Beta-lactoglobulin (beta-LG), one of the most investigated proteins, is a major bovine milk protein with a predominantly beta structure. The structural function of the only alpha-helix with three turns at the C-terminus is unknown. Vitamin D(3) binds to the central calyx formed by the beta-strands. Whether there are two vitamin D binding-sites in each beta-LG molecule has been a subject of controversy. Here, we report a second vitamin D(3) binding site identified by synchrotron X-ray diffraction (at 2.4 A resolution). In the central calyx binding mode, the aliphatic tail of vitamin D(3) clearly inserts into the binding cavity, where the 3-OH group of vitamin D(3) binds externally. The electron density map suggests that the 3-OH group interacts with the carbonyl of Lys-60 forming a hydrogen bond (2.97 A). The second binding site, however, is near the surface at the C-terminus (residues 136-149) containing part of an alpha-helix and a beta-strand I with 17.91 A in length, while the span of vitamin D(3) is about 12.51 A. A remarkable feature of the second exosite is that it combines an amphipathic alpha-helix providing nonpolar residues (Phe-136, Ala-139, and Leu-140) and a beta-strand providing a nonpolar (Ile-147) and a buried polar residue (Arg-148). They are linked by a hydrophobic loop (Ala-142, Leu-143, Pro-144, and Met-145). Thus, the binding pocket furnishes strong hydrophobic force to stabilize vitamin D(3) binding. This finding provides a new insight into the interaction between vitamin D(3) and beta-LG, in which the exosite may provide another route for the transport of vitamin D(3) in vitamin D(3) fortified dairy products. Atomic coordinates for the crystal structure of beta-LG-vitamin D(3) complex described in this work have been deposited in the PDB (access code 2GJ5).

Limited structural information of drug targets, cellular toxicity possessed by lead compounds, and large amounts of potential leads are the major issues facing the design-oriented approach of discovering new leads. In an attempt to tackle these issues, we have developed a process of virtual screening based on the observation that conformational rearrangements of the dengue virus envelope protein are essential for the mediation of viral entry into host cells via membrane fusion. Screening was based solely on the structural information of the Dengue virus envelope protein and was focused on a target site that is presumably important for the conformational rearrangements necessary for viral entry. To circumvent the issue of lead compound toxicity, we performed screening based on molecular docking using structural databases of medical compounds. To enhance the identification of hits, we further categorized and selected candidates according to their novel structural characteristics. Finally, the selected candidates were subjected to a biological validation assay to assess inhibition of Dengue virus propagation in mammalian host cells using a plaque formation assay. Among the 10 compounds examined, rolitetracycline and doxycycline significantly inhibited plaque formation, demonstrating their inhibitory effect on dengue virus propagation. Both compounds were tetracycline derivatives with IC50s estimated to be 67.1 µM and 55.6 µM, respectively. Their docked conformations displayed common hydrophobic interactions with critical residues that affected membrane fusion during viral entry. These interactions will therefore position the tetracyclic ring moieties of both inhibitors to bind firmly to the target and, subsequently, disrupt conformational rearrangement and block viral entry. This process can be applied to other drug targets in which conformational rearrangement is critical to function.

We previously identified a novel inhibitor of influenza virus in mouse saliva that halts the progression of susceptible viruses from the upper to the lower respiratory tract of mice in vivo and neutralizes viral infectivity in MDCK cells. Here, we investigated the viral target of the salivary inhibitor by using reverse genetics to create hybrid viruses with some surface proteins derived from an inhibitor-sensitive strain and others from an inhibitor-resistant strain. These viruses demonstrated that the origin of the viral neuraminidase (NA), but not the hemagglutinin or matrix protein , was the determinant of susceptibility to the inhibitor. Comparison of the NA sequences of a panel of H3N2 viruses with differing sensitivities to the salivary inhibi-tor revealed that surface residues 368 to 370 (N2 numbering) outside the active site played a key role in resistance. Resistant viruses contained an EDS motif at this location , and mutation to either EES or KDS, found in highly susceptible strains, significantly increased in vitro susceptibility to the inhibitor and reduced the ability of the virus to progress to the lungs when the viral inoculum was initially confined to the upper respiratory tract. In the presence of saliva, viral strains with a susceptible NA could not be efficiently released from the surfaces of infected MDCK cells and had reduced enzymatic activity based on their ability to cleave substrate in vitro. This work indicates that the mouse has evolved an innate inhibitor similar in function, though not in mechanism, to what humans have created synthetically as an antiviral drug for influenza virus. IMPORTANCE Despite widespread use of experimental pulmonary infection of the laboratory mouse to study influenza virus infection and pathogenesis, to our knowledge , mice do not naturally succumb to influenza. Here, we show that mice produce their own natural form of neuraminidase inhibitor in saliva that stops the virus from reaching the lungs, providing a possible mechanism through which the species may not experience severe influenza virus infection in the wild. We show that the murine salivary inhibitor targets the outer surface of the influenza virus neuraminidase, possibly occluding entry to the enzymatic site rather than binding within the active site like commercially available neuraminidase inhibitors. This knowledge sheds light on how the natural inhibitors of particular species combat infection.

Interest in bringing p- and n-type monolayer semiconducting transition metal dichalcogenides (TMD) into contact to form rectifying pn diode has thrived since it is crucial to control the electrical properties in two-dimensional (2D) electronic and optoelectronic devices. Usually this involves vertically stacking different TMDs with pn heterojunction or, laterally manipulating carrier density by gate biasing. Here, by utilizing a locally reversed ferroelectric polarization, we laterally manipulate the carrier density and created a WSe2 pn homojunction on the supporting ferroelectric BiFeO3 substrate. This non-volatile WSe2 pn homojunction is demonstrated with optical and scanning probe methods and scanning photoelectron micro-spectroscopy. A homo-interface is a direct manifestation of our WSe2 pn diode, which can be quantitatively understood as a clear rectifying behavior. The non-volatile confinement of carriers and associated gate-free pn homojunction can be an addition to the 2D electron–photon toolbox and pave the way to develop laterally 2D electronics and photonics.

The strong coupling between antiferromagnetism and ferroelectricity at room temperature found in BiFeO3 generates high expectations for the design and development of technological devices with novel functionalities. However, the multi-domain nature of the material tends to nullify the properties of interest and complicates the thorough understanding of the mechanisms that are responsible for those properties. Here we report the realization of a BiFeO3 material in thin film form with single-domain behaviour in both its magnetism and ferroelectricity: the entire film shows its antiferromagnetic axis aligned along the crystallographic b axis and its ferroelectric polarization along the c axis. With this we are able to reveal that the canted ferromagnetic moment due to the Dzyaloshinskii–Moriya interaction is parallel to the a axis. Furthermore, by fabricating a Co/BiFeO3 heterostructure, we demonstrate that the ferromagnetic moment of the Co film does couple directly to the canted moment of BiFeO3.