Dynamics of Co-translational Membrane Protein Integration and Translocation via the Sec Translocon

Abstract

An important aspect of cellular function is the correct targeting and delivery of newly synthesized proteins. Central to this task is the machinery of the Sec translocon, a transmembrane channel that is involved in both the translocation of nascent proteins across cell membranes and the integration of proteins into the membrane. Considerable experimental and computational effort has focused on the Sec translocon and its role in nascent protein biosynthesis, including the correct folding and expression of integral membrane proteins. However, the use of molecular simulation methods to explore Sec-facilitated protein biosynthesis is hindered by the large system sizes and long (i.e., minute) timescales involved. In this work, we describe the development and application of a coarse-grained simulation approach that addresses these challenges and allows for direct comparison with both in vivo and in vitro experiments. The method reproduces a wide range of experimental observations, providing new insights into the underlying molecular mechanisms, predictions for new experiments, and a strategy for the rational enhancement of membrane protein expression levels.

Coordination of -1 Programmed Ribosomal Frameshifting by Transcript and Nascent Chain Features Revealed by Deep Mutational Scanning

“…Nevertheless, it should also be noted that the translation of this segment occurs as TM2 begins to emerge from the exit tunnel and interact with the translocon and/ or membrane. Modifications that alter the translation kinetics within this region could therefore also impact the membrane integration of TM2 and pulling forces on the nascent chain (Niesen et al, 2020). To evaluate the potential role of translational kinetics, we calculated mKate: eGFP intensity ratios associated with each individual codon substitution, then sorted the values for each codon according to the relative abundance of their decoding tRNAs in HEK293 cells (see Methods ).…”

“…Mutations that increase the relative abundance of decoding tRNAs and accelerate translation could potentially decrease - 1PRF by reducing the efficiency of translocon-mediated membrane integration (Niesen et al, 2020). To assess how translational kinetics impact the membrane integration of TM2, we carried out a series of CGMD simulations of polyprotein biosynthesis in which the rate of translation was varied.…”

“…However, we find that -1PRF is also sensitive to numerous mutations within the ~160 base region preceding the slip-site, which encode a pair of transmembrane (TM) domains that occupy the ribosomal exit tunnel and translocon during frameshifting. In conjunction with a suite of atomistic and coarse-grained molecular dynamics (CGMD) simulations (Niesen et al, 2020), we show that many mutations within this region attenuate -1PRF through their effects on a folding intermediate that forms during translocon-mediated membrane integration of the nascent polyprotein. We also find that -1PRF appears to be sensitive to changes in the translation kinetics within the region immediately preceding the slip-site, which influences the pulling forces generated by the cotranslational folding of the nascent chain.…”

Molecular Modeling of Signal Peptide Recognition by Eukaryotic Sec Complexes

“…Since the structural changes of the translocon are essential for translocon-assisted protein insertion, many MD simulation studies focused on the conformational changes of the prokaryotic and eukaryotic Sec channels [ 15 , 36 , 37 , 38 , 39 , 40 ]. Furthermore, MD simulation studies explored how the Sec61 channel recognizes substrate peptides and directs them either to membrane insertion or translocation [ 41 , 42 , 43 , 44 , 45 , 46 ]. The contribution of thermodynamics and kinetics and the detailed role of the translocon in the exit mechanism were also explored by 2D and 3D coarse-grained simulations [ 47 , 48 ].…”

“…Their simulation results also demonstrated that increasing the length of the C-terminal loop of the TM segment raises the probability that the TM segment adopts a type II topology (N /C ). Their studies helped to elucidate the effects of sequence, translation rate, and external forces on the probability of nascent-chain integration into the membrane and to rationalise the resulting orientation of TMDs with respect to the membrane [ 44 ]. Besides, by combining site-directed mutagenesis in the yeast Sec61 translocon (in vivo) and MD simulations of SecY, it was shown that the membrane insertion propensity of signal-anchor sequences strongly depends on the positions of hydrophobic residues in the sequence segment and on its interaction with the six pore ring residues [ 41 ].…”