Mapping Functionally Important Residues in the Na⁺/Dicarboxylate Cotransporter, NaDC1

Abstract

Transporters from the SLC13 family couple the transport of two to four Na ions with a di- or tricarboxylate, such as succinate or citrate. We have previously modeled mammalian members of the SLC13 family, including the Na/dicarboxylate cotransporter NaDC1 (SLC13A2), based on a structure of the bacterial homologue VcINDY in an inward-facing conformation with one sodium ion bound at the Na1 site. In the study presented here, we modeled the outward-facing conformation of rabbit and human NaDC1 (rbNaDC1 and hNaDC1, respectively) using an outward-facing model of VcINDY as a template and identified residues in or near the putative Na2 and Na3 cation binding sites. Guided by the structural models in both conformations, we performed site-directed mutagenesis in rbNaDC1 for residues proposed to be in the Na or substrate binding sites. Cysteine substitution of T474 in the predicted Na2 binding site results in an inactive protein. The M539C mutant has a low apparent affinity for both sodium and lithium cations, suggesting that M539 may form part of the putative Na3 binding site. The Y432C and T86C mutants have increased K values for succinate, supporting their proposed location in the outward-facing substrate binding site. In addition, cysteine labeling by MTSEA-biotin shows that Y432C is accessible from the outside of the cell, and the accessibility changes in the presence or absence of Na. The results of this study improve our understanding of substrate and ion recognition in the mammalian members of the SLC13 family and provide a framework for developing conformationally specific inhibitors against these transporters.

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

“…Analogous to the situation with Glt Ph and its homologues, binding of Na + is likely required for high-affinity binding of succinate, and binding of the succinate molecule is required to stabilize the “closed” conformation of the re-entrant loop, which allows the elevator-like conformational change to take place ( 23 , 44 , 45 , 46 , 47 ). The suggestion that the re-entrant hairpins undergo conformational changes in response to succinate binding is strengthened by the observations made during a series of solvent accessibility studies on the eukaryotic DASS transporter, NaDC1 ( 20 , 48 , 49 ). In concordance with our work presented here, the binding of Na + and succinate had substantial and separable effects on the solvent accessibility of residues in the arm of re-entrant hairpin 2 in NaDC1 (in the same region and Ser-381, Leu-384, and Val-388 in VcINDY), suggestive of discrete conformational changes upon each Na + - and succinate-binding event ( 20 , 48 , 49 ).…”

“…The suggestion that the re-entrant hairpins undergo conformational changes in response to succinate binding is strengthened by the observations made during a series of solvent accessibility studies on the eukaryotic DASS transporter, NaDC1 ( 20 , 48 , 49 ). In concordance with our work presented here, the binding of Na + and succinate had substantial and separable effects on the solvent accessibility of residues in the arm of re-entrant hairpin 2 in NaDC1 (in the same region and Ser-381, Leu-384, and Val-388 in VcINDY), suggestive of discrete conformational changes upon each Na + - and succinate-binding event ( 20 , 48 , 49 ). In contrast to our observations with VcINDY, the HP2 re-entrant loop residues become more accessible in the presence of Na + alone, and then less accessible upon the addition of succinate ( 48 ).…”

“…The model VcINDY substrate, succinate, is transported in its dianionic form coupled to the transport of three Na + ions ( 15 , 19 ). Our laboratory and others have revealed that VcINDY shares structural and functional characteristics with its mammalian homologues, suggesting that insight derived from the mechanism of VcINDY is directly applicable to the mammalian transporters ( 15 , 19 , 20 ).…”

Substrate-dependent accessibility changes in the Na+-dependent C4-dicarboxylate transporter, VcINDY, suggest differential substrate effects in a multistep mechanism

“…The model VcINDY substrate, succinate, is transported in its dianionic form coupled to the transport of three Na + ion 17,20 . Our lab and others have revealed that VcINDY shares structural and functional characteristics with its mammalian homologues, suggesting that insight derived from the mechanism of VcINDY is directly applicable to the mammalian transporters 17,20,21 . The x-ray structures of VcINDY reveal that it forms a homodimer and each protomer consists of two domains; the scaffold domain that forms dimer interface contacts, and the transport domain that houses key substrate binding residue ( Fig.…”

Molecular Modeling of Drug–Transporter Interactions—An International Transporter Consortium Perspective

“…This fact can be utilized to efficiently derive alternative conformations (79), and has been used to model transporters belonging to a variety of families and folds (80) (Figure 4). For example, the Vibrio Cholerae Na+/succinate transporter VcINDY structure was determined in the inward conformation (81), and has been modeled in the outward conformation using this technique (Figure 4) (82), providing a template for modeling the human homologs of the SLC13 family in this conformation (83).…”