25 research outputs found
Monomeric ephrinB2 binding induces allosteric changes in Nipah virus G that precede its full activation.
Nipah virus is an emergent paramyxovirus that causes deadly encephalitis and respiratory infections in humans. Two glycoproteins coordinate the infection of host cells, an attachment protein (G), which binds to cell surface receptors, and a fusion (F) protein, which carries out the process of virus-cell membrane fusion. The G protein binds to ephrin B2/3 receptors, inducing G conformational changes that trigger F protein refolding. Using an optical approach based on second harmonic generation, we show that monomeric and dimeric receptors activate distinct conformational changes in G. The monomeric receptor-induced changes are not detected by conformation-sensitive monoclonal antibodies or through electron microscopy analysis of G:ephrinB2 complexes. However, hydrogen/deuterium exchange experiments confirm the second harmonic generation observations and reveal allosteric changes in the G receptor binding and F-activating stalk domains, providing insights into the pathway of receptor-activated virus entry.Nipah virus causes encephalitis in humans. Here the authors use a multidisciplinary approach to study the binding of the viral attachment protein G to its host receptor ephrinB2 and show that monomeric and dimeric receptors activate distinct conformational changes in G and discuss implications for receptor-activated virus entry
Recommended from our members
Angular Mapping of Protein Structure Using Nonlinear Optical Measurements
Proteins are inherently dynamic, flexible molecules that execute precise conformational changes to perform their functions, but existing techniques to directly measure relevant structural changes in solution at room temperature remain limited. Here, we demonstrate a structural technique using second-harmonic generation and two-photon fluorescence under single-laser excitation to map both the mean angular orientation and the distribution width of a probe at various sites throughout the protein with high sensitivity. Our work resolves distinct dihydrofolate reductase (DHFR) ligand-protein conformations, allows interrogation of regions unresolvable by other techniques, and reveals structural differences between DHFR and a point mutant (DHFR-G121V). The technique, angular mapping of protein structure, enables direct and rapid determination of previously unseen aspects of protein structure in a benchtop optical system
Detection of Ligand-Induced Conformational Changes in Oligonucleotides by Second-Harmonic Generation at a Supported Lipid Bilayer Interface
There is a high demand for characterizing
oligonucleotide structural
changes associated with binding interactions as well as identifying
novel binders that modulate their structure and function. In this
study, second-harmonic generation (SHG) was used to study RNA and
DNA oligonucleotide conformational changes associated with ligand
binding. For this purpose, we developed an avidin-based biotin capture
surface based on a supported lipid bilayer membrane. The technique
was applied to two well-characterized aptamers, both of which undergo
conformational changes upon binding either a protein or a small molecule
ligand. In both cases, SHG was able to resolve conformational changes
in these oligonucleotides sensitively and specifically, in solution
and in real time, using nanogram amounts of material. In addition,
we developed a competition assay for the oligonucleotides between
the specific ligands and known, nonspecific binders, and we demonstrated
that intercalators and minor groove binders affect the conformation
of the DNA and RNA oligonucleotides in different ways upon binding
and subsequently block specific ligand binding in all cases. Our work
demonstrates the broad potential of SHG for studying oligonucleotides
and their conformational changes upon interaction with ligands. As
SHG offers a powerful, high-throughput screening approach, our results
here also open an important new avenue for identifying novel chemical
probes or sequence-targeted drugs that disrupt or modulate DNA or
RNA structure and function
Identification of inactive conformationâselective interleukinâ2âinducible Tâcell kinase (ITK) inhibitors based on secondâharmonic generation
Many clinically approved protein kinase inhibitors stabilize an inactive conformation of their kinase target. Such inhibitors are generally highly selective compared to active conformation inhibitors, and consequently, general methods to identify inhibitors that stabilize an inactive conformation are much sought after. Here, we have applied a highâthroughput, secondâharmonic generation (SHG)âbased conformational approach to identify small molecule stabilizers of the inactive conformation of interleukinâ2âinducible Tâcell kinase (ITK). A singleâsite cysteine mutant of the ITK kinase domain was created, labeled with an SHGâactive dye, and tethered to a supported lipid bilayer membrane. Fourteen tool compounds, including stabilizers of the inactive and active conformations as well as nonbinders, were first examined for their effect on the conformation of the labeled ITK protein in the SHG assay. As a result, inactive conformation inhibitors were clearly distinguished from active conformation inhibitors by the intensity of SHG signal. Utilizing the SHG assay developed with the tool compounds described above, we identified the mechanism of action of 22 highly selective, inactive conformation inhibitors within a group of 105 small molecule inhibitors previously identified in a highâthroughput biochemical screen. We describe here the first use of SHG for identifying and classifying inhibitors that stabilize an inactive vs. an active conformation of a protein kinase, without the need to determine costructures by Xâray crystallography. Our results suggest broad applicability to other proteins, particularly with singleâsite labels reporting on specific protein movements associated with selectivity
Small Molecules Detected by Second-Harmonic Generation Modulate the Conformation of Monomeric \u3b1-Synuclein and Reduce Its Aggregation in Cells
Proteins are structurally dynamic molecules that perform specialized functions through unique conformational changes accessible in physiological environments. An ability to specifically and selectively control protein function via conformational modulation is an important goal for development of novel therapeutics and studies of protein mechanism in biological networks and disease. Here we applied a second-harmonic generation-based technique for studying protein conformation in solution and in real time to the intrinsically disordered, Parkinson disease related protein \u3b1-synuclein. From a fragment library, we identified small molecule modulators that bind to monomeric \u3b1-synuclein in vitro and significantly reduce \u3b1-synuclein aggregation in a neuronal cell culture model. Our results indicate that the conformation of \u3b1-synuclein is linked to the aggregation of protein in cells. They also provide support for a therapeutic strategy of targeting specific conformations of the protein to suppress or control its aggregation