boAP3D1, BLV Env and recombinant proteins physicochemical properties.

<p>boAP3D1, BLV Env and recombinant proteins physicochemical properties.</p

<i>In silico</i> and <i>in vitro</i> analysis of boAP3d1 protein interaction with bovine leukaemia virus gp51 - Fig 1

<p><b><a href="http://web.expasy.org/protscale/pscale/Hphob.Doolittle.html" target="_blank">Kyte & Doolittle</a> hydrophobicity for BLV Env (A) and boAP3D1 (B) proteins.</b> Despite differences regarding the amount of aa in both proteins, their physicochemical characteristics were comparable.</p

boAP3D1 and gp51 docking.

<p>5A. Overview. 5B. Asn170 (gp51) and Lys925 (boAP3D1). 5C. Trp127 (gp51) and Asp807 (boAP3D1). 5D. His115 (gp51) and Asp695 (boAP3D1), hydrogen bonds, non-bonded contacts. 5E. Ala97, Ser98, Glu128 (gp51) and Arg800 (boAP3D1), hydrogen bonds, non-bonded contacts and salt bridges.</p

gp51 recombinant fragment purification.

<p>Lanes 1, 3 and 5 show Western blot detection with an anti-his monoclonal antibody. Lanes 2, 4 and 6 show Coomassie blue stained purified recombinant proteins. The proteins’ molecular weight marker is indicated in the first lane (molecular masses for the three recombinants agreed with expected ones: 30, 16 and 14 kDa for rgp51, rNgp51 and rCgp51, respectively).</p

Binding free energy results for gp51 and boAP3D1 interaction.

<p>Binding free energy results for gp51 and boAP3D1 interaction.</p

gp51 recombinant protein binding assays.

<p>8A. rgp51 MDBK cell binding. Low rgp51 protein concentration (black bars) gave greater MDBK cell binding; however, rgp51 and rCgp51 binding did not change when protein concentration was duplicated (blue bars), whilst rNgp51 increased. 8B. MDBK cell interaction with rNgp51. The black bars represent enzymatically-treated MDBK and rNgp51 binding to the proposed receptor (AP3D1), followed by trypsin and chymotrypsin treatment, resulting in reduced in rNgp51 (98%) and MDBK binding (80%).</p

ClustalOmega sequence alignment of boAP3D1 and huAP3D1.

<p>“-” represents identical aa, the red line represents the BLVR domain.</p

Schematic representation of Env BLV and boAP3D1 proteins.

<p>3A. BLV Env protein. Signal peptide 1 to 34, SU (gp51) 34 to 438, TM (gp30) 439 to 460 and cytoplasmic region 461 to 515. Binding sites are shown by yellow circles (48, 50, 77, 98, 99, 112, 113, 120, 122, 135, 136, 187, 292 and 298), N-glycosylation sites (129, 203, 230, 251, 256, 271, 287, 351 and 398). 3B. boAP3D1 protein has 1,207 aa with two domains: one from aa 32 to 583 for adaptin (dark green) and a second (BLVR) from aa 661 to 807 (red). AP3D1 has 2 binding sites in positions 1 and 474 (yellow dots) and 16 conserved sites in the BLVR domain (D661, E662, S686, S688, K726, E728, E729, K739, D767, E779, E783, A785, L786, S788, D797 and A801).</p

ClustalOmega sequence alignment of different BLV gp51 sequences (genotypes 1 to 10).

<p>“-” represents identical aa. The six key amino acids in the interaction are shown in yellow (Ala97, Ser98, His115, Trp127, Glu128 and Asn170).</p

Modelling boAP3D1 and Env proteins.

<p>4A. boAP3D1 structure is coloured green, and in red the BLVR domain. 4B. The BLVR domain (red) with conserved sites highlighted in yellow (D661, E662, S686, S688, K726, E728, E729, K739, D767, E779, E783, A785, L786, S788, D797 and A801). 4C. BLV Env protein structure predicted by I-TASSER, gp51 (SU) is shown in magenta, gp30 (TM) in cyan and cytoplasmic domain in orange. 4D. The COPI coat triad structure (5A1U in green) overlaps with our BLV Env protein model.</p