Marginally hydrophobic transmembrane α-helices shaping membrane protein folding

Abstract

Cells have developed an incredible machinery to facilitate the insertion of membrane proteins into the membrane. While we have a fairly good understanding of the mechanism and determinants of membrane integration, more data is needed to understand the insertion of membrane proteins with more complex insertion and folding pathways. This review will focus on marginally hydrophobic transmembrane helices and their influence on membrane protein folding. These weakly hydrophobic transmembrane segments are by themselves not recognized by the translocon and therefore rely on local sequence context for membrane integration. How can such segments reside within the membrane? We will discuss this in the light of features found in the protein itself as well as the environment it resides in. Several characteristics in proteins have been described to influence the insertion of marginally hydrophobic helices. Additionally, the influence of biological membranes is significant. To begin with, the actual cost for having polar groups within the membrane may not be as high as expected; the presence of proteins in the membrane as well as characteristics of some amino acids may enable a transmembrane helix to harbor a charged residue. The lipid environment has also been shown to directly influence the topology as well as membrane boundaries of transmembrane helices-implying a dynamic relationship between membrane proteins and their environment.

Keywords: aquaporin 1; hydrophobicity; marginally hydrophobic transmembrane helices; membrane protein folding.

Figure 1

Membrane protein insertion, topology and folding A) In the two-stage model, individual transmembrane segments are partitioned into the membrane one after an other in a sequential manner. B) In non-sequential membrane integration, some transmembrane segments are not initially recognized as such (depicted in red). As a consequence, some transmembrane helices enter the membrane in an opposite orientation and the protein adopts an intermediate topology.

Figure 2

Structure of a phospholipid. A) The structure of phospholipids is illustrate with phosphatidylcholine. The head group consists of the glycerol backbone (in green), linked through a phosphate (in cyan) to a polar or charged head group (in magenta). Here this head group is choline, but it can be replaced by for instance ethanolamine in phosphatidylethanolamine. The two remaining carbons of the glycerol are attached to two fatty acids through ester linkage. The fatty acids can be saturated or unsaturated. The saturation status as well as the properties of the head group determines the physiochemical properties of a phospholipid. B) Schematic representation of lipids with different overall shapes and on how the fatty acid structure influences membrane curvature.

Figure 3

Protein machinery involved in the membrane integration of α-helical membrane proteins. A) A cartoon over the SRP-dependent cotranslational pathway. When the N-terminal signal sequence (orange) emerges, it is recognized by the Signal Recognition Particle (SRP, in green). The ribosome-nascent chain complex is brought to the translocon (cyan) through the interaction between SRP and SR-receptor (red). B) Bottom view of the translocon heterotrimer (pdb 1rh5). The alpha subunit (grey) forms a clam-shell like structure with the gamma subunit (purple) associated at its back. The beta subunit is shown in red. C) The loop between the 5th and 6th TMHs form a hinge, which allows the lateral opening of the channel. The plug (green) resides in the center of the channel, maintaining membrane permeability. The lateral gate formed by the 2nd (blue) and the 7th (gold) TMHs is highlighted.

Figure 4

Topological rearrangements in AQP1 due to a mTMH. AQP1 is initially inserted into the membrane as a four-helix intermediate (top). TMH2 (in red) cannot be inserted into the membrane initially resulting in the wrong orientation of TMH3 (in blue). Further, TMH4 can now not insert into the membrane due to a strong positive-charge bias. For AQP1 to obtain its six transmembrane domain topology (bottom), the reorientation of TMH3 is required. This reorientation requires residue Arg93 (marked with red circle) to cross the membrane. The middle panel shows a proposed “R1-H3 shift”; according to this model, TMH3 can spontaneously shift out of the membrane core, initiating topological realignment and folding of AQP1.

Figure 5

Local sequence context can aid in the insertion of a mTMH (in red). A) Positive charges in cytoplasmic loops can contribute toward the free energy for membrane integration and promote membrane insertion of a mTMH. B) Orientational preference of a subsequent TMH (in blue), induced by for example positive charges, can pull a mTMH into the membrane. C) Specific interactions (stars) between a mTMH and another transmembrane helix can allow insertion of the helix-pair. D) Repositioning can allow a segment of lower hydrophobicity to enter the membrane, even though a more hydrophobic part is initially recognized by the translocon.