Mode of Interaction of the Signal-Transducing Protein EIIA^Glc with the Maltose ABC Transporter in the Process of Inducer Exclusion

Abstract

Enzyme IIA(Glc) (EIIA(Glc)) of the phosphoenolpyruvate phosphotransferase system for the uptake of glucose in Escherichia coli and Salmonella inhibits the maltose ATP-binding cassette transporter (MalE-FGK2) by interaction with the nucleotide-binding and -hydrolyzing subunit MalK, a process termed inducer exclusion. We have investigated binding of EIIA(Glc) to the MalK dimer by cysteine cross-linking in proteoliposomes. The results prove that the binding site I of EIIA(Glc) is contacting the N-terminal subdomain of MalK while the binding site II is relatively close to the C-terminal (regulatory) subdomain, in agreement with a crystal structure [ Chen , S. , Oldham , M. L. , Davidson , A. L. , and Chen , J. ( 2013 ) Nature 499 , 364 - 368 ]. Moreover, EIIA(Glc) was found to bind to the MalK dimer regardless of its conformational state. Deletion of the amphipathic N-terminal peptide of EIIA(Glc), which is required for inhibition, reduced formation of cross-linked products. Using a spin-labeled transporter variant and EPR spectroscopy, we demonstrate that EIIA(Glc) arrests the transport cycle by inhibiting the ATP-dependent closure of the MalK dimer.

An integrated transport mechanism of the maltose ABC importer

“…5; section 4). This model is in accordance with EPR studies that employed double-spin-labeled versions of MalK (V17C and E128C variants) to analyze their inter-distance distributions in MalFGK 2 in nanodisc in the presence of maltose-bound MalE [132]. These results confirmed the hypothesis that EIIA glc reduces transport activity by inhibiting ATP hydrolysis, without affecting the binding affinity of ATP for MalFGK 2 .…”

“…This membrane interaction is mediated by the first 18 N-terminal amino acids of EIIA glc , which form a α-helix upon proximity to the negatively charged headgroups of PG (and other phospholipids); the interaction occurs mainly thanks to the presence of lysine residues in this region of the protein. This interaction has been observed in structural analyses [85] and in co-sedimentation and detergent versus proteoliposomes/nanodiscs functional assays that employed N-truncated versions of EIIA glc [85], [129], [132]. Taken together, these studies suggest that the N-terminal amphipathic α-helix plays an essential role in inducer exclusion of the MalEFGK 2 transporter, essentially by stabilizing the interaction with MalK.…”

Contributions of SpoT Hydrolase, SpoT Synthetase, and RelA Synthetase to Carbon Source Diauxic Growth Transitions in Escherichia coli

“…Prevention of their uptake excludes what otherwise would constitute induction of non-PTS sugar operons and is therefore termed inducer exclusion ( Deutscher, 2008 ). Biochemical details of EIIA glc inhibition of lacY permease and malK transporter are available ( Hariharan et al, 2015 ; Wuttge et al, 2016 ).…”

Removal of a Membrane Anchor Reveals the Opposing Regulatory Functions of Vibrio cholerae Glucose-Specific Enzyme IIA in Biofilms and the Mammalian Intestine

“…coli EIIA Glc binds to MalK, a component of the maltose-specific ABC transporter, to inhibit maltose entry into the cell (16, 17). This process, known as inducer exclusion, prevents maltose from inducing transcription of genes required for its transport and catabolism (4, 18). To test whether this regulatory process is dependent on the EIIA Glc AH in V.…”