title: Successful prediction of substrate-binding pocket in SLC17 transporter sialin.
abstract: Secondary active transporters from the SLC17 protein family are required for excitatory and purinergic synaptic transmission, sialic acid metabolism, and renal function, and several members are associated with inherited neurological or metabolic diseases. However, molecular tools to investigate their function or correct their genetic defects are limited or absent. Using structure-activity, homology modeling, molecular docking, and mutagenesis studies, we have located the substrate-binding site of sialin (SLC17A5), a lysosomal sialic acid exporter also recently implicated in exocytotic release of aspartate. Human sialin is defective in two inherited sialic acid storage diseases and is responsible for metabolic incorporation of the dietary nonhuman sialic acid N-glycolylneuraminic acid. We built cytosol-open and lumen-open three-dimensional models of sialin based on weak, but significant, sequence similarity with the glycerol-3-phosphate and fucose permeases from Escherichia coli, respectively. Molecular docking of 31 synthetic sialic acid analogues to both models was consistent with inhibition studies. Narrowing the sialic acid-binding site in the cytosol-open state by two phenylalanine to tyrosine mutations abrogated recognition of the most active analogue without impairing neuraminic acid transport. Moreover, a pilot virtual high-throughput screening of the cytosol-open model could identify a pseudopeptide competitive inhibitor showing >100-fold higher affinity than the natural substrate. This validated model of human sialin and sialin-guided models of other SLC17 transporters should pave the way for the identification of inhibitors, glycoengineering tools, pharmacological chaperones, and fluorescent false neurotransmitters targeted to these proteins.
authors.affiliation: Centre National de la Recherche Scientifique, UMR 8601, Université Paris Descartes, Sorbonne Paris Cité, 45 rue des Saints-Pères, F-75006 Paris, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Pietrancosta
authors.forename: Christine
authors.initials: C
authors.lastname: Anne
authors.forename: Horst
authors.initials: H
authors.lastname: Prescher
authors.forename: Raquel
authors.initials: R
authors.lastname: Ruivo
authors.forename: Corinne
authors.initials: C
authors.lastname: Sagné
authors.forename: Cécile
authors.initials: C
authors.lastname: Debacker
authors.forename: Hugues-Olivier
authors.initials: HO
authors.lastname: Bertrand
authors.forename: Reinhard
authors.initials: R
authors.lastname: Brossmer
authors.forename: Francine
authors.initials: F
authors.lastname: Acher
authors.forename: Bruno
authors.initials: B
authors.lastname: Gasnier
mesh_terms: D001381:Azepines / Q000378:metabolism; D001665:Binding Sites; D019295:Computational Biology; D004353:Drug Evaluation, Preclinical; D057809:HEK293 Cells; D006801:Humans; D007211:Indoles / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D027361:Organic Anion Transporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010865:Pilot Projects; D011485:Protein Binding; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D012794:Sialic Acids / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D027981:Symporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D001381:Azepines; D007211:Indoles; D027361:Organic Anion Transporters; D012794:Sialic Acids; D027981:Symporters; C421224:sialic acid transport proteins; C079574:FR 139317