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Edited by Ernest M. Wright, David Geffen School of Medicine at UCLA, Los Angeles, CA, and approved April 30, 2018 (received for review January 12, 2018)
Article Figures & SI
Figures
- Fig. 1.
Cross-linking of bcMalT(T280C-E54C). (A) SDS/PAGE analysis of bcMalT(T280C-E54C) under various conditions. Lanes: 1, molecular weight standard; 2, bcMalT(T280C-E54C) without any treatment; 3, bcMalT(T280C-E54C) treated with Hg2+. Cross-linked bcMalT migrates faster, and the band is marked as X-linked; 4, bcMalT(T280C-E54C), first treated with 2 mM N-ethyl maleimide (NEM) and then incubated with 1:1 molar ratio of Hg2+; 5, bcMalT(T280C-E54C) in the presence of 2 mM β-mercaptoethanol (β-ME); 6, bcMalT(T280C-E54C) treated with Hg2+ and then incubated with 2 mM β-ME. (B) Time course of Hg2+-induced cross-linking of bcMalT(T280C-E54C).
- Fig. 2.
Side-by-side comparison of bcMalT and bcMalT-X. Crystal structures of bcMalT wild type (Left, PDB accession number 5IWS) and bcMalT-X (Right) are shown in cartoon (A) and surface (B) representations. In A, the two protomers are colored blue and green, respectively. Within each protomer, the dimerization domain is colored darker than the substrate-binding domain. In B, the electrostatic surface was calculated by the Adaptive Poisson–Boltzmann Solver plugin in Pymol (34); the bar represents electrostatic potential from −61.1 to +61.1 kBT/eC. (C) Location of T280 and E54 on a single protomer in the outward-facing conformation (Left) and inward-facing conformation (Right). TM1 is removed for clarity. Cβ of T280 (magenta) and E54 (orange) are shown as spheres. (D) Superposition of the dimerization domain (Left) and substrate-binding domain (Right). Green, bcMalT wild type; blue, bcMalT-X.
- Fig. 3.
Stereoview of the maltose-binding site in bcMalT and bcMalT-X. (A) bcMalT wild type; (B) bcMalT-X. Maltose is shown as sticks with carbon in yellow and oxygen in red. Protein–maltose interactions are marked as dotted lines between the sugar and residues from HP1 (blue), HP2 (raspberry), and TM6,7,8 (light green). The C6-OH on the nonreducing end of maltose where phosphorylation happens is marked with a star.
- Fig. 4.
Conformational changes of AH2. One protomer of bcMalT (A) and bcMalT-X (B) is shown as cartoon representation. The dimerization domain is colored dark blue and the substrate-binding domain light blue. TM5 is colored green; AH2 is colored orange and TM6 is colored red. The hinge point between TM5 and AH2, P199, is marked as a magenta sphere.
- Fig. 5.
Backbone rmsd of bcMalT-X structure during all-atom MD simulations. (A) Average backbone rmsd between the simulated structure and bcMalT-X structure. (B) Average backbone rmsd between the simulated structure and bcMalT structure. Each line is the average of three independent simulations. Red, bcMalT-X; blue, bcMalT-X with the cross-linking between T280 and E54 removed; black, bcMalT CVSMD (23).
- Fig. 6.
smFRET on bcMalT. (A) bcMalT (Left) and bcMalT-X (Right) are shown as cartoon and colored according to Fig. 2A. Cβ of N288 and M340 are shown as spheres and colored red and purple respectively. The distances between the two symmetry-related residues are marked on the dotted lines. Histograms of smFRET efficiency are shown for N288C (B) and M340C (C). Solid lines are fitting of the histograms with two Gaussian functions, and the mean value of each Gaussian function (appEFRET) is tabulated in SI Appendix, Table S2.
Data supplements
Supporting Information
- Download Appendix (PDF)
- Download Movie_S01 (MP4) - Conformational changes of bcMalT from the outward-facing to the inwardfacing conformation. Crystal structures of bcMalT in outward-facing and inward-facing states are aligned by their dimerization domains. Using these two structures as ending states, a morph is generated showing the conformational changes of bcMalT between the two states feature a large rigid body movement of the substrate binding domain. The two protomers of bcMalT are colored blue and green, respectively. Within each protomer, the interface domain is colored dark blue or dark green while the transport domain light blue and light green.
Structure of an EIIC sugar transporter trapped in an inward-facing conformation
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- Biochemistry
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