Manami Nishizawa

Molecular Dynamics Simulation of Kv Channel Voltage Sensor Helix in a Lipid Membrane with Applied Electric Field

In this article, we present the results of the molecular dynamics simulations of amphiphilic helix peptides of 13 amino-acid residues, placed at the lipid-water interface of dipalmitoylphosphatidylcholine bilayers. The peptides are identical with, or are derivatives of, the N-terminal segment of the S4 helix of voltage-dependent K channel KvAP, containing four voltage-sensing arginine residues (R1-R4). Upon changing the direction of the externally applied electric field, the tilt angle of the wild-type peptide changes relative to the lipid-water interface, with the N-terminus heading up with an outward electric field. These movements were not observed using an octane membrane in place of the dipalmitoylphosphatidylcholine membrane, and were markedly suppressed by 1), substituting Phe located one residue before the first arginine (R1) with a hydrophilic residue (Ser, Thr); or 2), changing the periodicity rule of Rs from at-every-third to at-every-fourth position; or 3), replacing R1 with a lysine residue (K). These and other findings suggest that the voltage-dependent movement requires deep positioning of Rs when the resting (inward) electric field is present. Later, we performed simulations of the voltage sensor domain (S1-S4) of Kv1.2 channel. In simulations with a strong electric field (0.1 V/nm or above) and positional restraints on the S1 and S2 helices, S4 movement was observed consisting of displacement along the S4 helix axis and a screwlike axial rotation. Gating-charge-carrying Rs were observed to make serial interactions with E183 in S1 and E226 in S2, in the outer water crevice. A 30-ns-backward simulation started from the open-state model gave rise to a structure similar to the recent resting-state model, with S4 moving vertically approximately 6.7 A. The energy landscape around the movement of S4 appears very ragged due to salt bridges formed between gating-charge-carrying residues and negatively charged residues of S1, S2, and S3 helices. Overall, features of S3 and S4 movements are consistent with the recent helical-screw model. Both forward and backward simulations show the presence of at least two stable intermediate structures in which R2 and R3 form salt bridges with E183 or E226, respectively. These structures are the candidates for the states postulated in previous gating kinetic models, such as the Zagotta-Hoshi-Aldrich model, to account for more than one transition step per subunit for activation.

A DNA sequence evolution analysis generalized by simulation and the markov chain monte carlo method implicates strand slippage in a majority of insertions and deletions.

To study the mechanisms for local evolutionary changes in DNA sequences involving slippage-type insertions and deletions, an alignment approach is explored that can consider the posterior probabilities of alignment models. Various patterns of insertion and deletion that can link the ancestor and descendant sequences are proposed and evaluated by simulation and compared by the Markov chain Monte Carlo (MCMC) method. Analyses of pseudogenes reveal that the introduction of the parameters that control the probability of slippage-type events markedly augments the probability of the observed sequence evolution, arguing that a cryptic involvement of slippage occurrences is manifested as insertions and deletions of short nucleotide segments. Strikingly, ?80% of insertions in human pseudogenes and ?50% of insertions in murids pseudogenes are likely to be caused by the slippage-mediated process, as represented by BC in ABCD ? ABCBCD. We suggest that, in both human and murids, even very short repetitive motifs, such as CAGCAG, CACACA, and CCCC, have ?10- to 15-fold susceptibility to insertions and deletions, compared to nonrepetitive sequences. Our protocol, namely, indel-MCMC, thus seems to be a reasonable approach for statistical analyses of the early phase of microsatellite evolution.

Effects of Lys to Glu mutations in GsMTx4 on membrane binding, peptide orientation, and self-association propensity, as analyzed by molecular dynamics simulations.

GsMTx4, a gating modifier peptide acting on cationic mechanosensitive channels, has a positive charge (+5e) due to six Lys residues. The peptide does not have a stereospecific binding site on the channel but acts from the boundary lipids within a Debye length of the pore probably by changing local stress. To gain insight into how these Lys residues interact with membranes, we performed molecular dynamics simulations of Lys to Glu mutants in parallel with our experimental work. In silico, K15E had higher affinity for 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine bilayers than wild-type (WT) peptide or any other mutant tested, and showed deeper penetration than WT, a finding consistent with the experimental data. Experimentally, the inhibitory activities of K15E and K25E were most compromised, whereas K8E and K28E inhibitory activities remained similar to WT peptide. Binding of WT in an interfacial mode did not influence membrane thickness. With interfacial binding, the direction of the dipole moments of K15E and K25E was predicted to differ from WT, whereas those of K8E and K28E oriented similarly to that of WT. These results support a model in which binding of GsMTx4 to the membrane acts like an immersible wedge that serves as a membrane expansion buffer reducing local stress and thus inhibiting channel activity. In simulations, membrane-bound WT attracted other WT peptides to form aggregates. This may account for the positive cooperativity observed in the ion channel experiments. The Lys residues seem to fine-tune the depth of membrane binding, the tilt angle, and the dipole moments.

Local‐scale repetitiveness in amino acid use in eukaryote protein sequences: A genomic factor in protein evolution

We showed previously that the use of arginine versus lysine residues in eukaryote proteins is correlated positively with local GC content of the genome within ≈50 residues. Cumulative analyses show that the tendency for self‐clustering (or repetitive use) generally is the case for all types of amino acids except for certain hydrophobic types. The degree to which each of the amino acids is used recurrently is weak for ancient proteins (or protein domains), those that are conserved through both eukaryotes and prokaryotes, but strong for modern proteins, which are unique to organisms of particular phyla. These findings support the idea that repetitiveness occurs due to a propensity of genomic DNA to cause tandem genomic duplication. A protein sequence with high repetitiveness tends to be unique in the homology search, which may indicate the weaker constraints and, hence, more arbitrary use of amino acids. Simulation analyses suggest that tandem gene duplications on a very small scale (1 or 2 codons) is an important causal factor in maintaining repetitiveness in the presence of concomittant occurrence of substitutive point mutation. For yeast proteins, ≈1.3 duplication events per 1,000 residues on average are likely to occur, whereas 10 events of substitution mutation occur. It also is suggested that duplication enhances the probability of occurrence of some peptide motifs, such as those found in zinc fingers and segments with extreme physicochemical characteristics, and, thus, that local repetitiveness is a genomic factor influencing the evolution of eukaryote proteins. Proteins 1999;37:284–292. ©1999 Wiley‐Liss, Inc.

Potential of mean force analysis of the self-association of leucine-rich transmembrane α-helices: Difference between atomistic and coarse-grained simulations

Interaction of transmembrane (TM) proteins is important in many biological processes. Large-scale computational studies using coarse-grained (CG) simulations are becoming popular. However, most CG model parameters have not fully been calibrated with respect to lateral interactions of TM peptide segments. Here, we compare the potential of mean forces (PMFs) of dimerization of TM helices obtained using a MARTINI CG model and an atomistic (AT) Berger lipids-OPLS/AA model (AT(OPLS)). For helical, tryptophan-flanked, leucine-rich peptides (WL15 and WALP15) embedded in a parallel configuration in an octane slab, the AT(OPLS) PMF profiles showed a shallow minimum (with a depth of approximately 3 kJ/mol; i.e., a weak tendency to dimerize). A similar analysis using the CHARMM36 all-atom model (AT(CHARMM)) showed comparable results. In contrast, the CG analysis generally showed steep PMF curves with depths of approximately 16-22 kJ/mol, suggesting a stronger tendency to dimerize compared to the AT model. This CG > AT discrepancy in the propensity for dimerization was also seen for dilauroylphosphatidylcholine (DLPC)-embedded peptides. For a WL15 (and WALP15)/DLPC bilayer system, AT(OPLS) PMF showed a repulsive mean force for a wide range of interhelical distances, in contrast to the attractive forces observed in the octane system. The change from the octane slab to the DLPC bilayer also mitigated the dimerization propensity in the CG system. The dimerization energies of CG (AALALAA)3 peptides in DLPC and dioleoylphosphatidylcholine bilayers were in good agreement with previous experimental data. The lipid headgroup, but not the length of the lipid tails, was a key causative factor contributing to the differences between octane and DLPC. Furthermore, the CG model, but not the AT model, showed high sensitivity to changes in amino acid residues located near the lipid-water interface and hydrophobic mismatch between the peptides and membrane. These findings may help interpret CG and AT simulation results on membrane proteins.

On Slippage-Like Mutation Dynamics Within Genes: A Study of Pseudogenes and 3′UTRs

Recent analyses show that even very short repetitive sequences are prone to slippage-like mutations. Characterization of the dynamics of such mutations should increase our knowledge about the background frequencies of extension and contractions commonly occurring within genes, allowing the effect of the selections on particular repetitive motifs to be assessed. Consideration of the slippage-like changes may also help the reconstruction of the phylogenetic tree of a gene. In our previous report (Nishizawa and Nishizawa 2002), we described a method which finds the best alignment between two sequences by performing simulations of various types of changes including slippage-like ones. The method can be used to optimize the ‘‘slippage-controlling parameters’’ so that they explain the observed evolution of sequences well. While we believe that the presented framework was reasonable, we acknowledge that the accuracy of the parameter estimation in the paper was limited due to a dearth of informative pseudogenes at the time. The purpose of this letter is to present (1) the result of the pseudogene analysis that supplements our previous report and, in addition, (2) the results of 3¢UTR analyses, which have implications for evolutionary changes of 3¢UTR sequences. We extended the analyses to 160 human and 120 mouse pseudogenes, which were randomly chosen from the recent database (Echols et al. 2002; Zhang et al. 2004). The total numbers of insertions and deletions (‘‘the indel-total’’) that we estimated to have occurred for human and mouse pseudogene/functional-gene pairs were 627 and 931, respectively. Based on the simple binominal model, these numbers can be considered to produce only the negligible sampling errors, and therefore the p-values in the following primarily reflect the potential error in our likelihood estimations. Our method simulates the changes of the sequences in a manner such that the various patterns of insertions and deletions can be examined (Nishizawa and Nishizawa 2002). We have categorized the slippage-like changes into the three types of changes that are accounted for by the parameters f1, f2, and f3, respectively. The f1 parameter indicates the probability, for the insertion events scheduled during the simulation, that each insertion becomes a duplication (instead of an insertion of random nucleotides), as represented by the example ATCAGC fi ATCA CAGC or ATCAGCGC, where the introduced 2-nt segment is underlined. (If, for example, f1 is set to 0.6, the simulation is performed such that 60% of insertions [of any lengths] are duplicative, where 30% are the duplications of the upstream nucleotides and the remaining 30% are those of the downstream nucleotides.) Our previous results suggested that human pseudogenes are more likely to undergo the f1-type slippages than those of murids. In fact, further analyses show no difference between human and murids. For both the human and the mouse pseudogenes, the likelihood profile for f1 shows peak at f1 = 0.5, or 50% are duplicative, indicating that pseudogenes of human and mouse do not have an appreciable difference in f1 -mutations. The f2 and f3 parameters specify the ‘‘extra’’ probability (compared with nonrepetitive sequences) that the very short repetitive sequences, such as GGGG and CACACA, are subjected to the extension and contraction of the repeat, respectively. (For Correspondence to: Manami Nishizawa; email: nmanami@aol.com J Mol Evol (2005) 60:274–275 DOI: 10.1007/s00239-004-0109-5

Why have cholesterol and saturated fatty acids acquired proinflammatory roles during evolution? – A hypothesis from atomistic simulations showing sequence-nonspecific stabilization of peptide dimers in lipid raft-like bilayers

Increased levels of cholesterol and saturated fatty acids (FAs) in immune cell membranes have proinflammatory effects. As opposed to specific effects of lipids mediated by certain lipid-protein interactions, non-specific and indirect effects of such lipids that regulate protein dynamics, regardless of their sequence, could have played influential roles in the early stages of evolution of life. Our recent atomistic simulations showed that, compared to bilayers with abundant unsaturated acyl chains, raft-like bilayers rich in cholesterol and saturated FA chains exert an effect to stabilize the dimeric state of transmembrane helical peptides with simple sequences. The energy cost associated with desolvation of the peptides from lipids upon dimerization was less in the raft-like bilayers compared to unsaturated FArich bilayers, suggesting that solvation (or fitting) of peptides by lipids is important for the dimer-stabilizing effects of such bilayers. In our simulations, acyl chains of phospholipids, but not cholesterol, directly solvated peptides. It is hypothesized that the peptide dimer-stabilizing effect may be the origin of the pro-inflammatory effects of cholesterol and saturated FAs. In this commentary, we mainly discuss our observations in atomistic simulations with some considerations on related experiments and computations as well as on recent knowledge on the properties of various membrane microdomains. Correspondence to: Kazuhisa Nishizawa, Teikyo University School of Medical Technology, Kaga, Itabashi, Tokyo, 173-8605 Japan, Tel: +81-3-3964-1211; Fax: +81-3-5944-3354. Received: March 24, 2017; Accepted: April 28, 2017; Published: May 02, 2017 Specific and nonspecific effects of cholesterol and saturated fatty acids The effects of cholesterol and saturated fatty acids (FAs) have become widely recognized and imply the existence of complex interactions. Proinflammatory effects of these lipids species are considered to involve multilayered and multifaceted cooperative interactions of molecules involving proteins that can recognize specific lipid species [1-4]. On the other hand, as alterations of concentrations of these lipids can change the physicochemical properties of membranes, it is plausible that these lipids impact protein functions thorough mechanisms that are not dependent on specific amino acid sequences of proteins, i.e., in a sequence-nonspecific manner. So, when one questions biochemists or immunologists about possible mechanisms for the proinflammatory effects of lipids, responses are destined to be diverse, depending on the researchers’ particular expertise. Similar to, but slightly different from, the ‘specific versus nonspecific’ division is the classification of ‘direct’ and ‘indirect’ effects of particular lipids. ‘Indirect’ effects correspond to those situations in which particular types of lipids impact membrane protein structure/ functions indirectly by modifying the bilayer environment, including its stiffness, fluidity, thickness, trans-bilayer hydrophobic mismatch, curvature, and lipid domains [5]. Such ‘indirect’ effects should basically be classified as ‘non-specific’ effects. While ‘specific’ effects should be largely mediated by ‘direct’ association of cholesterol or particular FAs with proteins, ‘non-specific’ effects may involve cases with diverse degrees of lipid-protein association, ranging from temporary contacts to relatively stable, prolonged binding. For example, ‘tilted peptides’ may be considered to have the ability to bind to cholesterol (enabling direct effects), defying sequence-based definition (nonspecific effects) [6]. We would like to refer readers to Lange and Steck [5] for diverse modalities of cholesterol-protein interactions. For both cholesterol and FAs, recent efforts have uncovered important instances of specific and direct effects. As an example, for specific effects of FAs, n-3 PUFAs (DHA and EPA), but not saturated FAs, have been shown to exert potent anti-inflammatory effects through GPR120 [7]. In the case of cholesterol, well-studied peptide motifs that recognize it involve the CRAC motif ((L/V)-X1-5–(Y)X1-5-(K/R)) and the CARC motif, which is similar to CRAC, but has an opposed orientation [6]. Other motifs involve the sterol-sensing domain (SSD), which has been identified in several enzyme and transport proteins important for cholesterol metabolism [5]. To our knowledge, the biological significance of these motifs is largely elusive. Nevertheless, more examples of specific cholesterol effects are likely to be elucidated in the future. In the case of the β−2 adrenergic receptor, cholesterol is likely to facilitate interaction between G proteincoupled receptors (GPCRs) [8]. Hanson and colleagues presented an X-ray crystallography model of human β−2 adrenergic receptor in which cholesterol is accommodated in the groove formed by four transmembrane (TM) α-helices [9] Using atomic force microscopybased single-molecule force spectroscopy, Zocher et al. [10] for Nishizawa M (2017) Why have cholesterol and saturated fatty acids acquired proinflammatory roles during evolution? – A hypothesis from atomistic simulations showing sequence-nonspecific stabilization of peptide dimers in lipid raft-like bilayers Biomed Res Clin Prac, 2017 doi: 10.15761/BRCP.1000137 Volume 2(2): 2-6 example, showed that cholesterol (cholesteryl hemisuccinate was used) considerably augmented the strength of interactions stabilizing structural segments of the β−2 adrenergic receptor. Besides GPCRs, the ability of cholesterol to modulate activities of several ion channels and transporters, including Na+, K+-ATPase, is well-established, as discussed in Lange et al. [5]. For Kir2 and heterotetrameric Kir3.1/ Kir3.4 channels, specific mutations abrogated cholesterol’s effects, supporting the concept of direct binding by cholesterol to a specific site of such channels [11]. Although in some cases (dopamine transporter of Drosophila melanogaster, and Na+, K+-ATPase from pig kidney), the cholesterol binding site was identified by crystallographic analysis, the involvement of indirect effects cannot be excluded from the observed physiological effects of cholesterol. With respect to nonspecific effects, Matsuzaki and associates demonstrated that addition of cholesterol stabilized the dimeric state of (AALALAA)3 peptide [12] using liposomes composed of palmitoyloleoylphosphatidylcholine (POPC). However, to our knowledge, fewer studies have focused on nonspecific effects. Nonetheless, nonspecific effects may have been important as the basis that determines or influences the direction of long-term evolution, which favored pro-inflammatory roles for these lipids. In other words, nonspecific physicochemical effects might have influenced the ‘evolutionary fate’ of cholesterol and saturated FAs as proinflammatory factors; in this view, newly evolved molecules (proteins) may have augmented or fine-tuned such effects of these lipids by elaborating specific protein-lipid interactions. TM dimerization/oligomerization in cell signaling Interactions between TM domains of membrane proteins have drawn researchers’ interest from biomedical and pharmaceutical perspectives. Association between TM helices is a widely-used strategy that directs the assembly of protein complexes and mediates signal transduction of hormone and cytokine receptors. At least for many single-pass TM proteins, disease-associated mutations in TM domains have been reported. It seems convenient to tentatively consider that there are two broad categories of interactions [13]. The first involves static interactions, in which the TM domains form relatively fixed contacts necessary for the assembly of a functional protein complex and for proper folding of proteins. The second category is that of dynamic conformational changes, in which these changes are propagated through the membrane via changes in the oligomerization state and/ or orientation of TM helices [13]. Of note, Toll-like receptor (TLR) activation is accompanied by dimerization of TM domains [14]. As a well-studied example, TM domains of TLR2 and 6 are known to be important for activation of these proteins, interacting with each other and forming heterodimers. An inhibition analysis using synthetic peptide from TLR2 TM domain supported this conclusion [15]. There is currently no universal consensus model for signaling mechanisms mediated by diverse single-span membrane proteins. In general, the dimeric state involves the active configuration/ conformation. The simplest model (‘canonical model’) for the signaling of cytokine receptors and receptor tyrosine kinases was an equilibrium between inactive monomers and ligand-bound dimers [16,17]. However, recent studies have extended this view; a substantial portion of receptors is forming inactive pre-dimers and, in addition to dimerization, subtle conformational changes are likely necessary for activation (‘pre-formed dimer model’) [18,19]. In the case of epidermal growth factor receptor (EGFR), a well-studied example of receptor tyrosine kinases, inactive symmetric intracellular kinase domains need to change to enzymatically active (asymmetric) dimers for EGFR signaling [20-22]. For human vascular endothelial growth factor receptor (VEGFR)-2, which represents another family of receptor tyrosine kinases, 30 to 60% of it was shown to exist in the dimeric form in the absence of ligand based on a Fluorescence resonance energy transfer (FRET) analysis, and ligand binding induces a conformational change in the TM domain dimer structure causing increased phosphorylation and further structural changes [19]. Human growth hormone receptor (hGHr), which, like cytokine receptors, utilizes JAK kinase for phosphorylation of the cytosolic domain, has also been shown to assume an inactive dimer conformation. A scissorlike motion of the TM domain of hGHr can switch the conformation of the intracellular domains, coupled with JAK2, between the active

Free energy of helical transmembrane peptide dimerization in OPLS-AA/Berger force field simulations: inaccuracy and implications for partner-specific Lennard-Jones parameters between peptides and lipids

Abstract Interactions between transmembrane (TM) peptides are important in biophysical chemistry, but there are few studies assessing atomistic simulation parameters concerning the energetics of interactions of TM helical peptides. Our potential of mean force analysis using OPLS-AA protein/Berger lipid force fields (FFs) shows that the dimerisation energy of (AALALAA)3 helical peptides in the dioleoylphosphatidylcholine bilayer is −4.4 kJ/mol, which was much smaller than the reported experimental value (−12.7 kJ/mol), thus calling for improvement of parameters of the combined FFs. As each of the FFs has been independently developed, we then tested the effects of downscaling the Lennard-Jones (LJ) energy terms between the OPLS-AA atoms and Berger lipid atoms, preserving the parameters within each FF. A 0.9-fold rescaling of the LJ energies was found to render the dimerisation energy close to the experimental value. Solvation of backbone atoms as well as side chain atoms in lipids is crucial for the TM helix interaction. In similar analyses, GROMOS 53A6 FF exhibited as weak dimerisation propensity (~−5.2 kJ/mol) as OPLS-AA/Berger, but CHARMM36 showed relatively accurate propensity (~−9.9 kJ/mol). Challenges and strategies in rendering the TM interaction energy realistic within the framework of current FFs are discussed.