Substrate-binding guides individual melibiose permeases MelB to structurally soften and to destabilize cytoplasmic middle-loop C3

• Published in: Structure (London) Vol. 31; no. 1; pp. 58 - 67.e4
• Main Authors: Blaimschein, Nina, Hariharan, Parameswaran, Manioglu, Selen, Guan, Lan, Müller, Daniel J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Ltd 05.01.2023
• Subjects: atomic force microscopy; Cations; co-substrate binding; functional states; Ion Transport; mechanical stability; MelB; Melibiose - chemistry; melibiose permease; Membrane Transport Proteins; population of states; single-molecule force spectroscopy; Sodium - metabolism; structural destabilization. stability of C3 loop; substrate binding; Symporters - metabolism; melibiose permease; substrate binding; functional states; co-substrate binding; mechanical stability; population of states; MelB; single-molecule force spectroscopy; atomic force microscopy; structural destabilization. stability of C3 loop
• ISSN: 0969-2126; 1878-4186; 1878-4186
• DOI: 10.1016/j.str.2022.11.011

The melibiose permease MelB is a well-studied Na+-coupled transporter of the major facilitator superfamily. However, the symport mechanism of galactosides and cations is still not fully understood, especially at structural levels. Here, we use single-molecule force spectroscopy to investigate substrate-induced structural changes of MelB from Salmonella typhimurium. In the absence of substrate, MelB equally populates two different states, from which one shows higher mechanical structural stability with additional stabilization of the cytoplasmic middle-loop C3. In the presence of either melibiose or a coupling Na+-cation, however, MelB increasingly populates the mechanically less stable state, which shows a destabilized middle-loop C3. In the presence of both substrate and co-substrate, this mechanically less stable state of MelB is predominant. Our findings describe how both substrates guide MelB transporters to populate two different mechanically stabilized states, and contribute mechanistic insights to the alternating-access action for the galactoside/cation symport catalyzed by MelB. [Display omitted] •Mechanical stability of structural regions within MelB analyzed•MelB stabilizes two different mechanically stable states•Upon substrate-binding, MelB populates one state higher•With bound substrate and co-substrate MelB populates predominantly one state Blaimschein et al. determine the mechanical stability of single melibiose permeases MelB of the major facilitator superfamily in the absence and presence of substrate(s). The findings describe how MelB transporters mechanically stabilize and populate different states and contribute mechanistic insights to the alternating-access model of MelB for the galactoside/cation symport.