PDBsum entry 6v2e

Membrane protein

PDB id: 6v2e

Contents

Protein chains

555 a.a.

16 a.a.

Ligands

GLC-GLC

FMT ×3

NH2

Waters ×602

PDB id:

6v2e

Name:

Membrane protein

Title:

Crystal structure of the human clr:ramp2 extracellular domain heterodimer with bound high-affinity adrenomedullin s45r/k46l/s48g/q50w variant

Structure:

Maltose/maltodextrin-binding periplasmic protein,receptor activity-modifying protein 2,calcitonin gene-related peptide type 1 receptor. Chain: a. Synonym: mmbp,maltodextrin-binding protein,maltose-binding protein, mbp,calcitonin-receptor-like receptor activity-modifying protein 2, crlr activity-modifying protein 2,cgrp type 1 receptor,calcitonin receptor-like receptor. Engineered: yes.

Source:

Escherichia coli (strain k12), homo sapiens. Human. Organism_taxid: 83333, 9606. Strain: k12. Gene: male, b4034, jw3994, ramp2, calcrl, cgrpr. Expressed in: escherichia coli. Expression_system_taxid: 562. Synthetic: yes. Homo sapiens.

Resolution:

1.83Å R-factor: 0.158 R-free: 0.190

Authors:

J.M.Booe,A.A.Pioszak

Key ref:

J.M.Booe et al. (2020). Picomolar Affinity Antagonist and Sustained Signaling Agonist Peptide Ligands for the Adrenomedullin and Calcitonin Gene-Related Peptide Receptors. ACS Pharmacol Transl Sci, 3, 759-772. PubMed id: 32832875 DOI: 10.1021/acsptsci.0c00031

Date:

22-Nov-19 Release date: 05-Aug-20

Protein chain

O60895  (RAMP2_HUMAN) -  Receptor activity-modifying protein 2 from Homo sapiens

Seq: 175 a.a.

Struc: 555 a.a.*

Protein chain

P0AEX9  (MALE_ECOLI) -  Maltose/maltodextrin-binding periplasmic protein from Escherichia coli (strain K12)

Seq: 396 a.a.

Struc: 555 a.a.*

Protein chain

Q16602  (CALRL_HUMAN) -  Calcitonin gene-related peptide type 1 receptor from Homo sapiens

Seq: 461 a.a.

Struc: 555 a.a.*

Protein chain

P35318  (ADML_HUMAN) -  Pro-adrenomedullin from Homo sapiens

Seq: 185 a.a.

Struc: 16 a.a.*

* PDB and UniProt seqs differ at 120 residue positions (black crosses)

Enzyme reactions

• Enzyme class: Chains A, B: E.C.?

DOI no: 10.1021/acsptsci.0c00031

ACS Pharmacol Transl Sci 3:759-772 (2020)

PubMed id: 32832875

Picomolar Affinity Antagonist and Sustained Signaling Agonist Peptide Ligands for the Adrenomedullin and Calcitonin Gene-Related Peptide Receptors.

J.M.Booe, M.L.Warner, A.A.Pioszak.

ABSTRACT

The calcitonin receptor-like class B G protein-coupled receptor (CLR) mediates adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) functions including vasodilation, cardioprotection, and nociception. Receptor activity-modifying proteins (RAMP1-3) form heterodimers with CLR and determine its peptide ligand selectivity through an unresolved mechanism. The CGRP (RAMP1:CLR) and AM (RAMP2/3:CLR) receptors are proven or promising drug targets, but short AM and CGRP plasma half-lives limit their therapeutic utility. Here, we used synthetic peptide combinatorial library and rational design approaches to probe the ligand selectivity determinants and develop truncated AM and CGRP antagonist variants with receptor extracellular domain binding affinities that were enhanced ∼1000-fold into the low nanomolar range. Receptor binding studies and a high-resolution crystal structure of a novel library-identified AM variant bound to the RAMP2-CLR extracellular domain complex explained the increased affinities and defined roles for AM Lys46 and RAMP modulation of CLR conformation in the ligand selectivity mechanism. In longer AM and CGRP scaffolds that also bind the CLR transmembrane domain, the variants generated picomolar affinity antagonists, one with an estimated 12.5 h CGRP receptor residence time, and sustained signaling agonists "ss-AM" and "ss-CGRP" that exhibited persistent cAMP signaling after ligand washout. Sustained signaling was demonstrated in primary human umbilical vein endothelial cells and the SK-N-MC cell line, which endogenously express AM and CGRP receptors, respectively. This work clarifies the RAMP-modulated CLR ligand selectivity mechanism and provides AM and CGRP variants that are valuable pharmacological tools and may have potential as long-acting therapeutics.