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Regulation of multispanning membrane protein topology via post-translational annealing.

Van Lehn RC, Zhang B, Miller TF - Elife (2015)

Bottom Line: This work reveals a mechanism for regulating membrane-protein topogenesis, in which initially misintegrated configurations of the proteins undergo post-translational annealing to reach fully integrated multispanning topologies.The energetic barriers associated with this post-translational annealing process enforce kinetic pathways that dictate the topology of the fully integrated proteins.The proposed mechanism agrees well with the experimentally observed features of EmrE topogenesis and provides a range of experimentally testable predictions regarding the effect of translocon mutations on membrane protein topogenesis.

View Article: PubMed Central - PubMed

Affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, United States.

ABSTRACT
The canonical mechanism for multispanning membrane protein topogenesis suggests that protein topology is established during cotranslational membrane integration. However, this mechanism is inconsistent with the behavior of EmrE, a dual-topology protein for which the mutation of positively charged loop residues, even close to the C-terminus, leads to dramatic shifts in its topology. We use coarse-grained simulations to investigate the Sec-facilitated membrane integration of EmrE and its mutants on realistic biological timescales. This work reveals a mechanism for regulating membrane-protein topogenesis, in which initially misintegrated configurations of the proteins undergo post-translational annealing to reach fully integrated multispanning topologies. The energetic barriers associated with this post-translational annealing process enforce kinetic pathways that dictate the topology of the fully integrated proteins. The proposed mechanism agrees well with the experimentally observed features of EmrE topogenesis and provides a range of experimentally testable predictions regarding the effect of translocon mutations on membrane protein topogenesis.

No MeSH data available.


Related in: MedlinePlus

The average loop positions from the EOT ensemble of six EmrE mutants is presented in terms of the fraction of configurations for which each loop occupies the cytoplasm.The first column (dark gray) shows loop positions from the control simulation protocol. To test the effect of the hydrophobicity of TMD1, the second column (blue) presents results in which all four beads in TMD1 are modeled with the very hydrophobic L CG bead-type. This modification is seen to have a very minor effect on the EOT ensemble. To test the effect of negatively charged EmrE residues in the simulation, the third column (red) presents results in which a charge of −2 is assigned to bead 4 in loop L2 and bead 3 in loop L4, thus matching the profile of negative charges in the EmrE sequence (Figure 1—figure supplement 1). The figure shows that including negative charges consistently shifts the positions of loop L4 toward the periplasm and L5 toward the cytoplasm. The small magnitude of these shifts and their uniformity across all mutants suggests that the negative charges in EmrE play a small role in understanding the shifts in topology of the mutants studied here.DOI:http://dx.doi.org/10.7554/eLife.08697.015
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fig5s1: The average loop positions from the EOT ensemble of six EmrE mutants is presented in terms of the fraction of configurations for which each loop occupies the cytoplasm.The first column (dark gray) shows loop positions from the control simulation protocol. To test the effect of the hydrophobicity of TMD1, the second column (blue) presents results in which all four beads in TMD1 are modeled with the very hydrophobic L CG bead-type. This modification is seen to have a very minor effect on the EOT ensemble. To test the effect of negatively charged EmrE residues in the simulation, the third column (red) presents results in which a charge of −2 is assigned to bead 4 in loop L2 and bead 3 in loop L4, thus matching the profile of negative charges in the EmrE sequence (Figure 1—figure supplement 1). The figure shows that including negative charges consistently shifts the positions of loop L4 toward the periplasm and L5 toward the cytoplasm. The small magnitude of these shifts and their uniformity across all mutants suggests that the negative charges in EmrE play a small role in understanding the shifts in topology of the mutants studied here.DOI:http://dx.doi.org/10.7554/eLife.08697.015

Mentions: In the CG model, each TMD is represented by four CG beads and each soluble loop is represented by five CG beads, as seen in Figure 1A. The CG beads assume one of four types as determined by the associated amino-acid residues in the nascent protein; these CG bead-types include V (moderately hydrophobic), L (very hydrophobic), Q (neutral-hydrophilic), and K (positively charged). Among these types, the CG beads vary with respect to their charge and their water/membrane transfer free energies (Appendix table 1). In the hydropathy profile, the N-terminal TMD (TMD1) is less hydrophobic than the other three TMDs, so its beads are assigned the V bead type. All other TMD beads are assigned the L bead type. Beads in each soluble loop are assigned to either the K or Q bead type, depending on the location of positive charges in the amino-acid sequence; positive charges are highlighted in red in the EmrE wild-type amino-acid sequence in Figure 1—figure supplement 1. Each K bead type is assigned a +2 charge, following previous work (Zhang and Miller, 2012a). Negative charges are excluded from the CG representation of EmrE, because EmrE exhibits a small number of such charges (Figure 1—figure supplement 1) and because the experimentally studied EmrE mutations focus only on the addition/removal of positively charged residues (Seppälä et al., 2010). Nonetheless, the effect of negatively charged residues in the CG simulation was explicitly tested in Figure 5—figure supplement 1 and was found to be minor. Similarly, the results of the simulations are robust with respect to changes in the modeling of TMD1 hydrophobicity (Figure 5—figure supplement 1) and loop length (Figure 3—figure supplement 3).


Regulation of multispanning membrane protein topology via post-translational annealing.

Van Lehn RC, Zhang B, Miller TF - Elife (2015)

The average loop positions from the EOT ensemble of six EmrE mutants is presented in terms of the fraction of configurations for which each loop occupies the cytoplasm.The first column (dark gray) shows loop positions from the control simulation protocol. To test the effect of the hydrophobicity of TMD1, the second column (blue) presents results in which all four beads in TMD1 are modeled with the very hydrophobic L CG bead-type. This modification is seen to have a very minor effect on the EOT ensemble. To test the effect of negatively charged EmrE residues in the simulation, the third column (red) presents results in which a charge of −2 is assigned to bead 4 in loop L2 and bead 3 in loop L4, thus matching the profile of negative charges in the EmrE sequence (Figure 1—figure supplement 1). The figure shows that including negative charges consistently shifts the positions of loop L4 toward the periplasm and L5 toward the cytoplasm. The small magnitude of these shifts and their uniformity across all mutants suggests that the negative charges in EmrE play a small role in understanding the shifts in topology of the mutants studied here.DOI:http://dx.doi.org/10.7554/eLife.08697.015
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4635508&req=5

fig5s1: The average loop positions from the EOT ensemble of six EmrE mutants is presented in terms of the fraction of configurations for which each loop occupies the cytoplasm.The first column (dark gray) shows loop positions from the control simulation protocol. To test the effect of the hydrophobicity of TMD1, the second column (blue) presents results in which all four beads in TMD1 are modeled with the very hydrophobic L CG bead-type. This modification is seen to have a very minor effect on the EOT ensemble. To test the effect of negatively charged EmrE residues in the simulation, the third column (red) presents results in which a charge of −2 is assigned to bead 4 in loop L2 and bead 3 in loop L4, thus matching the profile of negative charges in the EmrE sequence (Figure 1—figure supplement 1). The figure shows that including negative charges consistently shifts the positions of loop L4 toward the periplasm and L5 toward the cytoplasm. The small magnitude of these shifts and their uniformity across all mutants suggests that the negative charges in EmrE play a small role in understanding the shifts in topology of the mutants studied here.DOI:http://dx.doi.org/10.7554/eLife.08697.015
Mentions: In the CG model, each TMD is represented by four CG beads and each soluble loop is represented by five CG beads, as seen in Figure 1A. The CG beads assume one of four types as determined by the associated amino-acid residues in the nascent protein; these CG bead-types include V (moderately hydrophobic), L (very hydrophobic), Q (neutral-hydrophilic), and K (positively charged). Among these types, the CG beads vary with respect to their charge and their water/membrane transfer free energies (Appendix table 1). In the hydropathy profile, the N-terminal TMD (TMD1) is less hydrophobic than the other three TMDs, so its beads are assigned the V bead type. All other TMD beads are assigned the L bead type. Beads in each soluble loop are assigned to either the K or Q bead type, depending on the location of positive charges in the amino-acid sequence; positive charges are highlighted in red in the EmrE wild-type amino-acid sequence in Figure 1—figure supplement 1. Each K bead type is assigned a +2 charge, following previous work (Zhang and Miller, 2012a). Negative charges are excluded from the CG representation of EmrE, because EmrE exhibits a small number of such charges (Figure 1—figure supplement 1) and because the experimentally studied EmrE mutations focus only on the addition/removal of positively charged residues (Seppälä et al., 2010). Nonetheless, the effect of negatively charged residues in the CG simulation was explicitly tested in Figure 5—figure supplement 1 and was found to be minor. Similarly, the results of the simulations are robust with respect to changes in the modeling of TMD1 hydrophobicity (Figure 5—figure supplement 1) and loop length (Figure 3—figure supplement 3).

Bottom Line: This work reveals a mechanism for regulating membrane-protein topogenesis, in which initially misintegrated configurations of the proteins undergo post-translational annealing to reach fully integrated multispanning topologies.The energetic barriers associated with this post-translational annealing process enforce kinetic pathways that dictate the topology of the fully integrated proteins.The proposed mechanism agrees well with the experimentally observed features of EmrE topogenesis and provides a range of experimentally testable predictions regarding the effect of translocon mutations on membrane protein topogenesis.

View Article: PubMed Central - PubMed

Affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, United States.

ABSTRACT
The canonical mechanism for multispanning membrane protein topogenesis suggests that protein topology is established during cotranslational membrane integration. However, this mechanism is inconsistent with the behavior of EmrE, a dual-topology protein for which the mutation of positively charged loop residues, even close to the C-terminus, leads to dramatic shifts in its topology. We use coarse-grained simulations to investigate the Sec-facilitated membrane integration of EmrE and its mutants on realistic biological timescales. This work reveals a mechanism for regulating membrane-protein topogenesis, in which initially misintegrated configurations of the proteins undergo post-translational annealing to reach fully integrated multispanning topologies. The energetic barriers associated with this post-translational annealing process enforce kinetic pathways that dictate the topology of the fully integrated proteins. The proposed mechanism agrees well with the experimentally observed features of EmrE topogenesis and provides a range of experimentally testable predictions regarding the effect of translocon mutations on membrane protein topogenesis.

No MeSH data available.


Related in: MedlinePlus