Art E. Cho

Extension of QM/MM docking and its applications to metalloproteins

To overcome the limitation of conventional docking methods which assume fixed charge model from force field parameters, combined quantum mechanics/molecular mechanics (QM/MM) method has been applied to docking as a variable charge model and shown to exhibit improvement on the docking accuracy over fixed charge based methods. However, it has also been shown that there are a number of examples for which adoption of variable‐charge model fails to reproduce the native binding modes. In particular, for metalloproteins, previously implemented method of QM/MM docking failed most often. This class of proteins has highly polarized binding sites at which high‐coordinate‐numbered metal ions reside. We extend the QM/MM docking method so that protein atoms surrounding the binding site along with metal ions are included as quantum region, as opposed to only ligand atoms. This extension facilitates the required scaling of partial charges on metal ions leading to prediction of correct binding modes in metalloproteins.

Correlation between Performance of QM/MM Docking and Simple Classification of Binding Sites

Use of SiteMap for binding site classification and its connection to QM/MM (quantum mechanics/ molecular mechanics) docking performance were investigated. Using the hydrophilic/hydrophobic character values along with balance between them which SiteMap calculates, we sorted 455 cocrystal complexes available from protein data bank into three groups and tested how Glide, a conventional docking program, and QPLD, a QM/MM docking program, perform on them. QPLD showed improvements on all three groups over Glide but with varying degrees. Analysis of the results was carried out, and establishment of correlations between the classification of binding sites and QM/MM docking performance was attempted. It was found that QM/MM docking delivers the most improvements for primarily hydrophobic binding sites with substantial hydrophilic interactions. Based on our findings, we make suggestions for use of QM/MM docking and directions for further developments.

Influence of ionic liquid and ionic salt on protein against the reactive species generated using dielectric barrier discharge plasma

The presence of salts in biological solution can affect the activity of the reactive species (RS) generated by plasma, and so they can also have an influence on the plasma-induced sterilization. In this work, we assess the influence that diethylammonium dihydrogen phosphate (DEAP), an ionic liquid (IL), and sodium chloride (NaCl), an ionic salt (IS), have on the structural changes in hemoglobin (Hb) in the presence of RS generated using dielectric barrier discharge (DBD) plasma in the presence of various gases [O2, N2, Ar, He, NO (10%) + N2 and Air]. We carry out fluorescence spectroscopy to verify the generation of •OH with or without the presence of DEAP IL and IS, and we use electron spin resonance (ESR) to check the generation of H• and •OH. In addition, we verified the structural changes in the Hb structure after treatment with DBD in presence and absence of IL and IS. We then assessed the structural stability of the Hb in the presence of IL and IS by using molecular dynamic (MD) simulations. Our results indicate that the IL has a strong effect on the conservation of the Hb structure relative to that of IS against RS generated by plasma.

Quantum mechanical scoring for protein docking

We develop a docking protocol based on quantum mechanical/molecular mechanical calculations in which quantum mechanical energy is used as scoring. We test the protocol with three groups of examples with various binding site characteristics. The new docking method performs as well as or better than conventional docking methods in all three groups. In particular, for proteins with primarily hydrophobic binding sites, structural motifs with possible pi-pi interactions are often found and it is shown that these can be better modeled with quantum mechanical scoring docking than force field based methods. It seems that the new method performs in such cases to a great accuracy.

Molecular Insights into the Adsorption Mechanism of Human β-Defensin-3 on Bacterial Membranes.

Human β-defensin-3 (hBD3) is an endogenous antimicrobial peptide that exhibits broad-spectrum antibacterial activity without eukaryotic cytotoxicity. In this work, we carried out molecular dynamics (MD) simulations to explore its adsorption mechanism on, and the structural and thermodynamic contributions of individual residues to its antibacterial activity with both Gram-negative (GN) and Gram-positive (GP) bacterial membrane. Due to the strong electrostatic interaction of hBD3 with POPG lipids, which are more prevalent on the GP membrane, its adhesion to the GP membrane is stronger than to the GN membrane and stabilized more rapidly. On the surface of both bacterial membranes, the orientation of hBD3 is dominated by an electric dipole. We next analyzed the binding free energy decompositions of the hBD3-membrane complex using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method. The results of both the GN and the GP membrane simulations show that Arg17, Arg36, and Arg38 form both polar and nonpolar interactions and are potentially the key residues for hBD3 antibacterial activity. On the other hand, there was a significant difference in the energy contribution of Arg12 between the GP and GN membrane simulations, suggesting that Arg12 is a key factor in the toxicity of hBD3 to specifically GP bacteria. Our findings shed light on the antibacterial activity of hBD3 on bacterial membranes and yield insights useful for the design of potent antimicrobial peptides targeting multidrug resistant bacteria.

Variation in structure of proteins by adjusting reactive oxygen and nitrogen species generated from dielectric barrier discharge jet

Over the last few years, the variation in liquid chemistry due to the development of radicals generated by cold atmospheric plasma (CAP) has played an important role in plasma medicine. CAP direct treatment or CAP activated media treatment in cancer cells shows promising anticancer activity for both in vivo and in vitro studies. However, the anticancer activity or antimicrobial activity varies between plasma devices due to the different abilities among plasma devices to generate the reactive oxygen and nitrogen species (RONS) at different ratios and in different concentrations. While the generation of RONS depends on many factors, the feeding gas plays the most important role among the factors. Hence, in this study we used different compositions of feeding gas while fixing all other plasma characteristics. We used Ar, Ar-O2 (at different ratios), and Ar-N2 (at different ratios) as the working gases for CAP and investigated the structural changes in proteins (Hemoglobin (Hb) and Myoglobin (Mb)). We then analyzed the influence of RONS generated in liquid on the conformations of proteins. Additionally, to determine the influence of H2O2 on the Hb and Mb structures, we used molecular dynamic simulation.

Re-engineering the Immune Response to Metastatic Cancer: Antibody-Recruiting Small Molecules Targeting the Urokinase Receptor

Developing selective strategies to treat metastatic cancers remains a significant challenge. Herein, we report the first antibody-recruiting small molecule (ARM) that is capable of recognizing the urokinase-type plasminogen activator receptor (uPAR), a uniquely overexpressed cancer cell-surface marker, and facilitating the immune-mediated destruction of cancer cells. A co-crystal structure of the ARM-U2/uPAR complex was obtained, representing the first crystal structure of uPAR complexed with a non-peptide ligand. Finally, we demonstrated that ARM-U2 substantially suppresses tumor growth in vivo with no evidence of weight loss, unlike the standard-of-care agent doxorubicin. This work underscores the promise of antibody-recruiting molecules as immunotherapeutics for treating cancer.

Structural Basis of Novel Iron-Uptake Route and Reaction Intermediates in Ferritins from Gram-Negative Bacteria.

Iron and oxygen chemistry is mediated by iron proteins for many biological functions. Carboxylate-bridged diiron enzymes including ferritin have the common mechanism of oxygen activation via peroxodiferric intermediates. However, the route for iron uptake and the structural identification of intermediates still remain incomplete. The 4-fold symmetry channel of Helicobacter pylori ferritin was previously proposed as the iron-uptake route in eubacteria, but the amino acid residues at the 4-fold channel are not highly conserved. Here, we show evidence for a short path for iron uptake from His93 on the surface to the ferroxidase center in H. pylori ferritin and Escherichia coli ferritin. The amino acid residues along this path are highly conserved in Gram-negative bacteria and some archaea, and the mutants containing S20A and H93L showed significantly decreased iron oxidation. Surprisingly, the E. coli ferritin S20A crystal structure showed oxygen binding and side-on, symmetric μ-η2:η2 peroxodiferric and oxodiferric intermediates. The results provide the structural basis for understanding the chemical nature of intermediates in iron oxidation in bacteria and some of archaea.

Incorporating QM and solvation into docking for applications to GPCR targets

A great number of GPCR crystal structures have been solved in recent years, enabling GPCR-targeted drug discovery using structure-based approaches such as docking. GPCRs generally have wide and open entrances to the binding sites, which render the binding sites readily accessible to solvent. GPCRs are also populated with hydrophilic residues in the extracellular regions. Thus, including solvent and polarization effects can be important for accurate GPCR docking. To test this hypothesis, a new docking protocol which incorporates quantum mechanical/molecular mechanical (QM/MM) calculations along with an implicit solvent model is developed. The new docking method treats the ligands and the protein residues in the binding sites as QM regions and performs QM/MM calculations with implicit solvent. The results of a test on all solved GPCR cocrystals show a significant improvement over the conventional docking method.

Solution structure of the transmembrane 2 domain of the human melanocortin-4 receptor in sodium dodecyl sulfate (SDS) micelles and the functional implication of the D90N mutant

The melanocortin receptors (MCRs) are members of the G protein-coupled receptor (GPCR) 1 superfamily with seven transmembrane (TM) domains. Among them, the melanocortin-4 receptor (MC4R) subtype has been highlighted recently by genetic studies in obese humans. In particular, in a patient with severe early-onset obesity, a novel heterozygous mutation in the MC4R gene was found in an exchange of Asp to Asn in the 90th amino acid residue located in the TM 2 domain (MC4RD90N). Mutations in the MC4R gene are the most frequent monogenic causes of severe obesity and are described as heterozygous with loss of function. We determine solution structures of the TM 2 domain of MC4R (MC4RTM2) and compared secondary structure of Asp90 mutant (MC4RTM2-D90N) in a micelle environment by nuclear magnetic resonance (NMR) spectroscopy. NMR structure shows that MC4RTM2 forms a long α-helix with a kink at Gly98. Interestingly, the structure of MC4RTM2-D90N is similar to that of MC4RTM2 based on data from CD and NMR spectrum. However, the thermal stability and homogeneity of MC4RD90N is quite different from those of MC4R. The structure from molecular modeling suggests that Asp90(2.50) plays a key role in allosteric sodium ion binding. Our data suggest that the sodium ion interaction of Asp90(2.50) in the allosteric pocket of MC4R is essential to its function, explaining the loss of function of the MC4RD90N mutant.