deficiency. CONCLUSION: A high prevalence of XIAP deficiency was noted among children with refractory CD. Advanced functional studies decreased the subjectivity in the case-level interpretation of XIAP VUSs and directed consideration of haematopoietic stem cell transplantation.

Structural and molecular insight into the pH-induced low-permeability of the voltage-gated potassium channel Kv1.2 through dewetting of the water cavity.

Understanding the gating mechanism of ion channel proteins is key to understanding the regulation of cell signaling through these channels. Channel opening and closing are regulated by diverse environmental factors that include temperature, electrical voltage across the channel, and proton concentration. Low permeability in voltage-gated potassium ion channels (Kv) is intimately correlated with the prolonged action potential duration observed in many acidosis diseases. The Kv channels consist of voltage-sensing domains (S1-S4 helices) and central pore domains (S5-S6 helices) that include a selectivity filter and water-filled cavity. The voltage-sensing domain is responsible for the voltage-gating of Kv channels. While the low permeability of Kv channels to potassium ion is highly correlated with the cellular proton concentration, it is unclear how an intracellular acidic condition drives their closure, which may indicate an additional pH-dependent gating mechanism of the Kv family. Here, we show that two residues E327 and H418 in the proximity of the water cavity of Kv1.2 play crucial roles as a pH switch. In addition, we present a structural and molecular concept of the pH-dependent gating of Kv1.2 in atomic detail, showing that the protonation of E327 and H418 disrupts the electrostatic balance around the S6 helices, which leads to a straightening transition in the shape of their axes and causes dewetting of the water-filled cavity and closure of the channel. Our work offers a conceptual advancement to the regulation of the pH-dependent gating of various voltage-gated ion channels and their related biological functions.

Atomic insights into the effects of pathological mutants through the disruption of hydrophobic core in the prion protein.

Destabilization of prion protein induces a conformational change from normal prion protein (PrPC) to abnormal prion protein (PrPSC). Hydrophobic interaction is the main driving force for protein folding, and critically affects the stability and solvability. To examine the importance of the hydrophobic core in the PrP, we chose six amino acids (V176, V180, T183, V210, I215, and Y218) that make up the hydrophobic core at the middle of the H2-H3 bundle. A few pathological mutants of these amino acids have been reported, such as V176G, V180I, T183A, V210I, I215V, and Y218N. We focused on how these pathologic mutations affect the hydrophobic core and thermostability of PrP. For this, we ran a temperature-based replica-exchange molecular dynamics (T-REMD) simulation, with a cumulative simulation time of 28 µs, for extensive ensemble sampling. From the T-REMD ensemble, we calculated the protein folding free energy difference between wild-type and mutant PrP using the thermodynamic integration (TI) method. Our results showed that pathological mutants V176G, T183A, I215V, and Y218N decrease the PrP stability. At the atomic level, we examined the change in pair-wise hydrophobic interactions from valine-valine to valine-isoleucine (and vice versa), which is induced by mutation V180I, V210I (I215V) at the 180th-210th (176th-215th) pair. Finally, we investigated the importance of the π-stacking between Y218 and F175.

Structural insight into conformational change in prion protein by breakage of electrostatic network around H187 due to its protonation.

A conformational change from normal prion protein(PrPC) to abnormal prion protein(PrPSC) induces fatal neurodegenerative diseases. Acidic pH is well-known factors involved in the conformational change. Because the protonation of H187 is strongly linked to the change in PrP stability, we examined the charged residues R156, E196, and D202 around H187. Interestingly, there have been reports on pathological mutants, such as H187R, E196A, and D202N. In this study, we focused on how an acidic pH and pathological mutants disrupt this electrostatic network and how this broken network destabilizes PrP structure. To do so, we performed a temperature-based replica-exchange molecular dynamics (T-REMD) simulation using a cumulative 252 µs simulation time. We measured the distance between amino acids comprising four salt bridges (R156-E196/D202 and H187-E196/D202). Our results showed that the spatial configuration of the electrostatic network was significantly altered by an acidic pH and mutations. The structural alteration in the electrostatic network increased the RMSF value around the first helix (H1). Thus, the structural stability of H1, which is anchored to the H2-H3 bundle, was decreased. It induces separation of R156 from the electrostatic network. Analysis of the anchoring energy also shows that two salt-bridges (R156-E196/D202) are critical for PrP stability.

Author Correction: Molecular mechanism of K65 acetylation-induced attenuation of Ubc9 and the NDSM interaction.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

The C-Domain of the NAC Transcription Factor ANAC019 Is Necessary for pH-Tuned DNA Binding through a Histidine Switch in the N-Domain.

The affinity of transcription factors (TFs) for their target DNA is a critical determinant of gene expression. Whether the DNA-binding domain (DBD) of TFs alone can regulate binding affinity to DNA is an important question for identifying the design principle of TFs. We studied ANAC019, a member of the NAC TF family of proteins in Arabidopsis, and found a well-conserved histidine switch located in its DBD, which regulates both homodimerization and transcriptional control of the TF through H135 protonation. We found that the removal of a C-terminal intrinsically disordered region (IDR) in the TF abolished the pH-dependent binding of the N-terminal DBD to DNA. We propose a mechanism in which long-range electrostatic interactions between DNA and the negatively charged C-terminal IDR turns on the pH dependency of the DNA-binding affinity of the N-terminal DBD.

Molecular mechanism of K65 acetylation-induced attenuation of Ubc9 and the NDSM interaction.

The negatively charged amino acid-dependent sumoylation motif (NDSM) carries an additional stretch of acidic residues downstream of the consensus Ψ-K-x-E/D sumoylation motif. We have previously shown that acetylation of the SUMO E2 conjugase enzyme, Ubc9, at K65 downregulates its binding to the NDSM and renders a selective decrease in sumoylation of substrates with the NDSM motif. Here, we provide detailed structural, thermodynamic, and kinetics results of the interactions between Ubc9 and its K65 acetylated variant (Ac-Ubc9K65) with three NDSMs derived from Elk1, CBP, and Calpain2 to rationalize the mechanism beneath this reduced binding. Our nuclear magnetic resonance (NMR) data rule out a direct interaction between the NDSM and the K65 residue of Ubc9. Similarly, we found that NDSM binding was entropy-driven and unlikely to be affected by the negative charge by K65 acetylation. Moreover our NMR, mutagenesis and molecular dynamics simulation studies defined the sequence of the NDSM as Ψ-K-x-E/D-x1-x2-(x3/E/D)-(x4/E/D)-xn and determined that K74 and K76 were critical Ubc9 residues interacting with the negatively charged residues of the NDSM.

Polymorphism of fibrillar structures depending on the size of assembled Aß17-42 peptides.

The size of assembled Aß17-42 peptides can determine polymorphism during oligomerization and fibrillization, but the mechanism of this effect is unknown. Starting from separate random monomers, various fibrillar oligomers with distinct structural characteristics were identified using discontinuous molecular dynamics simulations based on a coarse-grained protein model. From the structures observed in the simulations, two characteristic oligomer sizes emerged, trimer and paranuclei, which generated distinct structural patterns during fibrillization. A majority of the simulations for trimers and tetramers formed non-fibrillar oligomers, which primarily progress to off-pathway oligomers. Pentamers and hexamers were significantly converted into U-shape fibrillar structures, meaning that these oligomers, called paranuclei, might be potent on-pathway intermediates in fibril formation. Fibrillar oligomers larger than hexamers generated substantial polymorphism in which hybrid structures were readily formed and homogeneous fibrillar structures appeared infrequently.

Probing the Folding-Unfolding Transition of a Thermophilic Protein, MTH1880.

The folding mechanism of typical proteins has been studied widely, while our understanding of the origin of the high stability of thermophilic proteins is still elusive. Of particular interest is how an atypical thermophilic protein with a novel fold maintains its structure and stability under extreme conditions. Folding-unfolding transitions of MTH1880, a thermophilic protein from Methanobacterium thermoautotrophicum, induced by heat, urea, and GdnHCl, were investigated using spectroscopic techniques including circular dichorism, fluorescence, NMR combined with molecular dynamics (MD) simulations. Our results suggest that MTH1880 undergoes a two-state N to D transition and it is extremely stable against temperature and denaturants. The reversibility of refolding was confirmed by spectroscopic methods and size exclusion chromatography. We found that the hyper-stability of the thermophilic MTH1880 protein originates from an extensive network of both electrostatic and hydrophobic interactions coordinated by the central ß-sheet. Spectroscopic measurements, in combination with computational simulations, have helped to clarify the thermodynamic and structural basis for hyper-stability of the novel thermophilic protein MTH1880.

Uncovering multiloci-ordering by algebraic property of Laplacian matrix and its Fiedler vector.

MOTIVATION: The loci-ordering, based on two-point recombination fractions for a pair of loci, is the most important step in constructing a reliable and fine genetic map. RESULTS: Using the concept from complex graph theory, here we propose a Laplacian ordering approach which uncovers the loci-ordering of multiloci simultaneously. The algebraic property for a Fiedler vector of a Laplacian matrix, constructed from the recombination fraction of the loci-ordering for 26 loci of barley chromosome IV, 846 loci of Arabidopsis thaliana and 1903 loci of Malus domestica, together with the variable threshold uncovers their loci-orders. It offers an alternative yet robust approach for ordering multiloci. AVAILABILITY AND IMPLEMENTATION: Source code program with data set is available as supplementary data and also in a software category of the website (http://biophysics.dgist.ac.kr) CONTACT: crkim@pusan.ac.kr or iksoochang@dgist.ac.kr SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Molecular Basis of the Membrane Interaction of the ß2e Subunit of Voltage-Gated Ca(2+) Channels.

The auxiliary ß subunit plays an important role in the regulation of voltage-gated calcium (CaV) channels. Recently, it was revealed that ß2e associates with the plasma membrane through an electrostatic interaction between N-terminal basic residues and anionic phospholipids. However, a molecular-level understanding of ß-subunit membrane recruitment in structural detail has remained elusive. In this study, using a combination of site-directed mutagenesis, liposome-binding assays, and multiscale molecular-dynamics (MD) simulation, we developed a physical model of how the ß2e subunit is recruited electrostatically to the plasma membrane. In a fluorescence resonance energy transfer assay with liposomes, binding of the N-terminal peptide (23 residues) to liposome was significantly increased in the presence of phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PIP2). A mutagenesis analysis suggested that two basic residues proximal to Met-1, Lys-2 (K2) and Trp-5 (W5), are more important for membrane binding of the ß2e subunit than distal residues from the N-terminus. Our MD simulations revealed that a stretched binding mode of the N-terminus to PS is required for stable membrane attachment through polar and nonpolar interactions. This mode obtained from MD simulations is consistent with experimental results showing that K2A, W5A, and K2A/W5A mutants failed to be targeted to the plasma membrane. We also investigated the effects of a mutated ß2e subunit on inactivation kinetics and regulation of CaV channels by PIP2. In experiments with voltage-sensing phosphatase (VSP), a double mutation in the N-terminus of ß2e (K2A/W5A) increased the PIP2 sensitivity of CaV2.2 and CaV1.3 channels by ∼3-fold compared with wild-type ß2e subunit. Together, our results suggest that membrane targeting of the ß2e subunit is initiated from the nonspecific electrostatic insertion of N-terminal K2 and W5 residues into the membrane. The PS-ß2e interaction observed here provides a molecular insight into general principles for protein binding to the plasma membrane, as well as the regulatory roles of phospholipids in transporters and ion channels.

Structural Conversion of Aß17-42 Peptides from Disordered Oligomers to U-Shape Protofilaments via Multiple Kinetic Pathways.

Discovering the mechanisms by which proteins aggregate into fibrils is an essential first step in understanding the molecular level processes underlying neurodegenerative diseases such as Alzheimer's and Parkinson's. The goal of this work is to provide insights into the structural changes that characterize the kinetic pathways by which amyloid-ß peptides convert from monomers to oligomers to fibrils. By applying discontinuous molecular dynamics simulations to PRIME20, a force field designed to capture the chemical and physical aspects of protein aggregation, we have been able to trace out the entire aggregation process for a system containing 8 Aß17-42 peptides. We uncovered two fibrillization mechanisms that govern the structural conversion of Aß17-42 peptides from disordered oligomers into protofilaments. The first mechanism is monomeric conversion templated by a U-shape oligomeric nucleus into U-shape protofilament. The second mechanism involves a long-lived and on-pathway metastable oligomer with S-shape chains, having a C-terminal turn, en route to the final U-shape protofilament. Oligomers with this C-terminal turn have been regarded in recent experiments as a major contributing element to cell toxicity in Alzheimer's disease. The internal structures of the U-shape protofilaments from our PRIME20/DMD simulation agree well with those from solid state NMR experiments. The approach presented here offers a simple molecular-level framework to describe protein aggregation in general and to visualize the kinetic evolution of a putative toxic element in Alzheimer's disease in particular.

Analysis of phosphoinositide-binding properties and subcellular localization of GFP-fusion proteins.

Specific protein-phosphoinositide (PI) interactions are known to play a key role in the targeting of proteins to specific cellular membranes. Investigation of these interactions would be greatly facilitated if GFP-fusion proteins expressed in mammalian cells and used for their subcellular localization could also be employed for in vitro lipid binding. In this study, we found that lysates of cells overexpressing GFP-fusion proteins could be used for in vitro protein-PI binding assays. We applied this approach to examine the PI-binding properties of Aplysia Sec7 protein (ApSec7) and its isoform ApSec7(VPKIS), in which a VPKIS sequence is inserted into the PH domain of ApSec7. EGFP-ApSec7 but not EGFP-ApSec7(VPKIS) did specifically bind to PI(3,4,5)P3 in an in vitro lipid-coated bead assay. Overexpression of EGFP-ApSec7 but not EGFP-ApSec7(VPKIS) did induce neurite outgrowth in Aplysia sensory neurons. Structure modeling analysis revealed that the inserted VPKIS caused misfolding around the PI(3,4,5)P3-binding pocket of ApSec7 and disturbed the binding of PI(3,4,5)P3 to the pleckstrin homology (PH) domain. Our data indicate that plasma membrane localization of EGFP-ApSec7 via the interaction between its PH domain and PI(3,4,5)P3 might play a key role in neurite outgrowth in Aplysia.

Impact of sequence on the molecular assembly of short amyloid peptides.

The goal of this work is to understand how the sequence of a protein affects the likelihood that it will form an amyloid fibril and the kinetics along the fibrillization pathway. The focus is on very short fragments of amyloid proteins since these play a role in the fibrillization of the parent protein and can form fibrils themselves. Discontinuous molecular dynamics simulations using the PRIME20 force field were performed of the aggregation of 48-peptide systems containing SNQNNF (PrP (170-175)), SSTSAA (RNaseA(15-20)), MVGGVV (Aß(35-40)), GGVVIA (Aß(37-42)), and MVGGVVIA (Aß(35-42)). In our simulations SNQQNF, SSTTSAA, and MVGGVV form large numbers of fibrillar structures spontaneously (as in experiment). GGVVIA forms ß-sheets that do not stack into fibrils (unlike experiment). The combination sequence MVGGVVIA forms less fibrils than MVGGVV, hindered by the presence of the hydrophobic residues at the C-terminal. Analysis of the simulation kinetics and energetics reveals why MVGGVV forms fibrils and GGVVIA does not, and why adding I and A to MVGGVVIA reduces fibrillization and enhances amorphous aggregation into oligomeric structures. The latter helps explain why Aß(1-42) assembles into more complex oligomers than Aß(1-40), a consequence of which is that it is more strongly associated with Alzheimer's disease.

Influence of temperature on formation of perfect tau fragment fibrils using PRIME20/DMD simulations.

We investigate the fibrillization process for amyloid tau fragment peptides (VQIVYK) by applying the discontinuous molecular dynamics method to a system of 48 VQIVYK peptides modeled using a new protein model/force field, PRIME20. The aim of the article is to ascertain which factors are most important in determining whether or not a peptide system forms perfect coherent fibrillar structures. Two different directional criteria are used to determine when a hydrogen bond occurs: the original H-bond constraints and a parallel preference H-bond constraint that imparts a slight bias towards the formation of parallel versus antiparallel strands in a ß-sheet. Under the original H-bond constraints, the resulting fibrillar structures contain a mixture of parallel and antiparallel pairs of strands within each ß-sheet over the whole fibrillization temperature range. Under the parallel preference H-bond constraints, the ß-sheets within the fibrillar structures are more likely to be parallel and indeed become perfectly parallel, consistent with X-ray crystallography, at a high temperature slightly below the fibrillization temperature. The high temperature environment encourages the formation of perfect fibril structures by providing enough time and space for peptides to rearrange during the aggregation process. There are two different kinetic mechanisms, template assembly with monomer addition at high temperature and merging/rearrangement without monomer addition at low temperature, which lead to significant differences in the final fibrillar structure. This suggests that the diverse fibril morphologies generally observed in vitro depend more on environmental conditions than has heretofore been appreciated.