Computational prediction and experimental characterization of a “size switch type repacking” during the evolution of dengue envelope protein domain III (ED3)

ANALYSIS OF DOMAIN SPECIFICITY OF THE PROTECTIVE CHIMERIC ANTIBODY ch14D5a AGAINST GLYCOPROTEIN E OF TICK-BORNE ENCEPHALITIS VIRUS

“…Это достаточно стабильный домен, до-полнительно стабилизированный одной консервативной дисульфидной связью. Рекомбинантные белки, представ-ляющие собой домены D3 некоторых флавивирусов, таких как вирус желтой лихорадки, вирус Денге, вирус западного Нила и вирус Лангат, были получены в бактериальных системах экспрессии как в виде телец включения с по-следующим рефолдингом белка (Volk et al, 2009;Elahi et al, 2014;Kulkarni et al, 2016), так и в растворимом виде в формате белков слияния (White et al, 2003;Volk et al, 2006;Maillard et al, 2008). В случае периплазматической экспрессии домены D3 вируса японского энцефалита и вируса лихорадки Денге были получены в нативной кон-формации без рефолдинга и отщепления белка-носителя (Wu et al, 2003;Lisova et al, 2007;Yang et al, 2012).…”

Folding of the Ig-Like Domain of the Dengue Virus Envelope Protein Analyzed by High-Hydrostatic-Pressure NMR at a Residue-Level Resolution

“…The solution structure of DEN4-ED3 was solved previously by NMR (Protein Data Bank (PDB) entry: 2h0p) using a complete set of { 1 H, 13 C, 15 N} triple-resonance experiments for chemical-shifts assignment (deposited at BRMB: 7087) [15]. This structure was determined at 25 • C and pH 7.5 (50 mM Tris buffer, 50 mM NaCl), under physical and chemical conditions slightly different than those used in the present denaturation study (pH 7, 10 • C, no salt, and 0.5 M GuHCl), justifying the reassignment of the amide group resonances.…”

“…The sequence of DEN4-ED3 was retrieved from UniProt (ID P09866, https://www.uniprot.org/) [33] and a synthetic gene encoding ED3 s sequence, a His6-tag and a thrombin cleavage site at the N-terminus, was cloned into a pET15b vector, as reported previously [13]. The ED3 domain was expressed in Escherichia coli BL21 (DE3) PLysS as inclusion bodies and purified as described [34,35].…”

“…Surprisingly, only eight amino acid mutations in ED3 separate the two most divergent DEN4 strains, among which five have been implicated in the emergence of the human DEN4 virus [11,12] (Supplementary Materials, Figure S1 and Table S1). Recent X-ray crystallography (DEN3-ED3, DEN4-ED3) [13,14] and nuclear magnetic resonance (NMR) (DEN4-ED3) [15] analysis demonstrated that dengue ED3 remains natively folded even when it is isolated from the rest of the E-protein. Moreover, previous studies indicated that residues in the hydrophobic core of ED3 were not fully conserved among the four serotypes [13] and that a rearrangement of the side-chains interdigitization ensured the stabilization of the protein core and thus of the natively folded structure of DEN-ED3 across serotypes [14].…”

“…Recent X-ray crystallography (DEN3-ED3, DEN4-ED3) [13,14] and nuclear magnetic resonance (NMR) (DEN4-ED3) [15] analysis demonstrated that dengue ED3 remains natively folded even when it is isolated from the rest of the E-protein. Moreover, previous studies indicated that residues in the hydrophobic core of ED3 were not fully conserved among the four serotypes [13] and that a rearrangement of the side-chains interdigitization ensured the stabilization of the protein core and thus of the natively folded structure of DEN-ED3 across serotypes [14]. More generally, the Ig-fold is a classical fold found in several proteins with unrelated functions, and it would be of interest to determine whether proteins with Ig-fold but different core packing exhibit similar folding properties at the residue level [16].…”