title: Insight into mutation-induced activation of the luteinizing hormone receptor: molecular simulations predict the functional behavior of engineered mutants at M398.
abstract: In this study, molecular simulations have been combined with site-directed mutagenesis experiments to explore M398(2.43), a LH (lutropin) receptor (LHR) site in helix 2 susceptible to spontaneous activating mutations, and to develop a computational tool for predicting the functionality (i.e. active or nonactive) of LHR mutants.Site-directed mutagenesis experiments engineered 15 different substitutions for M389(2.43), which resulted in variable levels of constitutive activity, inversely correlated with the size of the replacing amino acid. This inverse correlation is suggested to be mediated by I460(3.46), M571(6.37), and Y623(7.53), the tyrosine of the NPxxY motif. In fact, size reduction at position 398(2.43), which is concurrent with constitutive receptor activity, releases the van der Waals interactions found in the wild-type LHR between M398(2.43) and these three amino acids, resulting in structural modifications in the proximity to the E/DRY/W motif. An increment, above a threshold value, in the solvent accessibility of the cytosolic ends of helices 3 and 6 is the main structural feature shared by the active mutants of the LHR. This feature has been successfully used for predicting the functionality of the engineered mutants at M398(2.43), proving that molecular simulations can be useful for in silico screening of LHR mutants.
authors.affiliation: Dulbecco Telethon Institute and Department of Chemistry, University of Modena and Reggio Emilia, Modena, Italy. fanelli@unimo.it.
authors.forename: Francesca
authors.initials: F
authors.lastname: Fanelli
authors.forename: Miriam
authors.initials: M
authors.lastname: Verhoef-Post
authors.forename: Marianna
authors.initials: M
authors.lastname: Timmerman
authors.forename: Annelieke
authors.initials: A
authors.lastname: Zeilemaker
authors.forename: John W M
authors.initials: JW
authors.lastname: Martens
authors.forename: Axel P N
authors.initials: AP
authors.lastname: Themmen
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D017930:Genes, Reporter; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011974:Receptors, LH / Q000378:metabolism*; D012243:Rhodopsin / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012984:Software; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry
chemical_list: D018076:DNA, Complementary; D009841:Oligonucleotides; D011974:Receptors, LH; D014443:Tyrosine; D012243:Rhodopsin; D000242:Cyclic AMP