ИСТИНА

The nuclear export protein (NEP) of influenza virus is a crucial component for export of viral RNP through bilayer lipid membrane of the infected cell nucleus in complex with viral matrix protein (M1)/Exportin1/Nucleoporin. So, in this process NEP may be involved in some interactions as a constituent of supramolecular lipid-protein complexes. Earlier attempts to crystallize the full length NEP were unsuccessful: only the C-terminal domain (amino acids 64-121) was accessible for X-ray analysis. Insolubility of the protein at physiological conditions hampers its structural investigations. So we analyze the overall structure of NEP by synchrotron SAXS. The on-line size-exclusion chromatography (SEC-SAXS) experiments demonstrated a presence of two fractions of the protein in solution. The first fraction consisted of large aggregates, while the second one revealed formation of the NEP clusters, whose characteristics were determined by ab initio shape reconstruction method. It was shown that the clusters demonstrated ordered helix-like particles with the length of about 20 nm and the cross-section (diameter of the helix) ~ 5 nm. The helix-like structure consisted of small uniform particles with size of about 2 nm. These particles could be considered as individual NEP macromolecules which impossible to separate even by size-exclusion chromatography. They formed stable clusters, which are typical for the majority of viral proteins, especially for the membrane interacting proteins. To obtain additional information on the peculiar self-association properties of NEP we carried out searching for the α-helices in NEP structure, and used the concept of intrinsically disordered/unstructured proteins, and predicted the SSE´s with the Psipred software. The prediction of NEP N- domain 3D structure by Scratch Protein Predict program (SPP) and by Modeller 9.8 software resulted in contradictory information. Nevertheless, only the latter software (server RaptorX) led to almost an exact convergence program predicts of ordered (helix) and disordered fragments of the polypeptide chain.