Structure of the Mth ectodomain. (A) Ribbon diagram of the Mth structure (D1, red; D2, green; D3, blue). Ordered N-linked carbohydrates are shown in cyan in ball-and-stick representation and disulfide bonds are yellow. (B) (Upper) Topology diagrams for three extracellular domains of Mth. Potential N-linked glycosylation sites are labeled CHO, with parentheses denoting N-linked sites in which ordered carbohydrates were not observed. (Lower) Ribbon diagrams of the three domains of Mth. A disulfide bond (yellow) and an N-linked carbohydrate (cyan) are located at corresponding positions in D1 and D3. (C) Stereoview of a superposition of D1 (in red) and D3 (in blue) made by aligning the Cα atoms of the S2-S3-S4 β-sheet.

Relation of Mth to family B GPCRs and other Mth homologs. (A) Phylogenetic relationship of transmembrane regions of GPCR family B proteins. A phylogenetic tree was generated by using the neighbor-joining method based on an alignment of transmembrane domains generated with the clustalw program (25). The human β-2 adrenergic receptor (not shown) served as the outgroup. A subfamily consisting of ≈10 Mth-like proteins identified from the Drosophila genome project clusters with Mth. CIRL, calcium-independent receptor of α-latrotoxin; BAI-1, brain-specific angiogenesis inhibitor 1; HE6, human epididymal gene product 6; SECR, secretin receptor; VIPR, vasoactive intestinal peptide receptor; PTRR, parathyroid hormone-related peptide receptor; GIPR, gastric inhibitory peptide receptor; GLUCR, glucagon receptor; CALR, calcitonin receptor. (B) Sequence alignment of the Mth ectodomain with predicted homologs from the Drosophila genome. Residue numbering begins at the first residue of the mature protein. Crystallographically determined secondary-structural elements are shown above the sequences. The 10 cysteines that form five disulfide bonds in the Mth ectodomain are highlighted in yellow, and potential N-linked glycosylation sites are highlighted in cyan.

Possible ligand binding site on Mth. (A) Stereoview of a space-filling model of Mth showing the shallow groove between D1 and D2D3. Atoms are colored according to their temperature factor, with blue and red corresponding to low (relatively well-ordered) and high temperature factors, respectively. Trp-120 (yellow), suggested to be at a ligand binding surface (see text), has a solvent accessible surface area of 126 Ų [calculated by using a 1.4-Å probe radius with areaimol (26)] compared with 238 Ų for a fully exposed tryptophan. Examples of protein–protein complexes including critical tryptophan residues at the interface include HFE/transferrin receptor (HFE Trp-81; 115 Ų; second most exposed tryptophan in HFE) (27) and FcRn/Fc (FcRn Trp-133; 136 Ų; third most exposed tryptophan in FcRn) (17). Other receptor-ligand interfaces that include surface-exposed hydrophobic side chains on one of the binding partners are discussed in ref. 17 and references therein. (B) Stereoview of Mth showing residues that are greater than 70% conserved in known Drosophila Mth homologs (excluding conserved cysteines). Asp-121 (conserved in five of the Mth-like proteins and conservatively substituted for glutamate in three others) is involved in an interdomain hydrogen bonds (purple dotted lines) with Arg-57 (conserved in all Mth homologs). (C) Experimental electron density map (MAD solvent-flattened map contoured at 2 σ) in the region near Trp-120. (D) Model for the structure of intact Mth showing relative sizes of Mth ectodomain and the transmembrane region. The Mth transmembrane region is represented by the structure of rhodopsin (21), with adjustments in loop regions to reflect differences in loop lengths between rhodopsin and Mth. Dashed lines represent the seven residues following the Mth ectodomain C terminus and the predicted start of the first transmembrane helix. The Mth ectodomain is oriented such that the C terminus of the model derived from the crystal structure is closest to the membrane. In this orientation, the interdomain groove and Trp-120 are accessible for ligand binding, and two regions containing residues (Asp-46 and Phe-153) suggested to interact with the extracellular face of the seven pass transmembrane domain are positioned near the interhelical loops.