Combining Three-Dimensional Modeling with Artificial Intelligence to Increase Specificity and Precision in Peptide-MHC Binding Predictions.

The reliable prediction of the affinity of candidate peptides for the MHC is important for predicting their potential antigenicity and thus influences medical applications, such as decisions on their inclusion in T cell-based vaccines. In this study, we present a rapid, predictive computational approach that combines a popular, sequence-based artificial neural network method, NetMHCpan 4.0, with three-dimensional structural modeling. We find that the ensembles of bound peptide conformations generated by the programs MODELLER and Rosetta FlexPepDock are less variable in geometry for strong binders than for low-affinity peptides. In tests on 1271 peptide sequences for which the experimental dissociation constants of binding to the well-characterized murine MHC allele H-2Db are known, by applying thresholds for geometric fluctuations the structure-based approach in a standalone manner drastically improves the statistical specificity, reducing the number of false positives. Furthermore, filtering candidates generated with NetMHCpan 4.0 with the structure-based predictor led to an increase in the positive predictive value (PPV) of the peptides correctly predicted to bind very strongly (i.e., K d < 100 nM) from 40 to 52% (p = 0.027). The combined method also significantly improved the PPV when tested on five human alleles, including some with limited data for training. Overall, an average increase of 10% in the PPV was found over the standalone sequence-based method. The combined method should be useful in the rapid design of effective T cell-based vaccines.

Substrate-dependent transport mechanism in AcrB of multidrug resistant bacteria.

The multidrug resistance (MDR) system effectively expels antibiotics out of bacteria causing serious issues during bacterial infection. In addition to drug, indole, a common metabolic waste of bacteria, is expelled by MDR system of gram-negative bacteria for their survival. Experimental results suggest that AcrB, one of the key components of MDR system, undergoes large scale conformation changes during the pumping due to proton-motive process. However, due to extremely short time scale, it is difficult to observe (experimentally) those changes in the AcrB, which might facilitate the pumping process. Molecular simulations can shed light to understand the conformational changes for transport of indole in AcrB. Examination of conformational changes using all-atom simulation is, however, impractical. Here, we develop a hybrid coarse-grained force field to study the conformational changes of AcrB in presence of indole in the porter domain of monomer II. Using the coarse-grained force field, we investigated the conformational changes of AcrB for a number of model systems considering the effect of protonation in aspartic acid (Asp) residues Asp407 and Asp408 in the transmembrane domain of monomer II. Our results show that in the presence of indole, protonation of Asp408 or Asp407 residue causes conformational changes from binding state to extrusion state in monomer II, while remaining two monomers (I and III) approach access state in AcrB protein. We also observed that all three AcrB monomers prefer to go back to access state in the absence of indole. Steered molecular dynamics simulations were performed to demonstrate the feasibility of indole transport mechanism for protonated systems. Identification of indole transport pathway through AcrB can be very helpful in understanding the drug efflux mechanism used by the MDR bacteria.

Coarse-grained simulations of conformational changes in the multidrug efflux transporter AcrB.

The multidrug resistance (MDR) system actively pumps antibiotics out of cells causing serious health problems. During the pumping, AcrB (one of the key components of MDR) undergoes a series of large-scale and proton-motive conformational changes. Capturing the conformational changes through all-atom simulations is challenging. Here, we implement a hybrid coarse-grained force field to investigate the conformational changes of AcrB in the porter domain under different protonation states of Asp407/Asp408 in the trans-membrane domain. Our results show that protonation of Asp408 in monomer III (extrusion) stabilizes the asymmetric structure of AcrB; deprotonation of Asp408 induces clear opening of the entrance and closing of the exit leading to the transition from extrusion to access state. The structural changes in the porter domain of AcrB are strongly coupled with the proton translocation stoichiometry in the trans-membrane domain. Moreover, our simulations support the postulation that AcrB should adopt the symmetric resting state in a substrate-free situation.

Exploration of conformational changes in lactose permease upon sugar binding and proton transfer through coarse-grained simulations.

Escherichia coli lactose permease (LacY) actively transports lactose and other galactosides across cell membranes through lactose/H+ symport process. Lactose/H+ symport is a highly complex process that involves sugar translocation, H+ transfer, and large-scale protein conformational changes. The complete picture of lactose/H+ symport is largely unclear due to the complexity and multiscale nature of the process. In this work, we develop the force field for sugar molecules compatible with PACE, a hybrid and coarse-grained force field that couples the united-atom protein models with the coarse-grained MARTINI water/lipid. After validation, we implement the new force field to investigate the binding of a ß-d-galactopyranosyl-1-thio- ß-d-galactopyranoside (TDG) molecule to a wild-type LacY. Results show that the local interactions between TDG and LacY at the binding pocket are consistent with the X-ray experiment. Transitions from inward-facing to outward-facing conformations upon TDG binding and protonation of Glu269 have been achieved from ∼5.5 µs simulations. Both the opening of the periplasmic side and closure of the cytoplasmic side of LacY are consistent with double electron-electron resonance and thiol cross-linking experiments. Our analysis suggests that the conformational changes of LacY are a cumulative consequence of interdomain H-bonds breaking at the periplasmic side, interdomain salt-bridge formation at the cytoplasmic side, and the TDG orientational changes during the transition.

Decoupled Ion Transport in a Protein-Based Solid Ion Conductor.

Simultaneous achievement of good electrochemical and mechanical properties is crucial for practical applications of solid ion conductors. Conventional polymer conductors suffer from low conductivity, low transference number, and deteriorated mechanical properties with the enhancement of conductivity, resulting from the coupling between ion transport and polymer movement. Here we present a successful fabrication and fundamental understanding of a high performance soy protein-based solid conductor. The conductor shows ionic conductivity of ∼10-5 S/cm, transference number of 0.94, and modulus of 1 GPa at room temperature, and still remains flexible and easily processable. Molecular simulations indicate that this is due to appropriate manipulation of the protein structures for effective exploitation of protein functional groups. A decoupled transport mechanism, which is able to explain all results, is proposed. The new insights can be utilized to provide guidelines for design, optimization, and fabrication of high performance biosolid conductors.

Coarse-grained simulations of proton-dependent conformational changes in lactose permease.

During lactose/H(+) symport, the Escherichia coli lactose permease (LacY) undergoes a series of global conformational transitions between inward-facing (open to cytoplasmic side) and outward-facing (open to periplasmic side) states. However, the exact local interactions and molecular mechanisms dictating those large-scale structural changes are not well understood. All-atom molecular dynamics simulations have been performed to investigate the molecular interactions involved in conformational transitions of LacY, but the simulations can only explore early or partial global structural changes because of the computational limits (< 100 ns). In this work, we implement a hybrid force field that couples the united-atom protein models with the coarse-grained MARTINI water/lipid, to investigate the proton-dependent dynamics and conformational changes of LacY. The effects of the protonation states on two key glutamate residues (Glu325 and Glu269) have been studied. Our results on the salt-bridge dynamics agreed with all-atom simulations at early short time period, validating our simulations. From our microsecond simulations, we were able to observe the complete transition from inward-facing to outward-facing conformations of LacY. Our results showed that all helices have participated during the global conformational transitions and helical movements of LacY. The inter-helical distances measured in our simulations were consistent with the double electron-electron resonance experiments at both cytoplasmic and periplasmic sides. Our simulations indicated that the deprotonation of Glu325 induced the opening of the periplasmics side and partial closure of the cytoplasmic side of LacY, while protonation of the Glu269 caused a stable cross-domain salt-bridge (Glu130-Arg344) and completely closed the cytoplasmic side. Proteins 2016; 84:1067-1074. © 2016 Wiley Periodicals, Inc.