RESUMO
GsMTx4 is a spider venom peptide that inhibits cationic mechanosensitive channels (MSCs). It has six lysine residues that have been proposed to affect membrane binding. We synthesized six analogs with single lysine-to-glutamate substitutions and tested them against Piezo1 channels in outside-out patches and independently measured lipid binding. Four analogs had â¼20% lower efficacy than the wild-type (WT) peptide. The equilibrium constants calculated from the rates of inhibition and washout did not correlate with the changes in inhibition. The lipid association strength of the WT GsMTx4 and the analogs was determined by tryptophan autofluorescence quenching and isothermal calorimetry with membrane vesicles and showed no significant differences in binding energy. Tryptophan fluorescence-quenching assays showed that both WT and analog peptides bound superficially near the lipid-water interface, although analogs penetrated deeper. Peptide-lipid association, as a function of lipid surface pressure, was investigated in Langmuir monolayers. The peptides occupied a large fraction of the expanded monolayer area, but that fraction was reduced by peptide expulsion as the pressure approached the monolayer-bilayer equivalence pressure. Analogs with compromised efficacy had pressure-area isotherms with steeper slopes in this region, suggesting tighter peptide association. The pressure-dependent redistribution of peptide between "deep" and "shallow" binding modes was supported by molecular dynamics (MD) simulations of the peptide-monolayer system under different area constraints. These data suggest a model placing GsMTx4 at the membrane surface, where it is stabilized by the lysines, and occupying a small fraction of the surface area in unstressed membranes. When applied tension reduces lateral pressure in the lipids, the peptides penetrate deeper acting as "area reservoirs" leading to partial relaxation of the outer monolayer, thereby reducing the effective magnitude of stimulus acting on the MSC gate.
Assuntos
Canais Iônicos/antagonistas & inibidores , Canais Iônicos/metabolismo , Fenômenos Mecânicos , Peptídeos/farmacologia , Venenos de Aranha/farmacologia , Fenômenos Biomecânicos , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Ativação do Canal Iônico/efeitos dos fármacos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Peptídeos/metabolismo , Pressão , Venenos de Aranha/metabolismo , Estresse MecânicoRESUMO
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.
Assuntos
Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto , Peptídeos/química , Venenos de Aranha/química , Membrana Celular/química , Membrana Celular/metabolismo , Ácido Glutâmico/química , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Cinética , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lisina/química , Lisina/genética , Lisina/metabolismo , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Ligação Proteica , Venenos de Aranha/genética , Venenos de Aranha/metabolismo , TermodinâmicaRESUMO
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.
Assuntos
Membrana Celular/química , Dimerização , Leucina , Bicamadas Lipídicas/química , Proteínas de Membrana/química , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/química , Sequência de Aminoácidos , Membrana Celular/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Estrutura Secundária de Proteína , TermodinâmicaRESUMO
Membrane fusion often exhibits slow dynamics in electrophysiological experiments, involving prespike foot and fusion pore-flickering, but the structural basis of such phenomena remains unclear. Hemifusion intermediates have been implicated in the early phase of membrane fusion. To elucidate the dynamics of formation of membrane defects and pores within the hemifusion diaphragm (HD), atomistic and coarse-grained models of hemifusion intermediates were constructed using dipalmitoylphosphatidylcholine or dioleoylphosphatidylcholine membranes. The work necessary to displace a lipid molecule to the hydrophobic core of the bilayer was measured. For a lipid within the HD with radius of 4 nm, the work was â¼80 kJ/mol, similar to that in a planar bilayer. The work was much less (â¼40 kJ/mol) when the HD was surrounded by a steep stalk, i.e., stalk wings forming a large angle at the junction of three bilayers. In the latter case, the lipid displacement engendered formation of a pore contacting the HD rim. The work was similarly small (40 kJ/mol) for a small HD of 1.5 nm radius, where a pore formed and grew rapidly, quickly generating a toroidal structure (<40 ns). Combining the steep stalk and the small HD decreased the work further, although quantitative analysis was difficult because the latter system was not in a stable equilibrium state. Results suggest that fine tuning of fusion dynamics requires strict control of the HD size and the angle between the expanded stalk and HD. In additional free simulations, the steep stalk facilitated widening of a preformed pore contacting the HD rim.
Assuntos
Bicamadas Lipídicas/química , Fusão de Membrana , Simulação de Dinâmica Molecular , Membrana Celular/química , Membrana Celular/fisiologiaRESUMO
The recently determined crystal structure of a chimeric Kv1.2-Kv2.1 Kv channel at 2.4 A resolution motivated this molecular-dynamics simulation study of the chimeric channel and its mutants embedded in a DPPC membrane. For the channel protein, we used two types of C-terminus: E+ and Eo. E+ contains, and Eo lacks, the EGEE residue quartet located distal to the S6 helix. For both E+ and Eo, the following trend was observed: When S4 helices were restrained at the same position as in the x-ray structure (S4high), the S6 gate remained open for 12 ns. The results were similar when the S4 helices were pulled downward 7 A (S4low). However, S4middle (or S4low) facilitated the S6 gate-narrowing for the following mutated channels (shown in order of increasing effect): 1), E395W; 2), E395W-F401A-F402A; and 3), E395W-F401A-F402A-V478W. The amino acid numbering system is that used for the Shaker channel. Even though all four subunits were set at S4low, S6 gate-narrowing was often brought about by movements of only two opposing S6 helices toward the central axis of the pore, resulting in a twofold symmetry-like structure. A free-energy profile analysis over the ion conduction pathway shows that the two opposing S6 helices whose peptide backbones are approximately 10.4 A distant from each other lead to an energetic barrier of approximately 25 kJ/mol. S6 movement was coupled with translocation of the S4-S5 linker toward the central axis of the same subunit, and the coupling was mediated by salt bridges formed between the inner (intracellular side) end of S4 and that of S6. Simulations in which S4 of only one subunit was pulled down to S4low showed that a weak intersubunit coordination is present for S5 movement, whereas the coupling between the S4-S5 linker and S6 is largely an intrasubunit one. In general, whereas subunit-based behavior appears to be dominant and to permit heteromeric conformations of the pore domain, direct intersubunit coupling of S5 or S6 is weak. Therefore, the "concerted transition" of the pore domain that has been predicted based on electrophysiological analyses is likely to be mediated mainly by the dual effects of S4 and the S4-S5 linker; these segments of one subunit can interact with both S5 of the same subunit and that of the adjacent subunit.
Assuntos
Simulação por Computador , Canal de Potássio Kv1.2/química , Canal de Potássio Kv1.2/genética , Modelos Químicos , Canais de Potássio Shab/química , Canais de Potássio Shab/genética , Modelos Moleculares , Mutação , Conformação Proteica , Fatores de Tempo , Água/químicaRESUMO
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.
Assuntos
Bicamadas Lipídicas/química , Bicamadas Lipídicas/efeitos da radiação , Modelos Químicos , Modelos Moleculares , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/efeitos da radiação , Simulação por Computador , Campos Eletromagnéticos , Conformação Proteica/efeitos da radiação , Doses de RadiaçãoRESUMO
Voltage-sensing phosphatases (VSP) contain a voltage sensor domain (VSD) similar to that of voltage-gated ion channels but lack a pore-gate domain. A VSD in a VSP regulates the cytoplasmic catalytic region (CCR). However, the mechanisms by which the VSD couples to the CCR remain elusive. Here we report a membrane interface (named 'the hydrophobic spine'), which is essential for the coupling of the VSD and CCR. Our molecular dynamics simulations suggest that the hydrophobic spine of Ciona intestinalis VSP (Ci-VSP) provides a hinge-like motion for the CCR through the loose membrane association of the phosphatase domain. Electrophysiological experiments indicate that the voltage-dependent phosphatase activity of Ci-VSP depends on the hydrophobicity and presence of an aromatic ring in the hydrophobic spine. Analysis of conformational changes in the VSD and CCR suggests that the VSP has two states with distinct enzyme activities and that the second transition depends on the hydrophobic spine.
Assuntos
Citoplasma/genética , Ativação do Canal Iônico/genética , Membranas/química , Monoéster Fosfórico Hidrolases/química , Sequência de Aminoácidos/genética , Animais , Domínio Catalítico/genética , Ciona intestinalis/química , Citoplasma/química , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Monoéster Fosfórico Hidrolases/genética , Domínios ProteicosRESUMO
Our recent molecular dynamics simulation study of hanatoxin 1 (HaTx1), a gating modifier that binds to the voltage sensor of K(+) channels, has shown that HaTx1 has the ability to interact with carbonyl oxygen atoms of both leaflets of the lipid bilayer membrane and to be located at a deep position within the membrane. Here we performed a similar study of GsMTx4, a stretch-activated channels inhibitor, belonging to the same peptide family as HaTx1. Both toxins have an ellipsoidal shape, a belt of positively charged residues around the periphery, and a hydrophobic protrusion. Results show that, like HaTx1, GsMTx4 can interact with the membrane in two different ways. When all the positively charged residues interact with the outer leaflet lipid, GsMTx4 can assume a shallow binding mode. On the other hand, when the electrostatic interaction brings the positively charged groups of K-8 and K-28 into the vicinity of the carbonyl oxygen atoms of the inner leaflet lipids, the system exhibits a deep binding mode. This deep mode is accompanied by local membrane thinning. For both HaTx1 and GsMTx4, our mean force measurement analyses show that the deep binding mode is energetically favored over the shallow mode when a DPPC (dipalmitoyl-phosphatidylcholine) membrane is used at 310 K. In contrast, when a POPC (palmitooleoyl-phosphatidylcholine) membrane is used at 310 K, the two binding modes exhibited similar stability for both toxins. Similar analyses with DPPC membrane at 330 K led to an intermediary result between the above two results. Therefore, the structure of the lipid acyl chains appears to influence the location and the dynamics of the toxins within biological membranes. We also compared the behavior of an arginine and a lysine residue within the membrane. This is of interest because the arginine residue interaction with the lipid carbonyl oxygen atoms mediates the deep binding mode for HaTx1, whereas the lysine residue plays that role for GsMTx4. The arginine residue generally shows smoother dynamics near the lipid carbonyl oxygen atoms than the lysine residue. This difference between arginine and lysine may partly account for the functional diversity of the members of the toxin family.
Assuntos
Ativação do Canal Iônico , Bicamadas Lipídicas/química , Fluidez de Membrana , Modelos Químicos , Modelos Moleculares , Peptídeos/química , Fosfolipídeos/química , Venenos de Aranha/química , Simulação por Computador , Elasticidade , Peptídeos e Proteínas de Sinalização Intercelular , Conformação Molecular , Ligação Proteica , Estresse MecânicoRESUMO
Hanatoxin (HaTx) is an ellipsoidal-shaped peptide that binds to the voltage sensor of voltage-dependent channels. Of physicochemical interest, HaTx has a "ring" of charged residues around its periphery and a hydrophobic protrusion. It has previously been postulated that HaTx binds to and functions on the surface of membranes, but a recent fluorescent-quenching study has implied a fairly deep positioning of HaTx in the lipid bilayer membrane. We carried out numerous molecular dynamic simulations of HaTx1, a well-studied variant of HaTx, in fully hydrated phospholipid bilayers. The system reproduced the surface-binding mode of HaTx1, in which HaTx1 resided in the extracellular side (outer) of the water/membrane interface with the hydrophobic patch of HaTx1 facing the membrane interior. On the other hand, analyses with various parameter settings suggested that the surface-binding mode was unstable because of the substantial attractive electrostatic force between HaTx1 and the lipid head groups of the inner (opposite) leaflet. Compared with this electrostatic force, the energetic cost for membrane deformation involving meniscus formation appeared to be small. In an attempt to interpret the quenching data, we consider the possibility of dimpling (meniscus formation) that brings HaTx1 inward (only ~0.7-0.8 nm above the bilayer center), while accounting for the flexibility of both leaflets of the membrane and the long-range interaction between positively charged residues of the membrane-bound peptide and the polar head groups of the opposite leaflet of the membrane. It is suggested that molecular dynamics simulations taking into account the flexibility of the membrane surface is potentially useful in interpreting the fluorescence-quenching data.
Assuntos
Ativação do Canal Iônico , Bicamadas Lipídicas/química , Fluidez de Membrana , Modelos Químicos , Modelos Moleculares , Peptídeos/química , Canais de Potássio/química , Simulação por Computador , Cinética , Conformação Molecular , Movimento (Física) , Fosfolipídeos/químicaRESUMO
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, approximately 80% of insertions in human pseudogenes and approximately 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 approximately 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.