Structure-Based Sequence Alignment of the Transmembrane Domains of All Human GPCRs: Phylogenetic, Structural and Functional Implications

Abstract

The understanding of G-protein coupled receptors (GPCRs) is undergoing a revolution due to increased information about their signaling and the experimental determination of structures for more than 25 receptors. The availability of at least one receptor structure for each of the GPCR classes, well separated in sequence space, enables an integrated superfamily-wide analysis to identify signatures involving the role of conserved residues, conserved contacts, and downstream signaling in the context of receptor structures. In this study, we align the transmembrane (TM) domains of all experimental GPCR structures to maximize the conserved inter-helical contacts. The resulting superfamily-wide GpcR Sequence-Structure (GRoSS) alignment of the TM domains for all human GPCR sequences is sufficient to generate a phylogenetic tree that correctly distinguishes all different GPCR classes, suggesting that the class-level differences in the GPCR superfamily are encoded at least partly in the TM domains. The inter-helical contacts conserved across all GPCR classes describe the evolutionarily conserved GPCR structural fold. The corresponding structural alignment of the inactive and active conformations, available for a few GPCRs, identifies activation hot-spot residues in the TM domains that get rewired upon activation. Many GPCR mutations, known to alter receptor signaling and cause disease, are located at these conserved contact and activation hot-spot residue positions. The GRoSS alignment places the chemosensory receptor subfamilies for bitter taste (TAS2R) and pheromones (Vomeronasal, VN1R) in the rhodopsin family, known to contain the chemosensory olfactory receptor subfamily. The GRoSS alignment also enables the quantification of the structural variability in the TM regions of experimental structures, useful for homology modeling and structure prediction of receptors. Furthermore, this alignment identifies structurally and functionally important residues in all human GPCRs. These residues can be used to make testable hypotheses about the structural basis of receptor function and about the molecular basis of disease-associated single nucleotide polymorphisms.

Fig 1. TM domains of the available crystal structures.

Top: Two views of the 24 inactive crystal structures from classes A, B, C, and F (aligned to β₂) show the general GPCR fold of the transmembrane (TM) bundle. Class A in green, class B in blue (CRF1, GLR), class C in orange (MGLU1, MGLU5), class F in magenta (SMO). Bottom: Same views for only the 19 inactive class A structures showing the highly conserved class A TM fold. A detailed view of the conserved hydrogen bonding networks is shown in S1 Fig.

Fig 2. Conserved inter-helical contacts.

Top left: Diagram of 40 conserved inter-helical contacts (CHICOs) present in at least 23 out of 24 studied class A structures. The contacts common to all classes are shown in purple, and contacts present only in class A in orange. Top right: List of these contacts in Ballesteros-Weinstein numbering scheme. Bottom: Extracellular view of the same contacts in the β₂ crystal structure. The contacts in the inner and outer half of the membrane are shows on the left and right respectively.

Fig 3. Testing the robustness of the alignment of the Vomeronasal receptors with the other groups.

The table shows similarity between TMs averaged over all pairs of sequences formed from the two groups (red denotes high similarity, blue low similarity). For most TMs the optimal choices agree with the optimal alignment to Aα (full table in S5 Fig); all combinations are shown only for TM5. The same table but using the GPCRtm substitution matrix [74] instead of BLOSUM62 is shown in S7 Fig. GPCRtm was developed in particular for GPCR proteins, but in this case both matrices result in the same alignment.

Fig 4. Testing the robustness of the alignment of the Taste2 receptors with the other groups.

The table shows similarity between TMs averaged over all pairs of sequences formed from the two groups (red denotes high similarity, blue low similarity). For most TMs the optimal choices agree with the optimal alignment to Aα (full table in S6 Fig) only TM6 shows a second possible alignment at offset +4. The same table but using the GPCRtm substitution matrix instead of BLOSUM62 is in S8 Fig. Again, both matrices result in the same alignment.

Fig 5. TM 3 sequence alignment for the 25 crystal structures.

Other TMs are shown in Fig 6. The sequences are taken from the selected PDB files. The TM helix residues are colored in the Zappos scheme, which captures the chemical nature of each residue (e.g. helix breakers, proline and glycine, are shown in purple). The loop residues are shown in grey. The BW n.50 residue (numbering displayed below the sequences) is the most conserved within the class A. The consensus sequence is most similar to class A, because most sequences are from this class. The largest differences are for the last 5 sequences, which belong to the classes B, C, and F. The figure was prepared using Jalview.

Fig 6. Sequence alignments for TMs 1,2,4–7 for the 25 crystal structures.

Same caption as Fig 5, where TM3 is shown.

Fig 7. The phylogenetic tree based only on TM similarity using the GRoSS alignment (loops were ignored).

Color coding denotes the GPCR class. Proteins with known crystal structure are emphasized with a dot. The full resolution version of this figure is in S4 Fig.

Fig 8. Native activation “hot-spot” residues (NACHOs), which are contacts that change upon receptor activation.

The width of the green lines is proportional to the number of contacts common to all six structures (RHO, β₂AR, M2, and their active structures). Blue shows the contacts present only in inactive structures, and not in inactive structures; while red shows the opposite. The upper diagrams show contacts in the extracellular half of the membrane. We see that there is no systematic change common to the class A receptors in the conformation of the extracellular half of the TMs. This is not obvious, because there are conformational changes accompanying ligand binding. All the systematic changes, which enable G protein binding, occur in the intracellular half of the TMs. The list only contains 15 different residues in 15 different contacts. Thus many of the residues switch partners upon activation.

Fig 9. Magnitude of the rigid body moves of the helices necessary to map one structure to another.

All TMs 1–7 from all available structure pairs were compared and each symbol denotes which TM is the data point from. The coordinate system is defined in the text. The maximal observed deviation is approximately proportional to the sequence dissimilarity of the two compared TMs, and it follows the same trend within class A (blue symbols) and across the GPCR superfamily (green symbols). The red symbols, which correspond to the active-inactive structure pairs, show rigid body moves caused by receptor activation. S10 Fig has an analogous plot of residual RMSD vs. similarity for each helix after the best rigid body transformation. RMSD shows a similar trend as the plots in this figure.