Journal: Nature Communications

Article Title: Structural basis for negative regulation of the Escherichia coli maltose system

doi: 10.1038/s41467-023-40447-y

Figure Lengend Snippet: a Affinity-purified MalT and MalY proteins were further analyzed by gel filtration using a Superdex 200 Increase 10/300 GL column in the presence of ADP (left). When MalT and MalY were incubated in a 1:1 molar ratio, the two proteins formed a stable complex with a molecular weight of about 300 kDa. The 9-mL peak observed when MalT is filtered alone may contain protein aggregates or MalT protein that partially oligomerized at a high protein concentration (>3 mg/ml). The presence of MalT and MalY in fractionated samples was confirmed by SDS-PAGE analyses (right). The experiments have been repeated for three times with similar results. b Cryo-EM density map with 2.94 Å resolution (top) and the refined structure model (middle) of the MalT-MalY complex (PDB: 8BOB) shown in different orientations. Each MalT or MalY protomer is labelled. Residue number and colors of each protein domain are indicated (bottom). The N-terminal segment of NBD is highlighted in yellow. The C-terminal domains of MalT are not well defined in the final structure. DBD DNA-binding domain.

Article Snippet: The MalT-MalY inhibitory complex was reconstituted by incubating purified MalT (in buffer A with 0.1 mM ADP) and MalY (in buffer TKCl with 10 μM PLP) at a molar ratio of 1:1 on ice for 30 min, then purified by gel filtration using a Superdex 200 Increase 10/300 GL column (GE Healthcare) with buffer TKCl supplemented with 0.1 mM ADP and 10 μM PLP.

Techniques: Affinity Purification, Filtration, Incubation, Molecular Weight, Protein Concentration, SDS Page, Cryo-EM Sample Prep, Binding Assay

Journal: Nature Communications

Article Title: Structural basis for negative regulation of the Escherichia coli maltose system

doi: 10.1038/s41467-023-40447-y

Figure Lengend Snippet: a 2D classification from cryo-EM analyses of active MalT. The density of C-terminal domains is indicated by red empty arrows. Particles show a preferred orientation. b A model for MalT regulation. In the absence of substrate transport, the idling E. coli maltose transporter MalFGK 2 anchors MalT to the cytoplasmic membrane via MalK-mediated interactions. Meanwhile, MalT molecules that remain in the cytoplasm are sequestered by inhibitory proteins. Sugar transport triggers a conformational change in the MalK dimer which frees membrane-localized MalT into cytoplasm , . The inhibitory effect of MalY can be relieved by an increased concentration of maltotriose. Binding of inducer to the sensor domain of MalT (1) is followed by a high-affinity binding step involving both the sensor and arm domains , driving the MalT activation pathway towards dissociation of the MalT-MalY complex (2), opening of NOD (3) and oligomerization. Maltotriose binding and oligomerization together stabilize the C-terminal domains of MalT, allowing the activator binding to promoter DNA and RNA polymerase (RNAP) recruitment for subsequent transcription initiation. The NBD, HD, WHD, arm, sensor, and DNA-binding domains of MalT are colored in pink, purple, wheat, cyan, slate, and green, respectively. Domains are colored in gray if they are flexible. This figure was created with BioRender.

Article Snippet: The MalT-MalY inhibitory complex was reconstituted by incubating purified MalT (in buffer A with 0.1 mM ADP) and MalY (in buffer TKCl with 10 μM PLP) at a molar ratio of 1:1 on ice for 30 min, then purified by gel filtration using a Superdex 200 Increase 10/300 GL column (GE Healthcare) with buffer TKCl supplemented with 0.1 mM ADP and 10 μM PLP.

Techniques: Cryo-EM Sample Prep, Concentration Assay, Binding Assay, Activation Assay