title: Influenza virus hemagglutinin spike neck architectures and interaction with model enzymes evaluated by MALDI-TOF mass spectrometry and bioinformatics tools.
abstract: Interactions between model enzymes and the influenza virus hemagglutinin (HA) homotrimeric spike were addressed. We digested influenza virions (naturally occurring strains and laboratory reassortants) with bromelain or subtilisin Carlsberg and analyzed by MALDI-TOF mass spectrometry the resulting HA2 C-terminal segments. All cleavage sites, together with (minor) sites detected in undigested HAs, were situated in the linker region that connects the transmembrane domain to the ectodomain. In addition to cleavage at highly favorable amino acids, various alternative enzyme preferences were found that strongly depended on the HA subtype/type. We also evaluated the surface electrostatic potentials, binding cleft topographies and spatial dimensions of stem bromelain (homologically modeled) and subtilisin Carlsberg (X-ray resolved). The results show that the enzymes (∼45Å(3)) would hardly fit into the small (∼18-20Å) linker region of the HA-spike. However, the HA membrane proximal ectodomain region was predicted to be intrinsically disordered. We propose that its motions allow steric adjustment of the enzymes' active sites to the neck of the HA spike. The subtype/type-specific architectures in this region also influenced significantly the cleavage preferences of the enzymes.
authors.affiliation: Research Institute of Physical-Chemical Medicine, Federal Agency for Health Care and Social Development, Moscow, Russia.
authors.forename: Marina V
authors.initials: MV
authors.lastname: Serebryakova
authors.forename: Larisa V
authors.initials: LV
authors.lastname: Kordyukova
authors.forename: Tatiana A
authors.initials: TA
authors.lastname: Semashko
authors.forename: Alexander L
authors.initials: AL
authors.lastname: Ksenofontov
authors.forename: Irina A
authors.initials: IA
authors.lastname: Rudneva
authors.forename: Ekaterina A
authors.initials: EA
authors.lastname: Kropotkina
authors.forename: Irina Yu
authors.initials: IY
authors.lastname: Filippova
authors.forename: Michael
authors.initials: M
authors.lastname: Veit
authors.forename: Lyudmila A
authors.initials: LA
authors.lastname: Baratova
mesh_terms: D001963:Bromelains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019295:Computational Biology; D018360:Crystallography, X-Ray; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D008954:Models, Biological; D008958:Models, Molecular; D025941:Protein Interaction Mapping; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013381:Subtilisins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus; D001963:Bromelains; D013381:Subtilisins