title: Turnover and accessibility of a reentrant loop of the Na+-glutamate transporter GltS are modulated by the central cytoplasmic loop.
abstract: GltS of Escherichia coli is a secondary transporter that catalyzes Na+-glutamate symport. The structural model of GltS shows two homologous domains with inverted membrane topology that are connected by a central loop that resides in the cytoplasm. Each domain contains a reentrant loop structure. Accessibility of the Cys residues in two GltS mutants in which Pro351 and Asn356 in the reentrant loop in the C-terminal domain were replaced by Cys is demonstrated to be sensitive to the catalytic state supporting a role for the reentrant loops in catalysis. Saturating concentrations of the substrate L-glutamate protected both mutants against inactivation by thiol reagents, while the presence of the co-ion Na+ stimulated the inactivation of both mutants. Insertion of the 10 kDa biotin acceptor domain (BAD) of oxaloacetate decarboxylase of Klebsiella pneumoniae in the central cytoplasmic loop blocked the access pathway from the periplasmic side of the membrane to the cysteine residues in mutants P351C and N356C in the reentrant loop. Kinetically, insertion of BAD increased the maximal rate of uptake 2.7-fold while leaving the apparent affinity constants for L-glutamate and Na+ unaltered. The data suggests that insertion of BAD in the central loop results in conformational changes at the translocation site that lower the activation energy of the translocation step without affecting the access pathway from the periplasmic side for substrate and co-ions. It is concluded that changes in the central loop that connects the two domains may have a regulatory function on the activity of the transporter.
authors: Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands.
authors: Tomas
authors: T
authors: Krupnik
authors: Iwona
authors: I
authors: Sobczak-Elbourne
authors: Juke S
authors: JS
authors: Lolkema
mesh_terms: D026922:Amino Acid Transport Systems, Acidic / Q000737:chemistry* / Q000235:genetics; D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics; D002384:Catalysis; D002462:Cell Membrane / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000737:chemistry / Q000235:genetics; D026901:Membrane Transport Proteins / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D027981:Symporters / Q000737:chemistry* / Q000235:genetics
chemical_list: D026922:Amino Acid Transport Systems, Acidic; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D027981:Symporters; C066603:gltS protein, E coli; D018698:Glutamic Acid; D002262:Carboxy-Lyases; C021916:oxaloacetate decarboxylase