Helical arrangement and helical structure

Like other polytopic membrane proteins, GPCRs are partially buried in the non-polar environment of the lipid bilayer by forming a compact bundle of transmembrane helices. The correct orientation and integration of the polypeptide chain is guided by a complex translocation apparatus residing in the ER. Two different folding stages can be distinguished following an initial translocation of the receptor N terminus into the ER lumen. In stage I,

Extracellular

Extracellular

DRY (TMD3)

DRY (TMD3)

NPXXY (TMD7)

NPXXY (TMD7)

N(TMD1)

N(TMD1)

DRY (TMD3)

Fig. 1.3 Structure of Family 1 GPCRs. A two-dimensional model of Family 1 GPCRs (a) and its organization in the plasma membrane (b) are shown. The ring-like arranged seven TMDs assemble in a counterclockwise fashion as viewed from the extracellular surface. Some of the highly conserved residues are shown in enlarged circles (a). The el and the e2 loops, in some cases also the N terminus and the e3 loop, are linked by disulfide bonds (b). The recent crystal structure of rhodopsin (2.8Â resolution) shows the orientation of the TMDs relative to each other (Palczewski et al. 2000). Positions of key residues are indicated in the three-dimensional rhodopsin structure viewed from extracellular (c) or laterally (d). (See Plate 1.)

hydrophobic a-helices are established across the lipid bilayer, and protein folding is predominantly driven by the hydrophobic effect. The TMDs adopt a secondary structure in order to minimize the polar surface area exposed to the lipid environment with the result that hydrophobic amino acids face the lipid bilayer and that more hydrophilic amino acid residues are orientated towards the core crevice of the TMD bundle. In stage II, a functional tertiary structure is formed by establishing specific helix-helix interactions, leading to the tightly packed, ring-like structure of the TMD bundle.

In the early stage of GPCR structure/function analysis, investigators used the structure of bacteriorhodopsin, a prokaryotic ion pump with structural similarities to the GPCR super-family, as a scaffold for topographical models of the transmembrane core of GPCRs (Baldwin 1994; see Chapter 3 for more detail). The identification of specific interhelical contact sites was required to provide information about the relative orientation of the different helices towards each other. To determine the structural determinants which actually contribute to specific helix-helix interactions, chimeric receptors were generated. Studies with chimeric muscarinic receptors provided the first experimental evidence as to how TMD1 and TMD7 are oriented relative to each other and also strongly suggested that the TMD helices in muscarinic receptors are arranged in a counterclockwise fashion as viewed from the extracellular membrane surface (Liu et al. 1995). Functional analysis of artificial metal ion-binding sites (Elling et al. 1995) and disulfide bonds (Farrens et al. 1996) as well as spectroscopic approaches (Beck et al. 1998) allowed the identification of distinct amino acid residues that are involved in helix-helix contacts and the relative orientation of single TMDs to each other. Finally, the proposed helical arrangement was confirmed by structural data from rhodop-sin crystallography highlighting the feasibility of mutagenesis approaches. Interestingly, the overall a-helical character of TMDs is often disrupted by non-a-helical components, such as intrahelical kinks (often due to residues other than proline), 310-helices and n-helices (Riek etal. 2001).

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