Standard Binding Free Energy and Membrane Desorption Mechanism for a Phospholipase C.

Peripheral membrane proteins (PMPs) bind temporarily to cellular membranes and play important roles in signaling, lipid metabolism, and membrane trafficking. Obtaining accurate membrane-PMP affinities using experimental techniques is more challenging than for protein-ligand affinities in an aqueous solution. At the theoretical level, calculation of the standard protein-membrane binding free energy using molecular dynamics simulations remains a daunting challenge owing to the size of the biological objects at play, the slow lipid diffusion, and the large variation in configurational entropy that accompanies the binding process. To overcome these challenges, we used a computational framework relying on a series of potential-of-mean-force (PMF) calculations including a set of geometrical restraints on collective variables. This methodology allowed us to determine the standard binding free energy of a PMP to a phospholipid bilayer using an all-atom force field. Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (BtPI-PLC) was chosen due to its importance as a virulence factor and owing to the host of experimental affinity data available. We computed a standard binding free energy of -8.2 ± 1.4 kcal/mol in reasonable agreement with the reported experimental values (-6.6 ± 0.2 kcal/mol). In light of the 2.3-µs separation PMF calculation, we investigated the mechanism whereby BtPI-PLC disengages from interactions with the lipid bilayer during separation. We describe how a short amphipathic helix engages in transitory interactions to ease the passage of its hydrophobes through the interfacial region upon desorption from the bilayer.

Specificity of Loxosceles α clade phospholipase D enzymes for choline-containing lipids: Role of a conserved aromatic cage.

Spider venom GDPD-like phospholipases D (SicTox) have been identified to be one of the major toxins in recluse spider venom. They are divided into two major clades: the α clade and the ß clade. Most α clade toxins present high activity against lipids with choline head groups such as sphingomyelin, while activities in ß clade toxins vary and include preference for substrates containing ethanolamine headgroups (Sicarius terrosus, St_ßIB1). A structural comparison of available structures of phospholipases D (PLDs) reveals a conserved aromatic cage in the α clade. To test the potential influence of the aromatic cage on membrane-lipid specificity we performed molecular dynamics (MD) simulations of the binding of several PLDs onto lipid bilayers containing choline headgroups; two SicTox from the α clade, Loxosceles intermedia αIA1 (Li_αIA) and Loxosceles laeta αIII1 (Ll_αIII1), and one from the ß clade, St_ßIB1. The simulation results reveal that the aromatic cage captures a choline-headgroup and suggest that the cage plays a major role in lipid specificity. We also simulated an engineered St_ßIB1, where we introduced the aromatic cage, and this led to binding with choline-containing lipids. Moreover, a multiple sequence alignment revealed the conservation of the aromatic cage among the α clade PLDs. Here, we confirmed that the i-face of α and ß clade PLDs is involved in their binding to choline and ethanolamine-containing bilayers, respectively. Furthermore, our results suggest a major role in choline lipid recognition of the aromatic cage of the α clade PLDs. The MD simulation results are supported by in vitro liposome binding assay experiments.

The voltage-sensing domain of a hERG1 mutant is a cation-selective channel.

A cationic leak current known as an "omega current" may arise from mutations of the first charged residue in the S4 of the voltage sensor domains of sodium and potassium voltage-gated channels. The voltage-sensing domains (VSDs) in these mutated channels act as pores allowing nonspecific passage of cations, such as Li+, K+, Cs+, and guanidinium. Interestingly, no omega currents have been previously detected in the nonswapped voltage-gated potassium channels such as the human-ether-a-go-go-related (hERG1), hyperpolarization-activated cyclic nucleotide-gated, and ether-a-go-go channels. In this work, we discovered a novel omega current by mutating the first charged residue of the S4 of the hERG1, K525 to serine. To characterize this omega current, we used various probes, including the hERG1 pore domain blocker, dofetilide, to show that the omega current does not require cation flux via the canonical pore domain. In addition, the omega flux does not cross the conventional selectivity filter. We also show that the mutated channel (K525S hERG1) conducts guanidinium. These data are indicative of the formation of an omega current channel within the VSD. Using molecular dynamics simulations with replica-exchange umbrella sampling simulations of the wild-type hERG1 and the K525S hERG1, we explored the molecular underpinnings governing the cation flow in the VSD of the mutant. We also show that the wild-type hERG1 may form water crevices supported by the biophysical surface accessibility data. Overall, our multidisciplinary study demonstrates that the VSD of hERG1 may act as a cation-selective channel wherein a mutation of the first charged residue in the S4 generates an omega current. Our simulation uncovers the atomistic underpinning of this mechanism.

Phospholipids in Motion: High-Resolution 31P NMR Field Cycling Studies.

Diverse phospholipid motions are key to membrane function but can be quite difficult to untangle and quantify. High-resolution field cycling 31P NMR spin-lattice relaxometry, where the sample is excited at high field, shuttled in the magnet bore for low-field relaxation, then shuttled back to high field for readout of the residual magnetization, provides data on phospholipid dynamics and structure. This information is encoded in the field dependence of the 31P spin-lattice relaxation rate (R1). In the field range from 11.74 down to 0.003 T, three dipolar nuclear magnetic relaxation dispersions (NMRDs) and one due to 31P chemical shift anisotropy contribute to R1 of phospholipids. Extraction of correlation times and maximum relaxation amplitudes for these NMRDs provides (1) lateral diffusion constants for different phospholipids in the same bilayer, (2) estimates of how additives alter the motion of the phospholipid about its long axis, and (3) an average 31P-1H angle with respect to the bilayer normal, which reveals that polar headgroup motion is not restricted on a microsecond timescale. Relative motions within a phospholipid are also provided by comparing 31P NMRD profiles for specifically deuterated molecules as well as 13C and 1H field dependence profiles to that of 31P. Although this work has dealt exclusively with phospholipids in small unilamellar vesicles, these same NMRDs can be measured for phospholipids in micelles and nanodisks, making this technique useful for monitoring lipid behavior in a variety of structures and assessing how additives alter specific lipid motions.

Martini 3: a general purpose force field for coarse-grained molecular dynamics.

The coarse-grained Martini force field is widely used in biomolecular simulations. Here we present the refined model, Martini 3 ( http://cgmartini.nl ), with an improved interaction balance, new bead types and expanded ability to include specific interactions representing, for example, hydrogen bonding and electronic polarizability. The updated model allows more accurate predictions of molecular packing and interactions in general, which is exemplified with a vast and diverse set of applications, ranging from oil/water partitioning and miscibility data to complex molecular systems, involving protein-protein and protein-lipid interactions and material science applications as ionic liquids and aedamers.

Refinement of a cryo-EM structure of hERG: Bridging structure and function.

The human-ether-a-go-go-related gene (hERG) encodes the voltage-gated potassium channel (KCNH2 or Kv11.1, commonly known as hERG). This channel plays a pivotal role in the stability of phase 3 repolarization of the cardiac action potential. Although a high-resolution cryo-EM structure is available for its depolarized (open) state, the structure surprisingly did not feature many functionally important interactions established by previous biochemical and electrophysiology experiments. Using molecular dynamics flexible fitting (MDFF), we refined the structure and recovered the missing functionally relevant salt bridges in hERG in its depolarized state. We also performed electrophysiology experiments to confirm the functional relevance of a novel salt bridge predicted by our refinement protocol. Our work shows how refinement of a high-resolution cryo-EM structure helps to bridge the existing gap between the structure and function in the voltage-sensing domain (VSD) of hERG.

Allosteric Coupling Between Drug Binding and the Aromatic Cassette in the Pore Domain of the hERG1 Channel: Implications for a State-Dependent Blockade.

Human-ether-a-go-go-related channel (hERG1) is the pore-forming domain of the delayed rectifier K+ channel in the heart which underlies the IKr current. The channel has been extensively studied due to its propensity to bind chemically diverse group of drugs. The subsequent hERG1 block can lead to a prolongation of the QT interval potentially leading to an abnormal cardiac electrical activity. The recently solved cryo-EM structure featured a striking non-swapped topology of the Voltage-Sensor Domain (VSD) which is packed against the pore-domain as well as a small and hydrophobic intra-cavity space. The small size and hydrophobicity of the cavity was unexpected and challenges the already-established hypothesis of drugs binding to the wide cavity. Recently, we showed that an amphipathic drug, ivabradine, may favorably bind the channel from the lipid-facing surface and we discovered a mutant (M651T) on the lipid facing domain between the VSD and the PD which inhibited the blocking capacity of the drug. Using multi-microseconds Molecular Dynamics (MD) simulations of wild-type and M651T mutant hERG1, we suggested the block of the channel through the lipid mediated pathway, the opening of which is facilitated by the flexible phenylalanine ring (F656). In this study, we characterize the dynamic interaction of the methionine-aromatic cassette in the S5-S6 helices by combining data from electrophysiological experiments with MD simulations and molecular docking to elucidate the complex allosteric coupling between drug binding to lipid-facing and intra-cavity sites and aromatic cassette dynamics. We investigated two well-established hERG1 blockers (ivabradine and dofetilide) for M651 sensitivity through electrophysiology and mutagenesis techniques. Our electrophysiology data reveal insensitivity of dofetilide to the mutations at site M651 on the lipid facing side of the channel, mirroring our results obtained from docking experiments. Moreover, we show that the dofetilide-induced block of hERG1 occurs through the intracellular space, whereas little to no block of ivabradine is observed during the intracellular application of the drug. The dynamic conformational rearrangement of the F656 appears to regulate the translocation of ivabradine into the central cavity. M651T mutation appears to disrupt this entry pathway by altering the molecular conformation of F656.

Capturing Choline-Aromatics Cation-π Interactions in the MARTINI Force Field.

Cation-π interactions play an important role in biomolecular recognition, including interactions between membrane phosphatidylcholine lipids and aromatic amino acids of peripheral proteins. While molecular mechanics coarse grain (CG) force fields are particularly well suited to simulate membrane proteins in general, they are not parameterized to explicitly reproduce cation-π interactions. We here propose a modification of the polarizable MARTINI coarse grain (CG) model enabling it to model membrane binding events of peripheral proteins whose aromatic amino acid interactions with choline headgroups are crucial for their membrane binding. For this purpose, we first collected and curated a dataset of eight peripheral proteins from different families. We find that the MARTINI CG model expectedly underestimates aromatics-choline interactions and is unable to reproduce membrane binding of the peripheral proteins in our dataset. Adjustments of the relevant interactions in the polarizable MARTINI force field yield significant improvements in the observed binding events. The orientation of each membrane-bound protein is comparable to reference data from all-atom simulations and experimental binding data. We also use negative controls to ensure that choline-aromatics interactions are not overestimated. We finally check that membrane properties, transmembrane proteins, and membrane translocation potential of mean force (PMF) of aromatic amino acid side-chain analogues are not affected by the new parameter set. This new version "MARTINI 2.3P" is a significant improvement over its predecessors and is suitable for modeling membrane proteins including peripheral membrane binding of peptides and proteins.

Interfacial Aromatics Mediating Cation-π Interactions with Choline-Containing Lipids Can Contribute as Much to Peripheral Protein Affinity for Membranes as Aromatics Inserted below the Phosphates.

Membrane-binding interfaces of peripheral proteins are restricted to a small part of their exposed surface, so the ability to engage in strong selective interactions with membrane lipids at various depths in the interface, both below and above the phosphates, is an advantage. Driven by their hydrophobicity, aromatic amino acids preferentially partition into membrane interfaces, often below the phosphates, yet enthalpically favorable interactions with the lipid headgroups, above the phosphate plane, are likely to further stabilize high interfacial positions. Using free-energy perturbation, we calculate the energetic cost of alanine substitution for 11 interfacial aromatic amino acids from 3 peripheral proteins. We show that the involvement in cation-π interactions with the headgroups (i) increases the ΔΔGtransfer as compared with insertion at the same depth without cation-π stabilization and (ii) can contribute at least as much as deeper insertion below the phosphates, highlighting the multiple roles of aromatics in peripheral membrane protein affinity.

Cation-π Interactions between Methylated Ammonium Groups and Tryptophan in the CHARMM36 Additive Force Field.

Cation-π interactions between tryptophan and choline or trimethylated lysines are vital for many biological processes. The performance of the additive CHARMM36 force field against target quantum mechanical data is shown to reproduce QM equilibrium geometries but required modified Lennard-Jones potentials to accurately reproduce the QM interaction energies. The modified parameter set allows accurate modeling, including free energies, of cation-π indole-choline and indole-trimethylated lysines interactions relevant for protein-ligand, protein-membrane, and protein-protein interfaces.

Search and Subvert: Minimalist Bacterial Phosphatidylinositol-Specific Phospholipase C Enzymes.

Phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes from Gram-positive bacteria are secreted virulence factors that aid in downregulating host immunity. These PI-PLCs are minimalist peripheral membrane enzymes with a distorted (ßα)8 TIM barrel fold offering a conserved and stable scaffold for the conserved catalytic amino acids while membrane recognition is achieved mostly through variable loops. Decades of experimental and computational research on these enzymes have revealed the subtle interplay between molecular mechanisms of catalysis and membrane binding, leading to a semiquantitative model for how they find, bind, and cleave their respective substrates on host cell membranes. Variations in sequence and structure of their membrane binding sites may correlate with how enzymes from different Gram-positive bacteria search for their particular targets on the membrane. Detailed molecular characterization of protein-lipid interactions have been aided by cutting-edge methods ranging from 31P field-cycling NMR relaxometry to monitor protein-induced changes in phospholipid dynamics to molecular dynamics simulations to elucidate the roles of electrostatic and cation-π interactions in lipid binding to single molecule fluorescence measurements of dynamic interactions between PI-PLCs and vesicles. This toolkit is readily applicable to other peripheral membrane proteins including orthologues in Gram-negative bacteria and more recently discovered eukaryotic minimalist PI-PLCs.

Improving the Force Field Description of Tyrosine-Choline Cation-π Interactions: QM Investigation of Phenol-N(Me)4+ Interactions.

Cation-π interactions between tyrosine amino acids and compounds containing N,N,N-trimethylethanolammonium (N(CH3)3) are involved in the recognition of histone tails by chromodomains and in the recognition of phosphatidylcholine (PC) phospholipids by membrane-binding proteins. Yet, the lack of explicit polarization or charge transfer effects in molecular mechanics force fields raises questions about the reliability of the representation of these interactions in biomolecular simulations. Here, we investigate the nature of phenol-tetramethylammonium (TMA) interactions using quantum mechanical (QM) calculations, which we also use to evaluate the accuracy of the additive CHARMM36 and Drude polarizable force fields in modeling tyrosine-choline interactions. We show that the potential energy surface (PES) obtained using SAPT2+/aug-cc-pVDZ compares well with the large basis-set CCSD(T) PES when TMA approaches the phenol ring perpendicularly. Furthermore, the SAPT energy decomposition reveals comparable contributions from electrostatics and dispersion in phenol-TMA interactions. We then compared the SAPT2+/aug-cc-pVDZ PES obtained along various approach directions to the corresponding PES obtained with CHARMM, and we show that the force field accurately reproduces the minimum distances while the interaction energies are underestimated. The use of the Drude polarizable force field significantly improves the interaction energies but decreases the agreement on distances at energy minima. The best agreement between force field and QM PES is obtained by modifying the Lennard-Jones terms for atom pairs involved in the phenol-TMA cation-π interactions. This is further shown to improve the correlation between the occupancy of tyrosine-choline cation-π interactions obtained from molecular dynamics simulations of a bilayer-bound bacterial phospholipase and experimental affinity data of the wild-type protein and selected mutants.

A Role for Weak Electrostatic Interactions in Peripheral Membrane Protein Binding.

Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (BtPI-PLC) is a secreted virulence factor that binds specifically to phosphatidylcholine (PC) bilayers containing negatively charged phospholipids. BtPI-PLC carries a negative net charge and its interfacial binding site has no obvious cluster of basic residues. Continuum electrostatic calculations show that, as expected, nonspecific electrostatic interactions between BtPI-PLC and membranes vary as a function of the fraction of anionic lipids present in the bilayers. Yet they are strikingly weak, with a calculated ΔGel below 1 kcal/mol, largely due to a single lysine (K44). When K44 is mutated to alanine, the equilibrium dissociation constant for small unilamellar vesicles increases more than 50 times (∼2.4 kcal/mol), suggesting that interactions between K44 and lipids are not merely electrostatic. Comparisons of molecular-dynamics simulations performed using different lipid compositions reveal that the bilayer composition does not affect either hydrogen bonds or hydrophobic contacts between the protein interfacial binding site and bilayers. However, the occupancies of cation-π interactions between PC choline headgroups and protein tyrosines vary as a function of PC content. The overall contribution of basic residues to binding affinity is also context dependent and cannot be approximated by a rule-of-thumb value because these residues can contribute to both nonspecific electrostatic and short-range protein-lipid interactions. Additionally, statistics on the distribution of basic amino acids in a data set of membrane-binding domains reveal that weak electrostatics, as observed for BtPI-PLC, might be a less unusual mechanism for peripheral membrane binding than is generally thought.

Membrane Docking of the Synaptotagmin 7 C2A Domain: Computation Reveals Interplay between Electrostatic and Hydrophobic Contributions.

The C2A domain of synaptotagmin 7 (Syt7) is a Ca(2+) and membrane binding module that docks and inserts into cellular membranes in response to elevated intracellular Ca(2+) concentrations. Like other C2 domains, Syt7 C2A binds Ca(2+) and membranes primarily through three loop regions; however, it docks at Ca(2+) concentrations much lower than those required for other Syt C2A domains. To probe structural components of its unusually strong membrane docking, we conducted atomistic molecular dynamics simulations of Syt7 C2A under three conditions: in aqueous solution, in the proximity of a lipid bilayer membrane, and embedded in the membrane. The simulations of membrane-free protein indicate that Syt7 C2A likely binds three Ca(2+) ions in aqueous solution, consistent with prior experimental reports. Upon membrane docking, the outermost Ca(2+) ion interacts directly with lipid headgroups, while the other two Ca(2+) ions remain chelated by the protein. The membrane-bound domain was observed to exhibit large-amplitude swinging motions relative to the membrane surface, varying by up to 70° between a more parallel and a more perpendicular orientation, both during and after insertion of the Ca(2+) binding loops into the membrane. The computed orientation of the membrane-bound protein correlates well with experimental electron paramagnetic resonance measurements presented in the preceding paper ( DOI: 10.1021/acs.biochem.5b00421 ). In particular, the strictly conserved residue Phe229 inserted stably ∼4 Å below the average depth of lipid phosphate groups, providing critical hydrophobic interactions anchoring the domain in the membrane. Overall, the position and orientation of Syt7 C2A with respect to the membrane are consistent with experiments.

Quantifying transient interactions between Bacillus phosphatidylinositol-specific phospholipase-C and phosphatidylcholine-rich vesicles.

Bacillus thuringiensis secretes the virulence factor phosphatidylinositol-specific phospholipase C (BtPI-PLC), which specifically binds to phosphatidylcholine (PC) and cleaves GPI-anchored proteins off eukaryotic plasma membranes. To elucidate how BtPI-PLC searches for GPI-anchored proteins on the membrane surface, we measured residence times of single fluorescently labeled proteins on PC-rich small unilamellar vesicles (SUVs). BtPI-PLC interactions with the SUV surface are transient with a lifetime of 379 ± 49 ms. These data also suggest that BtPI-PLC does not directly sense curvature, but rather prefers to bind to the numerous lipid packing defects in SUVs. Despite this preference for defects, all-atom molecular dynamics simulations of BtPI-PLC interacting with PC-rich bilayers show that the protein is shallowly anchored with the deepest insertions ∼18 Å above the bilayer center. Membrane partitioning is mediated, on average, by 41 hydrophobic, 8 hydrogen-bonding, and 2 cation-π (between PC choline headgroups and Tyr residues) transient interactions with phospholipids. These results lead to a quantitative model for BtPI-PLC interactions with cell membranes where protein binding is mediated by lipid packing defects, possibly near GPI-anchored proteins, and the protein diffuses on the membrane for ∼100-380 ms, during which time it may cleave ∼10 GPI-anchored proteins before dissociating. This combination of short two-dimensional scoots followed by three-dimensional hops may be an efficient search strategy on two-dimensional surfaces with obstacles.