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

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 struc-tural 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.

Automated summary

In this study, single-molecule force spectroscopy was used to investigate the substrate-induced structural changes of MelB from Salmonella typhimurium. The results showed that in the absence of substrate, MelB equally populated two different states, with one state showing higher mechanical structural stability. However, in the presence of melibiose or a coupling Na+-cation, MelB increasingly populated a mechanically less stable state. In the presence of both substrate and co-substrate, this mechanically less stable state of MelB was predominant. These findings provide mechanistic insights into the symport action of galactosides and cations catalyzed by MelB.