Computational protocol: Receptor Activity-modifying Proteins 2 and 3 Generate Adrenomedullin Receptor Subtypes with Distinct Molecular Properties*

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Protocol publication

[…] The AM peptide structure () was modeled from the known structures of its component parts (the disulfide-bonded region, the helical region, and the ECD region). The key stages in this modeling involved (i) the use of an in-house multiple-reference sequence alignment method tailored for aligning helices with low sequence identity () and (ii) the comparative modeling capabilities of PLOP (). There is little structural information for full-length AM in its receptor-bound conformation, making structure-based sequence alignments difficult. Moreover, class B GPCR peptide ligands appear to lie in a number of distinct groups (), so sequence alignment is not trivial. Consequently, separate alignments of the glucagon, GLP-1, PTH, and AM sequences were generated by ClustalX (). The helical region of the AM peptide homologs, as indicated by the NMR structure (PDB code 2L7S) (), was aligned to those of the equivalent helical region in the glucagon/GLP-1/PTH family of peptides using an in-house multiple-reference method tailored for aligning helices with low sequence identity () that is a development of the methods of reference (). The alignment is given in A; the alignment scores shown in B (and C) give strong support for the proposed alignment over the only plausible alternative involving a shift left of the AM helix by 4 positions. The AM/CLR/RAMP2 (PDB code 4RWF) ECD (), the GLP-1/exendin-4 structure (PDB code 3C59) (), and the glucagon model structure () were structurally aligned using the SALIGN module of MODELER () (D), from which a template was constructed using Asp35–Tyr52 from the AM x-ray structure and Thr7–Tyr13 of the glucagon model peptide structure, which was preferred over the corresponding (Thr7)-Asp9-Gln13 of exendin-4 because the angle was more appropriate for peptide binding to the TM bundle. The missing loop was inserted using the comparative modeling, loop modeling, and minimization capabilities of PLOP () based on the alignment in F. The N terminus, taken from Woolley et al. (), was added by structural alignment of the common helical domain using VMD (), again using the alignment in A. The resulting peptide structure of AM(15–52) (structurally aligned to the CLR ECD) is shown in E. [...] Comparative AM1 and AM2 receptor models were generated using MODELER version 9.12 (), essentially from two x-ray structures, namely the AM CLR-RAMP2 ECD complex () (PDB code 4RWF) and the glucagon receptor (GCGR) TM domain () (PDB code 4L6R). The GCGR was preferred over the corticotropin-releasing factor 1 receptor (CRF1R) TM structure because of its overall conformation and compatibility with the full GCGR model (), but part of the superior quality CRF1R structure (as denoted by ERRAT ()) was used in subsequent refinement. In addition, model structures for the full GCGR model () containing only Ser8–Asp15 of glucagon (c.f. D), the full-length AM peptide (E), CGRP(1–7) docked to an active model of CLR (), and a model of the RAMP1 TM helix docked to TM7 were used (). The active character of the model was also imposed by including TM5-6 of an active CLR model derived from the β2-adrenergic receptor active complex (); this template also contained the C-terminal peptide of the G protein, Gs (Arg373–Leu394). Each of these structural templates contained information on part but not all of the desired structure and was linked via a global alignment (). In addition, we also included short N- and C-terminal extensions (6 and 5 residues, respectively) to the RAMP TM helix and the RAMP ECD to prevent the linker between them from becoming entangled in the bulk of the receptor. Within this alignment, the position of the gap in the CLR sequence between the ECD and TM1 relative to the longer human glucagon receptor sequence was determined by analysis of gaps in similar subsets within the glucagon multiple-sequence alignment (). Two thousand models were generated, and the model having the lowest (best) DOPE score was chosen for further refinement. ECL1 was refined using MODELER from TM1–4 of a CLR model derived from the CRF1R structure in which variability (, , ) was used to orient the CLR ECL1 helix, as in a recent GLP-1 receptor model (). The ECLs and the RAMP linker (here defined as the region connecting the extracellular helical domain and the TM helix (i.e. residues Val134–Leu147 for RAMP2 and Val106–Leu119 for RAMP3)) were refined using PLOP, which has been shown to perform well in GPCR loop modeling (); this refinement removed any bias introduced by the extensions. The final models were minimized using PLOP ().Druggability was assessed using the PockDrug (, ) and DoGSiteScorer Web servers (); pocket hull volumes (which include atoms within the druggable binding pockets) were also determined using PockDrug; distances were measured using the PyMOL Molecular Graphics System (version 1.7.4; Schrödinger, LLC, New York), which was also used for image generation. The models are available as supplemental models 1 and 2. […]

Pipeline specifications

Software tools ERRAT, PockDrug, ProteinsPlus, PyMOL
Application Protein interaction analysis
Chemicals Amino Acids