Abstract
The YidC family of proteins are membrane insertases that catalyze the translocation of the periplasmic domain of membrane proteins via a hydrophilic groove located within the inner leaflet of the membrane. All homologs have a strictly conserved, positively charged residue in the center of this groove. In Bacillus subtilis, the positively charged residue has been proposed to be essential for interacting with negatively charged residues of the substrate, supporting a hypothesis that YidC catalyzes insertion via an early-step electrostatic attraction mechanism. Here, we provide data suggesting that the positively charged residue is important not for its charge but for increasing the hydrophilicity of the groove. We found that the positively charged residue is dispensable for Escherichia coli YidC function when an adjacent residue at position 517 was hydrophilic or aromatic, but was essential when the adjacent residue was apolar. Additionally, solvent accessibility studies support the idea that the conserved positively charged residue functions to keep the top and middle of the groove sufficiently hydrated. Moreover, we demonstrate that both the E. coli and Streptococcus mutans YidC homologs are functional when the strictly conserved arginine is replaced with a negatively charged residue, provided proper stabilization from neighboring residues. These combined results show that the positively charged residue functions to maintain a hydrophilic microenvironment in the groove necessary for the insertase activity, rather than to form electrostatic interactions with the substrates.
Highlights
The YidC/Oxa1/Alb3 proteins are found in bacteria, mitochondria, and chloroplast where they play a pivotal role in membrane protein biogenesis [1, 2]
In 2014, the structures of YidC from Bacillus halodurans [18] and Escherichia coli [19] were solved at high resolution using X-ray crystallography. These structures showed that the YidC protein possesses a five TM segment core domain (TM2TM6 in the case of the E. coli YidC) with an unusual hydrophilic cavity located within the inner leaflet of the membrane that is accessible from the cytoplasm and lipid bilayer, but not from the periplasm
Similar results were seen with the Pf3-23Lep substrate, which has the leader peptidase (Lep) residues 23 to 323 fused after the TM segment of Pf3 coat and an arginine introduced after the TM of Pf3-23Lep to prevent translocation of the Lep C-terminal domain [24] (Fig. 1C)
Summary
The conserved positively charged residue R366 of E. coli YidC becomes functionally indispensable by mutation of tyrosine 517. We hypothesize that the positively charged residue is required to maintain a hydrophilic environment in the groove To test this hypothesis, we examined the solvent accessibility of nine previously determined groove residues under WT and mutated conditions in the intact bacterial cells at 30 C using a well-developed cysteine alkylation assay [25]. The top panel shows that the groove is mostly aqueous under WT condition, while a cluster of red and yellow residues can be observed at the dome of the groove under mutated condition as revealed by the bottom panel, implying that the top part of the groove is not hydrated in these cases To confirm these results, we performed electron paramagnetic resonance (EPR) power saturation studies to examine solvent accessibility of a spin label incorporated into the hydrophilic groove.
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