A dynamic anchor domain in slc13 transporters controls metabolite transport

Abstract

Metabolite transport across cellular membranes is required for bioenergetic processes and metabolic signaling. The solute carrier family 13 (slc13) transporters mediate transport of the metabolites succinate and citrate and hence are of paramount physiological importance. Nevertheless, the mechanisms of slc13 transport and regulation are poorly understood. Here, a dynamic structural slc13 model suggested that an interfacial helix, H4c, which is common to all slc13s, stabilizes the stationary scaffold domain by anchoring it to the membrane, thereby facilitating movement of the SLC13 catalytic domain. Moreover, we found that intracellular determinants interact with the H4c anchor domain to modulate transport. This dual function is achieved by basic residues that alternately face either the membrane phospholipids or the intracellular milieu. This mechanism was supported by several experimental findings obtained using biochemical methods, electrophysiological measurements in Xenopus oocytes, and fluorescent microscopy of mammalian cells. First, a positively charged and highly conserved H4c residue, Arg108, was indispensable and crucial for metabolite transport. Furthermore, neutralization of other H4c basic residues inhibited slc13 transport function, thus mimicking the inhibitory effect of the slc13 inhibitor, slc26a6. Our findings suggest that the positive charge distribution across H4c domain controls slc13 transporter function and is utilized by slc13-interacting proteins in the regulation of metabolite transport.

Structural basis for the reaction cycle of DASS dicarboxylate transporters

“…Such arm rigidity agrees with the conserved salt bridge, between Arg122 and Glu283, and bulky-residue interactions at the elbows connecting H4c and H9c to the scaffold domain (Figures 6a & b). The importance the Arg122 to Glu283 salt bridge is also consistentwith recent observations in NaCT, where transport activity was abolished by mutations of the equivalent arginine39 , probably by disrupting the conserved salt bridge between H4c and TM7. In contrast to the rigidity at the elbows, flexibility of the hinge loops L4-HP in and L9-HP out at the other end of the arm helices allows transport domain movement(Figure 4).…”

Functional analysis of a species-specific inhibitor selective for human Na+-coupled citrate transporter (NaCT/SLC13A5/mINDY)

“…In each set, the inter-helical loops of the hairpins (HP in and HP out ), and the intra-helical loops of the adjacent discontinuous TM5 and TM10 helices, respectively, form a cleft that opens toward the cytoplasm. Both human and mouse transporters show long loops in the cytoplasm, similar to previously published human NaCT models [16,[29][30][31][32][33].…”

Solvent accessibility changes in a Na+-dependent C4-dicarboxylate transporter suggest differential substrate effects in a multistep mechanism

“…In addition, these models identified residues in NaDC1 that potentially form a discrete OFS-binding site that would be formed by the action of the proposed elevator mechanism; mutation of the residues diminished Na + and succinate transport activity, supportive of a unified elevator mechanism for the DASS transport family. Furthermore, a recent study of human NaDC1 and NaCT identified a cluster of positively charged residues, conserved in the SLC13 family, that appear to strongly influence transport activity, with Arg-108 (Val-118 in VcINDY) proving to be indispensable for transport activity ( 52 ). These residues are predicted, based on the structures of VcINDY, to form a short helix on the cytoplasmic facade of the scaffold domain.…”

“…These residues are predicted, based on the structures of VcINDY, to form a short helix on the cytoplasmic facade of the scaffold domain. Whereas these residues do not appear to contribute directly to binding site formation, they are hypothesized to interact with HP1 during the elevator-like transport cycle ( 52 ), perhaps playing a role in stabilizing a particular ligand-bound state. We note with interest that the equivalent helix in VcINDY contains multiple charged residues and that the cysteine accessibility assay data presented here demonstrate that accessibility to a residue in this helix (Ala-120) is highly sensitive to the presence of Na + .…”