Significance of hydrophobic and charged sequence similarities in sodium-bile acid cotransporter and vitamin D-binding protein macrophage activating factor

Ibne Raihan Zunaid; Stefania Pacini; Marco Ruggiero

sequence of NTCP aligned with the corresponding sequence of DBP-MAF. It is worth noticing that the sequence of seven aminoacids preceding the 157-KGIVISLVL-165 stretch of NTCP, that is the stretch that binds preS1, shows four amino acids that are identical to those of the aligned sequence of DBP-MAF, and two that are conserved substitutions. Fig. 2 , panel C, shows that DBP-MAF presents a sequence that is very similar to that of domain IV of the L NH2-EC1 TM1 TM1 TM2 TM3 TM4 TM5 TM6 TM6 TM7 TM7 IC1 IC2 IC3 COOH-IC4 EC2 EC3 EC3 EC4 . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.03.975524 doi: bioRxiv preprint protein of HBV. Both sequences are rich in hydrophobic residues; the sequence of DBP-MAF is located in a shallow cleft of the proteins that facilitates its binding to fatty acids that are sandwiched between the protein and the plasma membrane as we proposed in 2013 (12) . The sequence of domain IV of the L protein of HBV is a transmembrane domain that participates in infectivity (11) . Fig. 2, panel D, shows the peculiar concentration of negatively charged residues in the sequence of DBP-MAF that is aligned with the sequence of NTCP that binds preS1. In DBP-MAF, there are two contiguous negatively charged amino acids (ED) and, in the preceding epta-peptide sequence, there are three negatively charged aminoacids (D-ED) for a total of five negatively charged residues, whereas in the corresponding sequence of NTCP there are only two (D--D) that are not contiguous. This concentration of negatively charged aminoacids may be involved in the binding and electrostatic neutralization of protein inserts characterized by high density of positively charges residues. In particular, it may be involved in binding the sequences TNGTKR, HKNNKS, RSYLTPGDSSSG, and QTNSPRRA that are characterized by a high surface concentration of positive charges interspersed with hydrophobic residues (10, 13) . It is worth noticing that neutralization of positively charged residues may represent a strategy that exploits electrostatic interactions between charges on the surface of molecules. Such as strategy may not be limited to proteins; negatively charged glycosaminoglycans such as lowmolecular-weight chrondroitin sulfate (14) might also serve the scope of binding and neutralizing those sequences. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.03.975524 doi: bioRxiv preprint Fig. 3 , shows the results of alignment of NTCP, DBP-MAF and NAGAB_HUMAN. This latter protein has enzymatic activity and directly interacts with DBP-MAF, more precisely, it binds to alpha-N-acetylgalactosamine that is attached to threonine in the sequence TPTELAK close to the carboxyl terminus of DBP-MAF (see the sequence within the red square in Fig. 3) . We chose to study this protein because of its role in DBP-MAF function and we found that there is a high degree of similarity with the other two proteins, DBP-MAF and NTCP, with particular reference to stretches of hydrophobic aminoacids. Based on these evidence, we asked an independent laboratory to perform in vitro experiments to assess the binding of DBP-MAF to NAGAB_HUMAN using, as ligands, purified DBP-MAF and PMF (6, 8) . Fig. 4 , shows that purified DBP-MAF, used as positive control, bound NAGAB_HUM