pH-Induced conformational switching is essential for functioning of diphtheria toxin, which undergoes a membrane insertion/translocation transition triggered by endosomal acidification as a key step of cellular entry. In order to establish the sequence of molecular rearrangements and side chain protonation accompanying the formation of the membrane-competent state of the toxin’s translocation (T) domain, we have developed and applied an integrated approach that combines multiple techniques of computational chemistry (e.g., long, µsec-range, all-atom molecular dynamics simulations; continuum electrostatics calculations; and thermodynamic integration) with several experimental techniques of fluorescence spectroscopy. Thermodynamic integration calculations indicate that protonation of H257 causes the greatest destabilization of the native structure (6.9 kcal/mole), which is consistent with our early mutagenesis results. Extensive equilibrium MD simulations with a combined length of over eight µsec demonstrate that histidine protonation, while not accompanied by the loss of structural compactness of the T-domain, nevertheless results in substantial molecular rearrangements characterized by the partial loss of secondary structure due to unfolding of helices TH1 and TH2, and the loss of close contact between the C- and N-terminal segments. The structural changes accompanying the formation of the membrane-competent state ensure an easier exposure of the internal hydrophobic hairpin formed by helices TH8 and TH9, in preparation for its subsequent transmembrane insertion.

Diphtheria toxin translocation (T) domain inserts in lipid bilayers upon acidification of the environment. Computational and experimental studies have suggested that low pH triggers a conformational change of the T-domain in solution preceding membrane binding. The refolded membrane-competent state was modeled to be compact and mostly retain globular structure. In the present work, we investigate how this refolded state interacts with membrane interfaces in the early steps of T-domain’s membrane association. Coarse-grained molecular dynamics (CG-MD) simulations suggest two distinct membrane-bound conformations of the T-domain in the presence of bilayers composed of a mixture of zwitteronic and anionic phospholipids (POPC:POPG with a 1:3 molar ratio). Both membrane-bound conformations show a common near parallel orientation of hydrophobic helices TH8-TH9 relative to the membrane plane. The most frequently observed membrane-bound conformation is stabilized by electrostatic interactions between the N-terminal segment of the protein and the membrane interface. The second membrane-bound conformation is stabilized by hydrophobic interactions between protein residues and lipid acyl chains, which facilitate deeper protein insertion in the membrane interface. A theoretical estimate of a free energy of binding of a membrane-competent T-domain to the membrane is provided.

Sugars are essential sources of energy and carbon, and also function as key signaling molecules in plants. Sugar Transport Proteins (STP) are proton-coupled symporters, responsible for uptake of glucose from the apoplast into plant cells. They are integral to organ development in symplastically isolated tissues such as seed, pollen and fruit. Additionally, STPs play a significant role in plant responses to stressors such as dehydration, and prevalent fungal infections like rust and mildew. Here we present a structure of Arabidopsis thaliana STP10 in the inward open conformation at 2.6 Å resolution and a structure of the outward occluded conformation at improved 1.8 Å resolution, both with glucose and protons bound. The two structures describe key states in the STP transport cycle. Together with Molecular Dynamics simulations that establish protonation states and biochemical analysis, they pinpoint structural elements, conserved in all STPs, that clarify the basis of proton-to-glucose coupling. The results advance our understanding of monosaccharide uptake, essential for plant organ development, and sets the stage for bioengineering strategies in crops.

The pH-triggered membrane insertion of the diphtheria toxin translocation domain (T domain) results in transferring the catalytic domain into the cytosol, which is relevant to potential biomedical applications as a cargo-delivery system. Protonation of residues is suggested to play a key role in the process, and residues E349, D352 and E362 are of particular interest because of their location within the membrane insertion unit TH8–TH9. We have used various spectroscopic, computational and functional assays to characterize the properties of the T domain carrying the double mutation E349Q/D352N or the single mutation E362Q. Vesicle leakage measurements indicate that both mutants interact with the membrane under less acidic conditions than the wild-type. Thermal unfolding and fluorescence measurements, complemented with molecular dynamics simulations, suggest that the mutant E362Q is more susceptible to acid destabilization because of disruption of native intramolecular contacts. Fluorescence experiments show that removal of the charge in E362Q, and not in E349Q/D352N, is important for insertion of TH8–TH9. Both mutants adopt a final functional state upon further acidification. We conclude that these acidic residues are involved in the pH-dependent action of the T domain, and their replacements can be used for fine tuning the pH range of membrane interactions.

For protein structure modeling in the CASP12 experiment, we have developed a new protocol based on our previous CASP11 approach. The global optimization method of conformational space annealing (CSA) was applied to 3 stages of modeling: multiple sequence-structure alignment, three-dimensional (3D) chain building, and side-chain re-modeling. For better template selection and model selection, we updated our model quality assessment (QA) method with the newly developed SVMQA (support vector machine for quality assessment). For 3D chain building, we updated our energy function by including restraints generated from predicted residue-residue contacts. New energy terms for the predicted secondary structure and predicted solvent accessible surface area were also introduced. For difficult targets, we proposed a new method, LEEab, where the template term played a less significant role than it did in LEE, complemented by increased contributions from other terms such as the predicted contact term. For TBM (template-based modeling) targets, LEE performed better than LEEab, but for FM targets, LEEab was better. For model refinement, we modified our CASP11 molecular dynamics (MD) based protocol by using explicit solvents and tuning down restraint weights. Refinement results from MD simulations that used a new augmented statistical energy term in the force field were quite promising. Finally, when using inaccurate information (such as the predicted contacts), it was important to use the Lorentzian function for which the maximal penalty arising from wrong information is always bounded.

Solution acidity measured by pH is an important environmental factor that affects protein structure. It influences the protonation state of protein residues, which in turn may be coupled to protein conformational changes, unfolding, and ligand binding. It remains difficult to compute and measure the pK a of individual residues, as well as to relate them to pH-dependent protein transitions. This paper presents a hierarchical approach to compute the pK a of individual protonatable residues, specifically histidines, coupled with underlying structural changes of a protein. A fast and efficient free energy perturbation (FEP) algorithm has also been developed utilizing a fast implementation of standard molecular dynamics (MD) algorithms. Specifically, a CUDA version of the AMBER MD engine is used in this paper. Eight histidine pK a’s are computed in a diverse set of pH stable proteins to demonstrate the proposed approach’s utility and assess the predictive quality of the AMBER FF99SB force field. A reference molecule is carefully selected and tested for convergence. A hierarchical approach is used to model pK a’s of the six histidine residues of the diphtheria toxin translocation domain (DTT), which exhibits a diverse ensemble of individual conformations and pH-dependent unfolding. The hierarchical approach consists of first sampling equilibrium conformational ensembles of a protein with protonated and neutral histidine residues via long equilibrium MD simulations (Flores-Canales, J. C.; et al. bioRxiv, 2019, 572040). A clustering method is then used to identify sampled protein conformations, and pK a’s of histidines in each protein conformation are computed. Finally, an ensemble averaging formalism is developed to compute weighted average histidine pK a’s. These can be compared with an apparent experimentally measured pK a of the DTT protein and thus allows us to propose a mechanism of pH-dependent unfolding of the DTT protein.

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.

Diphtheria toxin is a multi-domain protein that invades cells by using their own endocytosis mechanism. In endocytosis, an endosome, a lipid bilayer vesicle, is formed to encapsulate an extracellular molecule. Subsequent acidification of endosome internal solution induces conformational rearrangements and membrane insertion of such encapsulated diphtheria toxin translocation domain (T-domain). In solution at neutral pH, a stand-alone T-domain adopts an all alpha-helical globular structure; however, atomistic details of the pH-dependent conformational changes of the protein are not completely understood. We model structural rearrangements in T-domain in 18 µs long molecular dynamics (MD) simulations of neutral and low pH T-domain models in explicit solvent. At low pH, six histidine residues of the protein were protonated. Two independent MD trajectories resulted in partial protein unfolding at low pH, in which similar regions of the protein conformational subspace were explored. Notably, a pH induced unfolding transition was initiated by partial unfolding of helix TH4 followed by unfolding of helix TH1. Helix TH2 repeatedly unfolds in the low pH T-domain model, which is consequently predicted to be disordered by a consensus of disorder prediction algorithms.Protonation of histidines disrupted a hydrophobic core containing a putative transmembrane helix TH8, which is encircled by hydrophobic surfaces of helices TH3, TH5 and TH9. Afterwards, the low pH T-domain model was reorganized into an ensemble of partially unfolded structures with increased solvent exposure of hydrophobic and charged sites. Thus, MD simulations suggest the destabilizing role of protonation of histidines, in the neutral pH conformation in solution, which may facilitate the initial stages of T-domain membrane binding.The simulation at neutral pH samples conformations in the vicinity of the native structure of the