Structural Insights into Substrate Recognition and Product Expulsion in CTX-M Enzymes

β-Lactamase-mediated resistance to β-lactam antibiotics poses a major threat to our antibiotic armamentarium. Among β-lactamases, a significant threat comes from enzymes that hydrolyze extended-spectrum cephalosporins such as cefotaxime. Among the enzymes that exhibit this phenotype, the CTX-M family is found worldwide. These enzymes have a small active site, which makes it difficult to explain how they hydrolyze the bulky extended-spectrum cephalosporins into the binding site. We investigated noncovalent substrate recognition and product release in CTX-M enzymes using steered molecular dynamics simulation and X-ray diffraction. An arginine residue located far from the binding site favors the capture and tracking of substrates during entrance into the catalytic pocket. We show that the accommodation of extended-spectrum cephalosporins by CTX-M enzymes induced subtle changes in the active site and established a high density of electrostatic interactions. Interestingly, the product of the catalytic reaction initiates its own release because of steric hindrances and electrostatic repulsions. This suggests that there exists a general mechanism for product release for all members of the β-lactamase family and probably for most carboxypeptidases.     

Mechanical unbinding of abeta peptides from amyloid fibrils

Using the experimental structures of Abeta amyloid fibrils and all-atom molecular dynamics, we study the force-induced unbinding of Abeta peptides from the fibril. We show that the mechanical dissociation of Abeta peptides is highly anisotropic and proceeds via different pathways when force is applied in parallel or perpendicular direction with respect to the fibril axis. The threshold forces associated with lateral unbinding of Abeta peptides exceed those observed during the mechanical dissociation along the fibril axis. In addition, Abeta fibrils are found to be brittle in the lateral direction of unbinding and soft along the fibril axis. Lateral mechanical unbinding and the unbinding along the fibril axis load different types of fibril interactions. Lateral unbinding is primarily determined by the cooperative rupture of fibril backbone hydrogen bonds. The unbinding along the fibril axis largely depends on the interpeptide Lys-Asp electrostatic contacts and the hydrophobic interactions formed by the Abeta C terminal. Due to universality of the amyloid beta structure, the anisotropic mechanical dissociation observed for Abeta fibrils is likely to be applicable to other amyloid assemblies. The estimates of equilibrium forces required to dissociate Abeta peptide from the amyloid fibril suggest that these supramolecular structures are mechanically stronger than most protein domains.     

生物单分子研究的进展

大多数分子生物学实验都代表一种集体平均的测量,所记录的都是复杂系统中大量分子的行为,随着技术的发展,目前已能够对单分子观察、探测、操纵并研究其动态与构象变化。从单分子研究可得到新的信息,这些信息隐藏在集体测量之中,或已被平均掉。这一领域代表着本世纪结构生物学的一个新方向。     

Towards optimal packing and diffusion of fullerene molecules in the Pc-PBBA covalent organic framework

We studied the adsorption and diffusion of the prototypical n-type semiconducting fullerene molecule, C₆₀, inside the pores of the p-type semiconductor, phthalocyanine phenylene-bis(boronic acid) (Pc-PBBA), a member of the class of two-dimensional covalent organic framework (COF) materials. This C₆₀/Pc-PBBA system is an example of an ordered p–n heterojunction suitable for photovoltaic solar cell applications. We found that the small 1.7 Å lateral offset present between COF layers leads to discrete adsorption sites inside the pore, forming essentially a periodic ‘lattice’ on which the fullerene diffuses. By categorising the location of such lattice sites, we found the maximum theoretical packing density of fullerene in representative helical, zigzag and staircase Pc-PBBA stacks, as well as an average packing density in randomly stacked Pc-PBBA layers. All these simulated packing densities (51% of bulk) closely matched related experimental results by Dogru et al. [M. Dogru, M. Handloser, F. Auras, T. Kunz, D. Medina, A. Hartschuh, P. Knochel, T. Bein. A photoconductive thienothiophene-based covalent organic framework showing charge transfer towards included fullerene. Angew Chem Int Ed. 2013;52:2920–2924]. (49% of bulk) for a similar fullerene–COF system, [6,6]-phenyl-C₆₁-butyric acid methyl ester/thienothiophene–COF, which produced a low power conversion efficiency. This shows that there is little improvement to be made to the electron transport in terms of fullerene packing density. We present values of the barriers for all diffusion pathways between neighbouring lattice sites categorised uniquely by a set of symmetry rules. Knowledge of these barriers allows us to generalise fullerene transport mechanisms within the Pc-PBBA pore. We observe that diffusion along the (vertical) pore axis is faster than (lateral) diffusion perpendicular to the pore axis; this will facilitate pore filling.     

Neuraminidase inhibitor R-125489--a promising drug for treating influenza virus: steered molecular dynamics approach

Two neuraminidase inhibitors, oseltamivir and zanamivir, are important drug treatments for influenza. Oseltamivir-resistant mutants of the influenza virus A/H1N1 and A/H5N1 have emerged, necessitating the development of new long-acting antiviral agents. One such agent is a new neuraminidase inhibitor R-125489 and its prodrug CS-8958. An atomic level understanding of the nature of this antiviral agents binding is still missing. We address this gap in our knowledge by applying steered molecular dynamics (SMD) simulations to different subtypes of seasonal and highly pathogenic influenza viruses. We show that, in agreement with experiments, R-125489 binds to neuraminidase more tightly than CS-8958. Based on results obtained by SMD and the molecular mechanics-Poisson–Boltzmann surface area method, we predict that R-125489 can be used to treat not only wild-type but also tamiflu-resistant N294S, H274Y variants of A/H5N1 virus as its binding affinity does not vary much across these systems. The high correlation level between theoretically determined rupture forces and experimental data on binding energies for the large number of systems studied here implies that SMD is a promising tool for drug design.     

Donor-strand exchange in chaperone-assisted pilus assembly revealed in atomic detail by molecular dynamics

Adhesive multi-subunit fibres are assembled on the surface of many pathogenic bacteria via the chaperone-usher pathway. In the periplasm, a chaperone donates a β-strand to a pilus subunit to complement its incomplete immunoglobulin-like fold. At the outer membrane, this is replaced with a β-strand formed from the N-terminal extension (Nte) of an incoming pilus subunit by a donor-strand exchange (DSE) mechanism. This reaction has previously been shown to proceed via a concerted mechanism, in which the Nte interacts with the chaperone:subunit complex before the chaperone has been displaced, forming a ternary intermediate. Thereafter, the pilus and chaperone β-strands have been postulated to undergo a strand swap by a ‘zip-in-zip-out’ mechanism, whereby the chaperone strand zips out, residue by residue, as the Nte simultaneously zips in, although direct experimental evidence for a zippering mechanism is still lacking. Here, molecular dynamics simulations have been used to probe the DSE mechanism during formation of the Saf pilus from Salmonella enterica at the atomic level, allowing the direct investigation of the zip-in-zip-out hypothesis. The simulations provide an explanation of how the incoming Nte is able to dock and initiate DSE due to inherent dynamic fluctuations within the chaperone:subunit complex. In the simulations, the chaperone donor strand was seen to unbind from the pilus subunit, residue by residue, in direct support of the zip-in-zip-out hypothesis. In addition, an interaction of a residue towards the N-terminus of the Nte with a specific binding pocket (P*) on the adjacent pilus subunit was seen to stabilise the DSE product against unbinding, which also proceeded in the simulations by a zippering mechanism. Together, the study provides an in-depth picture of DSE, including the first atomistic insights into the molecular events occurring during the zip-in-zip-out mechanism.     

Single-Molecule and FRET Fluorescence Correlation Spectroscopy Analyses of Phage DNA Packaging: Colocalization of Packaged Phage T4 DNA Ends within the Capsid

Linear DNAs of any sequence can be packaged into empty viral procapsids by the phage T4 terminase with high efficiency in vitro. Packaging substrates of 5 kbp and 50 kbp, terminated by energy transfer dye pairs, were constructed from plasmid and λ phage DNAs. Nuclease and fluorescence correlation spectroscopy (FCS) assays showed that ∼ 20% of the substrate DNA was packaged and that the DNA dye ends of the packaged DNA were protected from nuclease digestion. Upon packaging, both 5-kbp and  50-kbp DNAs produced comparable fluorescence resonance energy transfer (FRET) between Cy5 and Cy5.5 double-dye terminated DNAs. Single-molecule FRET (sm-FRET) and photobleaching analysis shows that FRET is intramolecular rather than intermolecular upon packaging of most procapsids and demonstrates that single-molecule detection allows mechanistic analysis of packaging in vitro. FRET-FCS and sm-FRET measurements are comparable and show that both the 5-kbp and the  50-kbp packaged DNA ends are held within 8-9 nm of each other, within the dimensions of the long axis of the procapsid portal. The calculated distribution of FRET distances is relatively narrow for both FRET-FCS and sm-FRET, suggesting that the two packaged DNA ends are held at the same fixed distance relative to each other in most capsids. Because one DNA end is known to be positioned for ejection through the portal, it can be inferred that both DNAs ends are held in proximity to the portal entrance and ejection channel. The analysis suggests that a DNA loop, rather than a DNA end, is translocated by the packaging motor to fill the procapsid.     

Force-based analysis of multidimensional energy landscapes: application of dynamic force spectroscopy and steered molecular dynamics simulations to an antibody fragment-peptide complex

Multidimensional energy landscapes are an intrinsic property of proteins and define their dynamic behavior as well as their response to external stimuli. In order to explore the energy landscape and its implications on the dynamic function of proteins dynamic force spectroscopy and steered molecular dynamics (SMD) simulations have proved to be important tools. In this study, these techniques have been employed to analyze the influence of the direction of the probing forces on the complex of an antibody fragment with its peptide antigen. Using an atomic force microscope, experiments were performed where the attachment points of the 12 amino acid long peptide antigen were varied. These measurements yielded clearly distinguishable basal dissociation rates and potential widths, proving that the direction of the applied force determines the unbinding pathway. Complementary atomistic SMD simulations were performed, which also show that the unbinding pathways of the system are dependent on the pulling direction. However, the main barrier to be crossed was independent of the pulling direction and is represented by a backbone hydrogen bond between Gly^H-H40 of the antibody fragment and Glu^Oε-6_peptide of the peptide. For each pulling direction, the observed barriers can be correlated with the rupture of specific interactions, which stabilize the bound complex. Furthermore, although the SMD simulations were performed at loading rates exceeding the experimental rates by orders of magnitude due to computational limitations, a detailed comparison of the barriers that were overcome in the SMD simulations with the data obtained from the atomic force microscope unbinding experiments show excellent agreement.     

The polymorphism for endothelial nitric oxide synthase gene,the level of nitric oxide and the risk for pre-eclampsia: A meta-analysis

Endothelial NO, which is synthesized by endothelial nitric oxide synthase (eNOS), has been reported to be related with the occurrence of pre-eclampsia (PE). However, the polymorphisms of eNOS (− 786 T > C, 4b/a and G894T), the level of nitric oxide and the risk of PE remain unclear. Thus we performed this meta-analysis to determine the associations between them in order to predict the risk for PE and interference with PE development in the early period of antenatal care. All studies investigating the associations between PE risk and polymorphisms of eNOS, or PE risk and serum concentration of NO were reviewed. Finally, 29 studies were included, involving 11 for − 786 T > C, 11for 4b/a, and 22 for G894T polymorphisms and PE risk. In the overall analysis, − 786 T > C polymorphism was found to be related with increased PE risk in the dominant model (OR = 1.17, 95% CI = 1.02-1.35). a allele for 4b/a suffers the high risk of PE (OR = 1.46, 95% CI = 1.01–2.10). In the subgroup analysis, significantly increased risk was detected among Europeans for − 786 T > C polymorphism (OR = 1.40, 95%CI = 1.14–1.73).However, no significant association was detected for G894T polymorphism in the overall and subgroup analysis. The comprehensive evaluation of 9 available studies indicated that serum NO level was significantly decreased in case group (SMD = − 0.96 umol/mL, 95%CI = − 1.80, − 0.12 umol/mL).Hence, we concluded that eNOS gene − 786 T > C and 4b/a except for G894T polymorphisms were contributed significantly to PE risk, especially for Europeans, and a low NO concentration in serum increased the risk for PE.     

Water transport in human aquaporin-4: molecular dynamics (MD) simulations

Aquaporin-4 (AQP4) is the predominant water channel in the central nervous system, where it has been reported to be involved in many pathophysiological roles including water transport. In this paper, the AQP4 tetramer was modeled from its PDB structure file, embedded in a palmitoyl-oleoyl-phosphatidyl-choline (POPC) lipid bilayer, solvated in water, then minimized and equilibrated by means of molecular dynamics simulations. Analysis of the equilibrated structure showed that the central pore along the fourfold axis of the tetramers is formed with hydrophobic amino acid residues. In particular, Phe-195, Leu-191 and Leu-75, form the narrowest part of the pore. Therefore water molecules are not expected to transport through the central pore, which was confirmed by MD simulations. Each monomer of the AQP4 tetramers forms a channel whose walls consist mostly of hydrophilic residues. There are eight water molecules in single file observed in each of the four channels, transporting through the selectivity filter containing Arg-216, His-201, Phe-77, Ala-210, and the two conserved Asn-Pro-Ala (NPA) motifs containing Asn-213 and Asn-97. By using Brownian dynamics fluctuation–dissipation-theorem (BD-FDT), the overall free-energy profile was obtained for water transporting through AQP4 for the first time, which gives a complete map of the entire channel of water permeation.