Role of the Cytosolic Loop C2 and the C Terminus of YidC in Ribosome Binding and Insertion Activity

Structural Dynamics of the YidC:Ribosome Complex during Membrane Protein Biogenesis

“…YidC TM6 is positioned in close proximity to ribosomal H59, with a strong connecting density in between, that has been assigned to the C-terminal tail of YidC that builds a primary contact with the ribosome (Kedrov et al., 2013, Seitl et al., 2014). Another contact site is resolved between ribosomal proteins L23/L29 and YidC TM4-TM5 positioned underneath, and the short loop that connects these two TMs has recently been shown to modulate ribosome binding (Geng et al., 2015). Additionally, we observe an extension at the ribosomal protein L24, which is large enough to fit up to three α-helical turns.…”

“…FCS measurements were conducted using a home-built setup (Crevenna et al., 2013) as previously described (Geng et al., 2015), using fluorescent markers conjugated either to YidC or DOPE lipids. Each individual measurement was conducted for 100 s, so data from ∼100,000 to ∼300,000 diffusing molecules (average residence time ∼300 μs) were accumulated to build an auto-correlation trace, and each measurement was repeated at least three times.…”

“…Each individual measurement was conducted for 100 s, so data from ∼100,000 to ∼300,000 diffusing molecules (average residence time ∼300 μs) were accumulated to build an auto-correlation trace, and each measurement was repeated at least three times. Association of the YidC-ND (radius ∼5 nm) with RNC F o c (radius ∼13 nm) slowed the translational diffusion of nanodiscs and prolonged their average residence time within the laser confocal volume of the FCS setup (Kedrov et al., 2013, Geng et al., 2015), so YidC-ND:ribosome interactions caused a shift of the auto-correlation curve along the time axis. Binding efficiencies were estimated from two-component fitting of auto-correlation traces, and diffusion coefficients/residence times of free nanodiscs and ribosomes were measured and used as parameters for fitting as previously described (Kedrov et al., 2013, Wu et al., 2012).…”

“…Initial substrate recognition is believed to occur at the cytoplasmic helical hairpin CH1-CH2 that connects TM2 and TM3 in E. coli YidC and caps the hydrophilic groove of the idle insertase (Kumazaki et al., 2014a, Kumazaki et al., 2014b). Deletions or mutations within CH1-CH2 lead to the loss of cellular viability (Chen et al., 2014, Wickles et al., 2014, Geng et al., 2015). Similarly, deletions within TM2 and TM3 of YidC have lethal effects on cells (Jiang et al., 2003), and these TMs have been described as the functional core of the insertase that interacts with the substrate upon its insertion (Kumazaki et al., 2014a).…”

“…Similar to the SecYEG system, YidC specifically interacts with ribosomes that expose hydrophobic nascent chains (Kedrov et al., 2013, Wu et al., 2013). The positively charged C terminus and a short cytoplasmic loop connecting TM4 and TM5 facilitate this interaction (Geng et al., 2015), while the YidC variant lacking the C terminus (YidCΔC) is impaired in ribosome binding (Kedrov et al., 2013). …”