Microsecond Simulations of the Diphtheria Toxin Translocation Domain in Association with Anionic Lipid Bilayers

Diphtheria toxin translocation (T) domain undergoes conformational changes in acidic solution and associates with the lipid membranes, followed by refolding and transmembrane insertion of two nonpolar helices. This process is an essential step in delivery of the toxic catalytic domain of the diphtheria toxin to the infected cell, yet its molecular determinants are poorly characterized and understood. Therefore, an atomistic model of the T-domain–membrane interaction is needed to help characterize factors responsible for such association. In this work, we present atomistic model structures of T-domain membrane-bound conformations and investigate structural factors responsible for T-domain affinity with the lipid bilayer in acidic solution using all-atom molecular dynamics (MD) simulations. The initial models of the protein conformations and protein–membrane association that serve as starting points in the present work were developed using atomistic simulations of partial unfolding of the T-domain in acidic solution (Kurnikov, I. V.; et al. <i>J. Mol. Biol.</i> <b>2013</b>, <i>425</i>, 2752–2764), and coarse-grained simulations of the T-domain association with the membranes of various compositions (Flores-Canales, J. C.; et al. <i>J. Membr. Biol.</i> <b>2015</b>, <i>248</i>, 529–543). In this work we present atomistic level modeling of two distinct configurations of the T-domain in association with the anionic lipid bilayer. In microsecond-long MD simulations both conformations retain their compact structure and gradually penetrate deeper into the bilayer interface. One membrane-bound conformation is stabilized by the protein contacts with the lipid hydrophobic core. The second modeled conformation is initially inserted less deeply and forms multiple contacts with the lipid at the interface (headgroup) region. Such contacts are formed by the charged and hydrophilic groups of partially unfolded terminal helixes and loops. Neutralization of the acidic residues at the membrane interface allows for deeper insertion of the protein and reorientation of the protein at the membrane interface, which corroborates that acidic residue protonation as well as presence of the anionic lipids may play a role in the membrane association and further membrane insertion of the T-domain as implicated in experiments. All simulations reported in this work were performed using AMBER force-field on Anton supercomputer. To perform these reported simulations, we developed and carefully tested a force-field for the anionic 1-palmitoyl-2-oleoyl-phosphatidyl-glycerol (POPG) lipid, compatible with the Amber 99SB force-field and stable in microsecond-long MD simulations in isothermal–isobaric ensemble

Exploring Protein Stability by Comparative Molecular Dynamics Simulations of Homologous Hyperthermophilic, Mesophilic, and Psychrophilic Proteins

In the present studies, we analyzed the influence of temperature on the stability and dynamics of the α subunit of tryptophan synthase (TRPS) from hyperthermophilic, mesophilic, and psychrophilic homologues at different temperatures by molecular dynamics simulations. Employing different indicators such as root-mean-square deviations, root-mean-square fluctuations, principal component analysis, and free energy landscapes, this study manifests the diverse behavior of these homologues with changes in temperature. Especially, an enhancement in the collective motions, classified as representative motions, is observed at high temperature. Similarly, the criterion for the selection of electrostatic interactions in terms of their life span (duty cycle) has indeed helped in identifying the short- and long-lived electrostatic interactions and how they affect the protein’s overall stability at different temperatures. Rigidity and flexibility patterns of the homologous proteins are examined using FIRST software along with the calculation of duty cycles with various threshold limits at different temperatures. Rigid cluster decomposition in TRPS of psychrophilic, mesophilic, and hyperthermophilic origin identifies the flexible and rigid regions in the protein. Early loss of rigidity is observed in mesophilic TRPS via loss of contact between the major fragments of the protein compared with the other homologues. In spite of the high similarity of their three-dimensional structures, the overall responses of the three proteins to varying temperatures are significantly different

Targeting Electrostatic Interactions in Accelerated Molecular Dynamics with Application to Protein Partial Unfolding

Accelerated molecular dynamics (aMD) is a promising sampling method to generate an ensemble of conformations and to explore the free energy landscape of proteins in explicit solvent. Its success resides in its ability to reduce barriers in the dihedral and the total potential energy space. However, aMD simulations of large proteins can generate large fluctuations of the dihedral and total potential energy with little conformational changes in the protein structure. To facilitate wider conformational sampling of large proteins in explicit solvent, we developed a direct intrasolute electrostatic interactions accelerated MD (DISEI-aMD) approach. This method aims to reduce energy barriers within rapidly changing electrostatic interactions between solute atoms at short-range distances. It also results in improved reconstruction quality of the original statistical ensemble of the system. Recently, we characterized a pH-dependent partial unfolding of diphtheria toxin translocation domain (T-domain) using microsecond long MD simulations. In this work, we focus on the study of conformational changes of a low-pH T-domain model in explicit solvent using DISEI-aMD. On the basis of the simulations of the low-pH T-domain model, we show that the proposed sampling method accelerates conformational rearrangement significantly faster than multiple standard aMD simulations and microsecond long conventional MD simulations

Structural Basis for NHERF1 PDZ Domain Binding

The Na⁺/H⁺ exchange regulatory factor-1 (NHERF1) is a scaffolding protein that possesses two tandem PDZ domains and a carboxy-terminal ezrin-binding domain (EBD). The parathyroid hormone receptor (PTHR), type II sodium-dependent phosphate cotransporter (Npt2a), and β2-adrenergic receptor (β2-AR), through their respective carboxy-terminal PDZ-recognition motifs, individually interact with NHERF1 forming a complex with one of the PDZ domains. In the basal state, NHERF1 adopts a self-inhibited conformation, in which its carboxy-terminal PDZ ligand interacts with PDZ2. We applied molecular dynamics (MD) simulations to uncover the structural and biochemical basis for the binding selectivity of NHERF1 PDZ domains. PDZ1 uniquely forms several contacts not present in PDZ2 that further stabilize PDZ1 interactions with target ligands. The binding free energy (Δ<i>G</i>) of PDZ1 and PDZ2 with the carboxy-terminal, five-amino acid residues that form the PDZ-recognition motif of PTHR, Npt2a, and β2-AR was calculated and compared with the calculated Δ<i>G</i> for the self-association of NHERF1. The results suggest that the interaction of the PTHR, β2-adrenergic, and Npt2a involves competition between NHERF1 PDZ domains and the target proteins. The binding of PDZ2 with PTHR may also compete with the self-inhibited conformation of NHERF1, thereby contributing to the stabilization of an active NHERF1 conformation

Structural Basis for NHERF1 PDZ Domain Binding

The Na⁺/H⁺ exchange regulatory factor-1 (NHERF1) is a scaffolding protein that possesses two tandem PDZ domains and a carboxy-terminal ezrin-binding domain (EBD). The parathyroid hormone receptor (PTHR), type II sodium-dependent phosphate cotransporter (Npt2a), and β2-adrenergic receptor (β2-AR), through their respective carboxy-terminal PDZ-recognition motifs, individually interact with NHERF1 forming a complex with one of the PDZ domains. In the basal state, NHERF1 adopts a self-inhibited conformation, in which its carboxy-terminal PDZ ligand interacts with PDZ2. We applied molecular dynamics (MD) simulations to uncover the structural and biochemical basis for the binding selectivity of NHERF1 PDZ domains. PDZ1 uniquely forms several contacts not present in PDZ2 that further stabilize PDZ1 interactions with target ligands. The binding free energy (Δ<i>G</i>) of PDZ1 and PDZ2 with the carboxy-terminal, five-amino acid residues that form the PDZ-recognition motif of PTHR, Npt2a, and β2-AR was calculated and compared with the calculated Δ<i>G</i> for the self-association of NHERF1. The results suggest that the interaction of the PTHR, β2-adrenergic, and Npt2a involves competition between NHERF1 PDZ domains and the target proteins. The binding of PDZ2 with PTHR may also compete with the self-inhibited conformation of NHERF1, thereby contributing to the stabilization of an active NHERF1 conformation

Intra-/Intermolecular crosslinks identified by mass spectrometric studies of higher order oligomeric GlyBP bands.

<p>*the ΔMass is the maximum observed ppm difference between theoretical and observed m/z over N, the number of times this m/z peak was observed in 10 independent experiments. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102571#pone-0102571-g005" target="_blank"><b>Fig. 5</b></a> for corresponding assigned peak in representative MALDI-TOF spectrum.</p><p>Unique bands found in higher order oligomeric GlyBP bands (absent in monomeric bands) and assigned as intermolecular crosslinks are <i>italicized</i>, with <b><i>bold</i></b> indicating assignments that <b><i>cannot</i></b> be assigned as an intramolecular crosslinks.</p

Intramolecular crosslinks identified by mass spectrometric studies of monomeric GlyBP bands.

<p>*the ΔMass is the maximum observed ppm difference between theoretical and observed m/z over N, the number of times this m/z peak was observed in 10 independent experiments. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102571#pone-0102571-g004" target="_blank"><b>Fig. 4</b></a> for corresponding assigned peak in representative MALDI-TOF spectrum.</p

Intra-/inter-molecular crosslinks observed in GlyBP by MALDI-TOF MS analysis after crosslinking with DMS.

<p>Representative mass spectrum of tryptic digest of excised higher molecular weight GlyBP band is shown in the top panel. Mass peaks assigned as crosslinked peptides are labeled and further identified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102571#pone-0102571-t002" target="_blank"><b>Table 2</b></a>. Average C_α-C_α Lys-Lys distances measured along the MD trajectory of assigned crosslinks are provided in the panel below. The calculation of distances is averaged over all 5 subunits/interfaces over the 2 ns long MD trajectory. a and b indices distinguish adjacent GlyBP monomers in a pentamer. The positions of the Lys residues in two neighboring subunits of the GlyBP model are shown in bottom right. The protein structure is shown in grey and gold color in cartoon representation and C_α atoms of Lys residues are shown as colored spheres. * the range of distances reflect variations the average distance between subunits in the MD trajectory; while upper range distances are greater that the crosslinker length, the flexibility of the C loop in GlyBP brings the distances (underlined) well within the crosslinker arm length.</p

Model of GlyBP.

<p>A) Structure of a single GlyBP subunit is shown in a ribbon representation. Colors represent mobility of individual residues in MD simulations. Mobility is measured by root mean squared fluctuations (RMSF) with respect to an average structure obtained in a steady-state dynamics. The coloring scheme is as follows: RMSF <0.8 Å - blue, RMSF range 0.8–1.3 Å - green, 1.3–1.5 Å –yellow, 1.5–1.8 Å – orange, RMSF> 1.8 Å - red. B) Conformational diversity of subunits within the GlyBP pentamer is shown by a structural superposition of average monomer structures (last 2 ns of the trajectory) color coded by root mean squared deviation between subunits. Front (outer side) and back (inner side) are shown in the left and right panels, respectively.</p

Coverage map and CLUSTAL 2.1 multiple sequence alignment (after manual adjustments described in [24]) of L. stagnalis.

<p>A. californica, and the extracellular domain (ECD) of human glycine receptor alpha1 subunit. Loops 7 and 9 of GlyR ECD have been mutated to obtain GlyBP (grey highlights on the alignment). Sequence highlighted in red cumulatively marks peptides whose mass ions are detected in control studies. As described in the text, tryptic fingerprinting of GlyBP gel slices typically resulted in 55–80% coverage.</p