Abstract:The human β2-adrenergic receptor (β2AR) belongs to the G protein-coupled receptor (GPCR) family and due to its central role in bronchodilation, is an important drug target. The inter-individual variability in β2AR has been implicated in disease susceptibility and differential drug response. In this work, we identified nine potentially deleterious non-synonymous single nucleotide polymorphisms (nsSNPs) using a consensus approach. The deleterious nsSNPs were found to cluster near the ligand binding site and towa… Show more
“…1). Using in silico modeling techniques 24,25 , we derived structural models of the two mutated PFN1 proteins and predicted the consequences of these mutations (G118V and T109M) on the structure and function of PFN1 in complex with actin or PLP and in its isolated form.Figure 1Crystal structure of PFN1 complexes with actin and poly-L-proline peptide (PLP) fragment. The actin-binding site is on the lower part of PFN1 (as shown), while the PLP-binding site is on the top part of PFN1 where PLP is shown in dark blue.…”
Profilin-1 (PFN1) is a 140-amino-acid protein with two distinct binding sites―one for actin and one for poly-L-proline (PLP). The best-described function of PFN1 is to catalyze actin elongation and polymerization. Thus far, eight DNA mutations in the PFN1 gene encoding the PFN1 protein are associated with human amyotrophic lateral sclerosis (ALS). We and others recently showed that two of these mutations (Gly118Val or G118V and Cys71Gly or C71G) cause ALS in rodents. In vitro studies suggested that Met114Thr and Thr109Met cause the protein to behave abnormally and cause neurotoxicity. The mechanism by which a single amino acid change in human PFN1 causes the degeneration of motor neurons is not known. In this study, we investigated the structural perturbations of PFN1 caused by each ALS-associated mutation. We used molecular dynamics simulations to assess how these mutations alter the secondary and tertiary structures of human PFN1. Herein, we present our in silico data and analysis on the effect of G118V and T109M mutations on PFN1 and its interactions with actin and PLP. The substitution of valine for glycine reduces the conformational flexibility of the loop region between the α-helix and β-strand and enhances the hydrophobicity of the region. Our in silico analysis of T109M indicates that this mutation alters the shape of the PLP-binding site and reduces the flexibility of this site. Simulation studies of PFN1 in its wild type (WT) and mutant forms (both G118V and T109M mutants) revealed differential fluctuation patterns and the formation of salt bridges and hydrogen bonds between critical residues that may shed light on differences between WT and mutant PFN1. In particular, we hypothesize that the flexibility of the actin- and PLP-binding sites in WT PFN1 may allow the protein to adopt slightly different conformations in its free and bound forms. These findings provide new insights into how each of these mutations in PFN1 might increase its propensity for misfolding and aggregation, leading to its dysfunction.
“…1). Using in silico modeling techniques 24,25 , we derived structural models of the two mutated PFN1 proteins and predicted the consequences of these mutations (G118V and T109M) on the structure and function of PFN1 in complex with actin or PLP and in its isolated form.Figure 1Crystal structure of PFN1 complexes with actin and poly-L-proline peptide (PLP) fragment. The actin-binding site is on the lower part of PFN1 (as shown), while the PLP-binding site is on the top part of PFN1 where PLP is shown in dark blue.…”
Profilin-1 (PFN1) is a 140-amino-acid protein with two distinct binding sites―one for actin and one for poly-L-proline (PLP). The best-described function of PFN1 is to catalyze actin elongation and polymerization. Thus far, eight DNA mutations in the PFN1 gene encoding the PFN1 protein are associated with human amyotrophic lateral sclerosis (ALS). We and others recently showed that two of these mutations (Gly118Val or G118V and Cys71Gly or C71G) cause ALS in rodents. In vitro studies suggested that Met114Thr and Thr109Met cause the protein to behave abnormally and cause neurotoxicity. The mechanism by which a single amino acid change in human PFN1 causes the degeneration of motor neurons is not known. In this study, we investigated the structural perturbations of PFN1 caused by each ALS-associated mutation. We used molecular dynamics simulations to assess how these mutations alter the secondary and tertiary structures of human PFN1. Herein, we present our in silico data and analysis on the effect of G118V and T109M mutations on PFN1 and its interactions with actin and PLP. The substitution of valine for glycine reduces the conformational flexibility of the loop region between the α-helix and β-strand and enhances the hydrophobicity of the region. Our in silico analysis of T109M indicates that this mutation alters the shape of the PLP-binding site and reduces the flexibility of this site. Simulation studies of PFN1 in its wild type (WT) and mutant forms (both G118V and T109M mutants) revealed differential fluctuation patterns and the formation of salt bridges and hydrogen bonds between critical residues that may shed light on differences between WT and mutant PFN1. In particular, we hypothesize that the flexibility of the actin- and PLP-binding sites in WT PFN1 may allow the protein to adopt slightly different conformations in its free and bound forms. These findings provide new insights into how each of these mutations in PFN1 might increase its propensity for misfolding and aggregation, leading to its dysfunction.
“…For the β 2 -adrenoceptor receptor, the most evident mutation was Asn69 2.40 Ser, which belongs to the allosteric binding site. Furthermore, Asn69 2.40 Ser was also homozygous mutation (pathogenic) and was found to have detrimental effects on G-protein coupling [154] . In the β 2 -adrenoceptor ligand binding site, Asn301Ser and Phe193 45.52 Leu mutations were the most relevant.…”
“…The cMD and aMD simulations in the present study were performed using the PMEMD module of AMBER 14 49 . The AMBER FF99SB force field 50 was used for P2Y 1 R, the general AMBER force field (GAFF) 51 was used for 2MeSADP and MRS2500, and the amber lipid force field LIPID14 52 was used for POPC. A series of minimizations were carried out for each system ( i.e ., 2MeSADP-P2Y 1 R, apo-P2Y 1 R and MRS2500-P2Y 1 R).…”
The human P2Y1 receptor (P2Y1R) is a purinergic G-protein-coupled receptor (GPCR) that functions as a receptor for adenosine 5′-diphosphate (ADP). An antagonist of P2Y1R might potentially have antithrombotic effects, whereas agonists might serve as antidiabetic agents. On the basis of the antagonist-bound MRS2500-P2Y1R crystal structure, we constructed computational models of apo-P2Y1R and the agonist-receptor complex 2MeSADP-P2Y1R. We then performed conventional molecular dynamics (cMD) and accelerated molecular dynamics (aMD) simulations to study the conformational dynamics after binding with agonist/antagonist as well as the P2Y1R activation mechanism. We identified a new agonist-binding site of P2Y1R that is consistent with previous mutagenesis data. This new site is deeper than those of the agonist ADP in the recently simulated ADP-P2Y1R structure and the antagonist MRS2500 in the MRS2500-P2Y1R crystal structure. During P2Y1R activation, the cytoplasmic end of helix VI shifts outward 9.1 Å, the Ser1463.47-Tyr2375.58 hydrogen bond breaks, a Tyr2375.58-Val2626.37 hydrogen bond forms, and the conformation of the χ1 rotamer of Phe2696.44 changes from parallel to perpendicular to helix VI. The apo-P2Y1R system and the MRS2500-P2Y1R system remain inactive. The newly identified agonist binding site and activation mechanism revealed in this study may aid in the design of P2Y1R antagonists/agonists as antithrombotic/antidiabetic agents, respectively.
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