Molecular Modeling Reveals the Novel Inhibition Mechanism and Binding Mode of Three Natural Compounds to Staphylococcal α-Hemolysin

et al. (2013) Molecular Modeling Reveals the Novel Inhibition Mechanism and Binding Mode of Three Natural Compounds to Staphylococcal a-Hemolysin. PLoS ONE 8(11): e80197. doi:10.1371/journal.pone.0080197 Molecular Modeling Reveals the Novel Inhibition Mechanism and Binding Mode of Three Natural Compounds to Staphylococcal a-Hemolysin Jiazhang Qiu 0 Dacheng Wang 0 Yu Zhang 0 Jing Dong 0 Jianfeng Wang 0 Xiaodi Niu 0 Jie Zheng, University of Akron, United States of America 0 1 Department of Food Quality and Safety, Jilin University , Changchun , China , 2 Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University , Changchun , China a-Hemolysin (a-HL) is a self-assembling, channel-forming toxin that is produced as a soluble monomer by Staphylococcus aureus strains. Until now, a-HL has been a significant virulence target for the treatment of S. aureus infection. In our previous report, we demonstrated that some natural compounds could bind to a-HL. Due to the binding of those compounds, the conformational transition of a-HL from the monomer to the oligomer was blocked, which resulted in inhibition of the hemolytic activity of a-HL. However, these results have not indicated how the binding of the a-HL inhibitors influence the conformational transition of the whole protein during the oligomerization process. In this study, we found that three natural compounds, Oroxylin A 7-O-glucuronide (OLG), Oroxin A (ORA), and Oroxin B (ORB), when inhibiting the hemolytic activity of a-HL, could bind to the ''stem'' region of a-HL. This was completed using conventional Molecular Dynamics (MD) simulations. By interacting with the novel binding sites of a-HL, the ligands could form strong interactions with both sides of the binding cavity. The results of the principal component analysis (PCA) indicated that because of the inhibitors that bind to the ''stem'' region of a-HL, the conformational transition of a-HL from the monomer to the oligomer was restricted. This caused the inhibition of the hemolytic activity of a-HL. This novel inhibition mechanism has been confirmed by both the steered MD simulations and the experimental data obtained from a deoxycholate-induced oligomerization assay. This study can facilitate the design of new antibacterial drugs against S. aureus. - . These authors contributed equally to this work. Staphylococcus aureus is a significant human pathogen that is capable of causing a multitude of infections, many of which are life-threatening, such as toxic shock syndrome, bacteremia, endocarditis, sepsis, and pneumonia [1]. Since 1960, methicillinresistant S. aureus (MRSA) has been a worldwide challenge with limited therapeutic options for treatment [2]. For example, a 2005 survey indicated that over 18,000 deaths could be attributed to invasive MRSA infection in the United States alone [3]. Alphahemolysin is one of the major toxins endowed with hemolytic, cytotoxic, dermonecrotic, and lethal properties [4]. Upon binding to susceptible cell membranes, a-hemolysin monomers penetrate the plasma membrane to form cylindrical heptameric pores with a diameter of approximately 2 nm [5]. These pores result in cytoplasmic leaking and osmotic swelling, which ultimately leads to cell damage and death. Several lines of evidence validate a-hemolysin as a significant virulence target for the treatment of S. aureus infection: i) most S. aureus strains encode hla (the gene encoding alpha-hemolysin) [4]; ii) it is not essential for the survival of S. aureus; iii) a-hemolysin is a critical virulence factor that determines the severity of S. aureus infections when measured in mouse models [69]; and iiii) active or passive immunization with a-hemolysin mutant protein (H35L), anti-a-hemolysin antibody, and chemicals (b-cyclodextrin derivative) that block the heptameric pore, genetically disrupt disintegrin and metalloprotease 10 (the cellular receptor of a-hemolysin), and have shown significant protection against S. aureus infections [10 13]. Furthermore, our previous study demonstrated that some compounds could significantly reduce the mortality and tissue damage of S. aureus pneumonia in a mouse model by preventing the self-assembly of the a-hemolysin heptamer [1416]. Molecular dynamics (MD) [1719] is a useful computational tool that can offer insight into specific molecular interactions between proteins and inhibitors at the atomic level. For example, in our previous reports, we demonstrated that baicalin, a natural compound, could bind to the binding sites of Y148, P151 and F153 in a-hemolysin (a-HL) using Molecular Dynamics (MD) simulations and mutagenesis assays [14]. This binding interaction inhibits heptamer formation. In addition, through Molecular Dynamics (MD) simulations and free energy calculations, we confirmed that oroxylin A (ORO) and cyrtominetin (CTM) could inhibit the hemolytic activity of a-hemolysin (a-HL) by binding with the Loop region of a-hemolysin (a-HL), which is different from baicalin [15,16]. Because of the binding of ORO and CTM, the conformational transition of the critical Loop region from the monomeric a-HL to the oligomer was blocked. This resulted in inhibition of the hemolytic activity of the protein. In our study, we found that three natural compounds, Oroxylin A 7-O-glucuronide (OLG), Oroxin A (ORA) and Oroxin B (ORB), which have similar structures, can suppress the hemolytic activity of a-HL at very low concentrations. The structures are different from our previously identified compounds (e.g. Baicalin and cyrtominetin) that can block the self-assembly of a-HL heptamer [14,16]. Thus, it is reasonable to speculate that the binding sites and binding modes of Oroxylin A 7-O-glucuronide (OLG), oroxin A (ORA) and oroxin B (ORB) would be different from baicalin or cyrtominetin. In this paper, the mechanisms of these compounds on inhibiting the hemolytic activity of a-HL were investigated, this would benefit for our understanding on drug discovery that targets staphylococcal a-HL. To explore the inhibition mechanism at the new binding sites of a-HL, we have performed Ligand-residue interaction decomposition and mutagenesis assays of three of the a-HL-inhibitor complexes in an attempt to identify specific residues that are important to the binding of a-HL inhibitors. A principle component analysis (PCA) was performed to address the collective motions of free protein and complexes. Based on the principle component analysis (PCA) simulations, the motion modes of the free protein were compared with those of the complexes, which led to the conclusion that the binding of the inhibitors hides the motion of the a-HL from the monomer to the oligomer. This inhibition activity mechanism is confirmed by the relative binding free energies calculated for the complexes based on performed potential of mean force (PMF) and the available experimental data obtained from a deoxycholateinduced oligomeriaza (...truncated)