Long-timescale dynamics and regulation of Sec-facilitated protein translocation

Abstract

We present a coarse-grained modeling approach that spans the nanosecond- to minute-timescale dynamics of cotranslational protein translocation. The method enables direct simulation of both integral membrane protein topogenesis and transmembrane domain (TM) stop-transfer efficiency. Simulations reveal multiple kinetic pathways for protein integration, including a mechanism in which the nascent protein undergoes slow-timescale reorientation, or flipping, in the confined environment of the translocon channel. Competition among these pathways gives rise to the experimentally observed dependence of protein topology on ribosomal translation rate and protein length. We further demonstrate that sigmoidal dependence of stop-transfer efficiency on TM hydrophobicity arises from local equilibration of the TM across the translocon lateral gate, and it is predicted that slowing ribosomal translation yields decreased stop-transfer efficiency in long proteins. This work reveals the balance between equilibrium and nonequilibrium processes in protein targeting, and it provides insight into the molecular regulation of the Sec translocon.

Figure 1. Structural features of the co-translational Sec machinery

The ribosome (brown) is shown in complex with the Sec translocon (green). The CG model projects the protein nascent chain dynamics onto the plane (red) that intersects the translocon channel axis and that bisects the lateral gate (LG) helices (dark green). (Inset) The CG model includes beads for the translocon (green), the ribosome (brown), and the protein nascent chain. The LG helices are shown in dark green, the ribosome exit channel is shown in red, and the lipid membrane is shown in blue. The nascent chain is composed of beads for the SP (yellow and blue) and the mature domain (gray).

Figure 2. Kinetic pathways for Type II and Type III membrane integration of signal anchor proteins obtained from direct CG simulations

The coloring scheme is described in Figure 1.

Figure 3. CG simulation results for integral membrane protein topogenesis

(A–C) Fraction of Type II integration as a function of protein MDL, with data sets that vary with respect to (A) SP charge distribution, (B) SP hydrophobicity, and (C) ribosomal translation rate. (D) Fraction of CG trajectories that follow the Type II loop pathway (red), Type II flipping pathway (blue), and the Type III pathway for membrane integration (white). (E) The distribution of arrival times for CG trajectories at state f of Type II integration via the loop pathway (red) and the flipping pathway (blue). (F) MDL-dependence of the fraction of CG trajectories that follow each integration mechanism. Unless otherwise specified, error bars throughout the paper represent the standard deviation of the mean.

Figure 4. Kinetic pathways for co-translational protein translocation and membrane integration obtained from direct CG simulations

The H-domain of the protein nascent chain is shown in blue and yellow. The full N-terminal anchor domain of the protein nascent chain is not shown here; the full system is shown in Figure S21.

Figure 5. CG simulation results for TM partitioning

(A) Stop-transfer efficiency as a function of H-domain hydrophobicity. (B) Dependence of stop-transfer efficiency upon (B₁) slowing ribosomal translation rate from 24 res/s to 6 res/s, (B₂) including explicit lumenal BiP binding, (B₃) increasing the CTL from 75 residues to 105 residues, and (B₄) replacing the hydrophobic beads in the protein C-terminal domain with hydrophilic beads; in each sub-panel, the dashed line corresponds to the sigmoidal fit of the data in (A). (C) Equilibrium transition rates between the states in Figure 4 as a function of H-domain hydrophobicity. For each color, the forward rate is indicated with the solid line, and the reverse rate is indicated with dashed line. (D) Dependence of stop-transfer efficiency on CTL and the ribosomal translation rate, obtained for protein sequences with H-domain transfer FE of ΔG = −1.25k_BT.