Secondary transporters of the 2HCT family contain two homologous domains with inverted membrane topology and trans re‐entrant loops

The 2‐hydroxycarboxylate transporter (2HCT) family of secondary transporters belongs to a much larger structural class of secondary transporters termed ST3 which contains about 2000 transporters in 32 families. The transporters of the 2HCT family are among the best studied in the class. Here we detect weak sequence similarity between the N‐ and C‐terminal halves of the proteins using a sensitive method which uses a database containing the N‐ and C‐terminal halves of all the sequences in ST3 and involves blast searches of each sequence in the database against the whole database. Unrelated families of secondary transporters of the same length and composition were used as controls. The sequence similarity involved major parts of the N‐ and C‐terminal halves and not just a small stretch. The membrane topology of the homologous N‐ and C‐terminal domains was deduced from the experimentally determined topology of the members of the 2HCT family. The domains consist of five transmembrane segments each and have opposite orientations in the membrane. The N terminus of the N‐terminal domain is extracellular, while the N terminus of the C‐terminal domain is cytoplasmic. The loops between the fourth and fifth transmembrane segment in each domain are well conserved throughout the class and contain a high fraction of residues with small side chains, Gly, Ala and Ser. Experimental work on the citrate transporter CitS in the 2HCT family indicates that the loops are re‐entrant or pore loops. The re‐entrant loops in the N‐ and C‐terminal domains enter the membrane from opposite sides (trans‐re‐entrant loops). The combination of inverted membrane topology and trans‐re‐entrant loops represents a new fold for secondary transporters and resembles the structure of aquaporins and models proposed for Na+/Ca2+ exchangers.