Probing the structural determinants for the function of intracellular loop 2 in structurally cognate G-protein-coupled receptors.
Intracellular loop 2 (IL2) in G-protein-coupled receptors (GPCRs) is functionally important, e.g., in binding to G-protein and β-arrestin. Differences in secondary structure of IL2 in the crystal structures of the very similar β(1)- and β(2)-adrenergic receptors (β(1)AR and β(2)AR, respectively), i.e., an α-helix and an L-shaped strand, respectively, emphasize the need to understand the structural basis for IL2 functionality. We studied the properties of IL2 in the context of experimental data using a Monte Carlo-based ab initio method. The procedure was validated first by verifying that the IL2 structures in β(1)AR and β(2)AR crystals were correctly reproduced, even after conformational ensemble searches at >1200 K where most secondary structure had been lost. We found that IL2 in β(1)AR and β(2)AR sampled each other's conformation but adopted different energetically preferred conformations, consistent with the crystal structures. The results indicate a persistent contextual preference for the structure of IL2, which was conserved when the IL2 sequences were interchanged between the receptors. We conclude that the protein environment, more than the IL2 sequence, regulates the IL2 structures. We extended the approach to the molecular model of 5-HT(2A)R for which no crystal structure is available and found that IL2 is predominantly helical, similar to IL2 in β(1)AR. Because the P3.57A mutation in IL2 had been shown to decrease β-arrestin binding and internalization, we predicted the effects of the mutation and found that it decreased the propensity of IL2 to form helix, identifying the helical IL2 as a component of the GPCR active form.
Department of Physiology and Biophysics, Weill Cornell Medical College, Cornell University, New York, New York 10065, United States.
Jufang
J
Shan
Harel
H
Weinstein
Ernest L
EL
Mehler
D000465:Algorithms; D000818:Animals; D002417:Cattle; D006801:Humans; D008958:Models, Molecular; D009010:Monte Carlo Method; D017434:Protein Structure, Tertiary; D018342:Receptors, Adrenergic, beta-1 / Q000737:chemistry; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry*
D018342:Receptors, Adrenergic, beta-1; D018343:Receptors, Adrenergic, beta-2; D043562:Receptors, G-Protein-Coupled