Allosteric and transport modulation of human concentrative nucleoside transporter 3 at the atomic scale

Nucleosides are important precursors of nucleotide synthesis in cells, and nucleoside transporters play an important role in many physiological processes by mediating transmembrane transport and absorption. During nucleoside transport, such proteins undergo a significant conformational transition between the outward- and inward-facing states, which leads to alternating access of the substrate-binding site to either side of the membrane. In this work, a variety of molecular simulation methods have been applied to comparatively investigate the motion modes of human concentrative nucleoside transporter 3 (hCNT3) in three states, as well as global and local cavity conformational changes; and finally, a possible elevator-like transport mechanism consistent with experimental data was proposed. The results of the Gaussian network model (GNM) and anisotropic network model (ANM) show that hCNT3 as a whole tends to contract inwards and shift towards a membrane inside, exhibiting an allosteric process that is more energetically favorable than the rigid conversion. To reveal the complete allosteric process of hCNT3 in detail, a series of intermediate conformations were obtained by an adaptive anisotropic network model (aANM). One of the simulated intermediate states is similar to that of a crystal structure, which indicates that the allosteric process is reliable; the state with lower energy is slightly inclined to the inward-facing structure rather than the expected intermediate crystal structure. The final HOLE analysis showed that except for the outward-facing state, the transport channels were gradually enlarged, which was conductive to the directional transport of nucleosides. Our work provides a theoretical basis for the multistep elevator-like transportation mechanism of nucleosides, which helps to further understand the dynamic recognition between nucleoside substrates and hCNT3 as well as the design of nucleoside anticancer drugs.