Forces on Nascent Polypeptides during Membrane Insertion and Translocation via the Sec Translocon

Abstract

During ribosomal translation, nascent polypeptide chains (NCs) undergo a variety of physical processes that determine their fate in the cell. This study utilizes a combination of arrest peptide experiments and coarse-grained molecular dynamics to measure and elucidate the molecular origins of forces that are exerted on NCs during cotranslational membrane insertion and translocation via the Sec translocon. The approach enables deconvolution of force contributions from NC-translocon and NC-ribosome interactions, membrane partitioning, and electrostatic coupling to the membrane potential. In particular, we show that forces due to NC-lipid interactions provide a readout of conformational changes in the Sec translocon, demonstrating that lateral gate opening only occurs when a sufficiently hydrophobic segment of NC residues reaches the translocon. The combination of experiment and theory introduced here provides a detailed picture of the molecular interactions and conformational changes during ribosomal translation that govern protein biogenesis.

Figure 1

Characterization of the physical processes that drive integration of a hydrophobic transmembrane domain. (a) CGMD simulation setup used to calculate pulling forces acting on an engineered hydrophobic H-segment (orange) during cotranslational integration. Shown is a CGMD snapshot at $L=28$; the C-terminal bead is held fixed, and forces exerted by the nascent protein on that bead are calculated. (b) Experimental data reproduced from (26). Two peaks in the pulling-force profile are observed during the cotranslational integration of the hydrophobic H-segment. (c) CGMD data for H-segments of varying Leucine content. Vertical dashed lines indicate the position of the corresponding peaks in the experimental results. (d–f) Representative CGMD configurations at $L=28$ (d), $L=39$ (e), and $L=57$ (f). (g) CGMD pulling-force profiles for an H-segment with nine leucine residues with default interactions (orange), nonspecific lipid interactions (teal), and nonspecific channel interactions (purple). (h) The maximal value of $f_{\text{FL}}$ for the peak near $L=28$ (teal) and the peak near $L=39$ (purple), obtained from CGMD (solid lines) and experiment (26) (dashed lines). Fig. S2 provides an alternate version of this figure for which the simulated curves are scaled to enable easier comparison. Error bars indicate the mean ± SE. To see this figure in color, go online.

Figure 2

Forces exerted on hydrophobic segments of variable length. (a) An CGMD snapshot for an H-segment with eight leucine residues (orange), stalled at $L+n=46$. The pulling-force profile determined from CGMD (b) and from experiment (c) for poly-leucine H-segments with increasing numbers of leucine residues is shown. (d) A CGMD pulling-force profile for an H-segment with eight leucine residues with default interactions (orange), nonspecific lipid interactions (teal), and nonspecific channel interactions (purple). (e) The maximal value of $f_{\text{FL}}$ from CGMD in which the peaks were isolated as shown (d). (f) Location of the lipid-interaction peak in the CGMD pulling-force profile as a function of $nLeu$. For poly-leucine H-segments (purple) and for 19-residue H-segments consisting of alanine and leucine (black). The dashed lines correspond to the $L+n$ value at which the channel interaction peak (teal) and the lipid interaction peak (purple) are observed for the fully spanning transmembrane domains in the fixed-length assay. Error bars indicate the mean ± SE. To see this figure in color, go online.

Figure 3

Forces exerted on hydrophilic H-segments. The pulling-force profile determined from experiment (29) (a) and from CGMD (b) for negatively charged (D₅, orange), positively charged (K₅, purple), and neutral (Q₅, teal) five-residue H-segments. (c) As in (b), but for CGMD simulations without a membrane potential. (d–f) CGMD snapshot for a D₅ H-segment (orange), stalled at $L+n=31$ (d), $L+n=49$ (e), and $L+n=67$ (f). (g) The pulling-force profile for a D₅ H-segment with (orange) and without (black) ribosomal charges. (h) The pulling-force profile for a D₅ H-segment with the original C-terminal loop (orange) and with a mutated C-terminal loop that is more hydrophilic (purple). Error bars indicate the mean ± SE. To see this figure in color, go online.