The Role of a Crystallographically Unresolved Cytoplasmic Loop in Stabilizing the Bacterial Membrane Insertase YidC2

Abstract

YidC, a bacterial member of the YidC/Alb3/Oxa1 insertase family, mediates membrane protein assembly and insertion. Cytoplasmic loops are known to have functional significance in membrane proteins such as YidC. Employing microsecond-level molecular dynamics (MD) simulations, we show that the crystallographically unresolved C2 loop plays a crucial role in the structural dynamics of Bacillus halodurans YidC2. We have modeled the C2 loop and used all- atom MD simulations to investigate the structural dynamics of YidC2 in its apo form, both with and without the C2 loop. The C2 loop was found to stabilize the entire protein and particularly the C1 region. C2 was also found to stabilize the alpha-helical character of the C-terminal region. Interestingly, the highly polar or charged lipid head groups of the simulated membranes were found to interact with and stabilize the C2 loop. These findings demonstrate that the crystallographically unresolved loops of membrane proteins could be important for the stabilization of the protein despite the apparent lack of structure, which could be due to the absence of the relevant lipids to stabilize them in crystallographic conditions.

The Cytoplasmic Domains of Streptococcus mutans Membrane Protein Insertases YidC1 and YidC2 Confer Unique Structural and Functional Attributes to Each Paralog

“…In E. coli, incoming substrates have been hypothesized to interact initially with the YidC C1 loop followed by interaction with a conserved arginine residue within transmembrane domain TM2 for insertion ( Kol et al, 2008 ; Kumazaki et al, 2014a , c ). More recently, molecular dynamics simulation showed that the crystallographically unresolved cytoplasmic C2 loop of BhYidC2 plays an important role in stabilizing this insertase’s structure ( Harkey et al, 2019 ).…”

Mechanistic picture for chemo-mechanical couplings in a bacterial proton-coupled oligopeptide transporter from Streptococcus thermophilus

“…17,18,22,27 PepT St is the only POT symporter that has been crystallized in OCC, IF, and IF occ conformational states. 17,18,22,27 Hence, in this study we simulated all three available conformational states of PepT St via unbiased all-atom MD [30][31][32][33] with either E300 protonated or deprotonated. Additionally, we simulated the IF occ (LA) in all possible protonation combinations of E299, E300, and E400 residues, which resulted in a total of eight substrate bound systems (see methods for more details).…”

Table_1.DOCX

“…Unlike its C1 loop, the BhYidC2 C2 loop was not structurally resolved by X-ray crystallography (Kumazaki et al, 2014c). Molecular dynamic (MD) simulations, however, predicted a contribution of the C2 loop in stabilizing protein structure, especially in reducing flexibility of the C1 loop by formation of an inter-domain salt bridge between D205 (C2) and K109 (C1) (Harkey et al, 2019). Structural analysis of BhYidC2 also revealed that its C1 loop is assessable to the cytoplasm, while the smaller C2 loop appears to be embedded within the lipid bilayer and probably involved in an interaction with lipid heads.…”

“…In light of in silico structure predictions, combined with insight gained from phenotypic assays, we hypothesized that specific interactions between cytoplasmic domains were responsible for particular structure-function attributes. In fact, Moradi and coworkers had proposed that formation of a salt-bridge between the C1 and C2 loops of B. halodurans YidC2 appears to stabilize the flexible C1 loop (Harkey et al, 2019). When the SmYidC1 and SmYidC2 cytoplasmic domains were evaluated, both paralogs contained multiple oppositely charged residues within close enough proximity (<3 Å) to form salt-bridges (Supplementary Table 6).…”

“…In E. coli, incoming substrates have been hypothesized to interact initially with the YidC C1 loop followed by interaction with a conserved arginine residue within transmembrane domain TM2 for insertion (Kol et al, 2008;Kumazaki et al, 2014a,c). More recently, molecular dynamics simulation showed that the crystallographically unresolved cytoplasmic C2 loop of BhYidC2 plays an important role in stabilizing this insertase's structure (Harkey et al, 2019).…”