Regulatory role of charged clusters in the N-terminal domain of BetP from Corynebacterium glutamicum

Abstract

The trimeric transporter BetP counteracts hyperosmotic stress by a fast increase in transport rate in order to accumulate the compatible solute betaine. The positively charged α-helical C-terminal domain acts as an osmosensor perceiving the increase in the internal potassium (K+) concentration. A second, still unidentified stimulus originates from stress-induced changes in the physical state of the membrane and depends on the amount of negatively charged lipids. BetP possesses a 60-amino acid (aa)-long negatively charged N-terminal domain, which is predicted to adopt a partly helical fold affecting osmoregulation by an unknown mechanism. It is assumed that the C-terminal domain, the N-terminal domain, and negatively charged lipids interact during stress sensing and regulation. Here, we have investigated the regulatory role of negatively charged clusters in the N-terminal domain. We identified one cluster, Glu24Glu25, to be crucial for osmoregulation. Cross-linking studies revealed an interaction between the C- and N-terminal domains of adjacent protomers modulating transport activation. A regulatory partner-switching mechanism emerges in which the C-terminal domain changes its interaction with the N-terminal domain of its own promoter and negatively charged lipids to an interaction with the N-terminal domain of an adjacent protomer and lipids bound to the central cavity of the BetP trimer.

Tales of tails in transporters

“…A dynamic interaction between the C- and N-terminal domains of adjacent protomers has been shown to modulate transport activation. In brief, it has been proposed that the C-terminal domain changes its interaction with the N-terminal domain of its own promoter and negatively charged lipids to an interaction with the N-terminal domain of an adjacent protomer and lipids bound to the central cavity of the BetP trimer [165,166]. Another paradigm of transporter tails involved in functional interactions with PM lipids is that of the human dopamine transporter hDAT, which contains long intracellular N- and C-terminal domains that seem to be implicated in the transporter function, and possibly oligomerization, in vivo [48,167].…”

Identification of 121 variants of honey bee Vitellogenin protein sequences with structural differences at functional sites

“…A positively charged C‐terminal region in other proteins has been linked to signaling for recruitment and translocation, 72 protein assembly, 73 and sensing changes in the extracellular environment. 74 Honey bee Vg has been demonstrated to sense oxidative stress 75 and suggested protecting honey bees from reactive oxidative species. Our earlier study shows that two disulfide bridges are conserved in the C‐terminal region, which is proposed to coordinate Zn 2+ (Leipart et al 2021 in manuscript 44 ) (Figure 7a ).…”

“…We additionally provide new evidence showing a conserved positively charged surface (Figure 7c). A positively charged C‐terminal region in other proteins has been linked to signaling for recruitment and translocation, 72 protein assembly, 73 and sensing changes in the extracellular environment 74 . Honey bee Vg has been demonstrated to sense oxidative stress 75 and suggested protecting honey bees from reactive oxidative species.…”

Autoregulation of a trimeric transporter involves the cytoplasmic domains of both adjacent subunits

“…Secondary structure prediction methods suggest that this tail contains small segments of helix among regions that lack structural definition (9,13). Accordingly, in the protein construct required for crystallographic studies, the first 29 amino acids were truncated and three glutamates (residues [44][45][46] were mutated to alanine to increase the helical content of this region (5).…”