Binding between a Distal C-Terminus Fragment of Cannabinoid Receptor 1 and Arrestin-2

Internalization of G-protein coupled receptors is mediated by phosphorylation of the C-terminus, followed by binding with the cytosolic protein arrestin. To explore structural factors that may play a role in internalization of cannabinoid receptor 1 (CB1), we utilize a phosphorylated peptide derived from the distal C-terminus of CB1 (CB15P454-473). Complexes formed between the peptide and human arrestin-2 (wt-arr21-418) were compared to those formed with a truncated arrestin-2 mutant (tr-arr21-382) using isothermal titration calorimetry and nuclear magnetic resonance spectroscopy. The penta-phosphopeptide CB15P454-473 adopts a helix-loop conformation, whether binding to full-length arrestin-2 or its truncated mutant. This structure is similar to that of a hepta-phosphopeptide, mimicking the distal segment of the rhodopsin C-tail (Rh7P330-348), binding to visual arrestin, suggesting that this adopted structure bears functional significance. Isothermal titration calorimetry (ITC) experiments show that the CB15P454-473 peptide binds to tr-arr21-382 with higher affinity than to the full-length wt-arr21-418. As the observed structure of the bound peptides is similar in either case, we attribute the increased affinity to a more exposed binding site on the N-domain of the truncated arrestin construct. The transferred nOe data from the bound phosphopeptides are used to predict a model describing the interaction with arrestin, using the data driven HADDOCK docking program. The truncation of arrestin-2 provides scope for positively charged residues in the polar core of the protein to interact with phosphates present in the loop of the CB15P454-473 peptide

Selectivity of stop codon recognition in translation termination is modulated by multiple conformations of GTS loop in eRF1

Translation termination in eukaryotes is catalyzed by two release factors eRF1 and eRF3 in a cooperative manner. The precise mechanism of stop codon discrimination by eRF1 remains obscure, hindering drug development targeting aberrations at translation termination. By solving the solution structures of the wild-type N-domain of human eRF1 exhibited omnipotent specificity, i.e. recognition of all three stop codons, and its unipotent mutant with UGA-only specificity, we found the conserved GTS loop adopting alternate conformations. We propose that structural variability in the GTS loop may underline the switching between omnipotency and unipotency of eRF1, implying the direct access of the GTS loop to the stop codon. To explore such feasibility, we positioned N-domain in a pre-termination ribosomal complex using the binding interface between N-domain and model RNA oligonucleotides mimicking Helix 44 of 18S rRNA. NMR analysis revealed that those duplex RNA containing 2-nt internal loops interact specifically with helix α1 of N-domain, and displace C-domain from a non-covalent complex of N-domain and C-domain, suggesting domain rearrangement in eRF1 that accompanies N-domain accommodation into the ribosomal A site

An investigation of the structural elements that underlie the arrestin mediated desensitization and internalization of cannabinoid receptor 1

Arrestins are cytosolic proteins that mediate the desensitization and internalization of G-Protein Coupled Receptors (GPCRs). Ligands bind and stabilize conformations of GPCR cytosolic loops that can couple to and activate G-Proteins. Following activation, the GPCR C-terminus is phosphorylated by G-Protein Coupled Receptor Kinases (GRK) and subsequently interacts with arrestin leading to desensitization and internalization of GPCRs. Internalization is followed by trafficking of the receptor to pathways that couple to alternate signaling pathways, recycle it to the cell surface or degrade it. An emerging concept in the literature is that arrestins display more than one conformation when bound, leading to alternate complexes that have different functional outcomes. The precise detail of these events is not known. Concomitantly there is no direct structural information on the bound state of the GPCR C-terminus and conflicting evidence as to the importance of phosphorylation in directing the formation of the bound arrestin conformations. Recent biological evidence suggests that the disposition of the cytosolic face of the GPCR holds sway in the adoption of a specific conformation. ^ The aims of this research are to study conformations of synthetic peptides derived from segments of CB1 shown to be responsible for desensitization and internalization, effect of the phosphorylation pattern on the conformations of the peptides and binding of these peptides to arrestin-2.^ To shed further light on the interaction between these peptides and arrestin, we investigated their effects on the HSQC spectra of arrestin. In our study, we observed that phosphorylation of the peptide was necessary for binding and the formation of short helical segments in bound peptides was a common structural element. By comparing the binding of these different segments we conclude that while phosphorylation is necessary for affinity, the primary amino acid sequence of the peptides can affect the final bound conformation. The NOE data of the bound peptides and mutational studies of arrestin taken from the literature were used to predict models describing the interaction interface between arrestin and peptides using the docking program. This study will expand our knowledge of signal transduction and may provide avenues for novel methods of modulating GPCR function.

An investigation of the structural elements that underlie the arrestin mediated desensitization and internalization of cannabinoid receptor 1

Arrestins are cytosolic proteins that mediate the desensitization and internalization of G-Protein Coupled Receptors (GPCRs). Ligands bind and stabilize conformations of GPCR cytosolic loops that can couple to and activate G-Proteins. Following activation, the GPCR C-terminus is phosphorylated by G-Protein Coupled Receptor Kinases (GRK) and subsequently interacts with arrestin leading to desensitization and internalization of GPCRs. Internalization is followed by trafficking of the receptor to pathways that couple to alternate signaling pathways, recycle it to the cell surface or degrade it. An emerging concept in the literature is that arrestins display more than one conformation when bound, leading to alternate complexes that have different functional outcomes. The precise detail of these events is not known. Concomitantly there is no direct structural information on the bound state of the GPCR C-terminus and conflicting evidence as to the importance of phosphorylation in directing the formation of the bound arrestin conformations. Recent biological evidence suggests that the disposition of the cytosolic face of the GPCR holds sway in the adoption of a specific conformation. ^ The aims of this research are to study conformations of synthetic peptides derived from segments of CB1 shown to be responsible for desensitization and internalization, effect of the phosphorylation pattern on the conformations of the peptides and binding of these peptides to arrestin-2.^ To shed further light on the interaction between these peptides and arrestin, we investigated their effects on the HSQC spectra of arrestin. In our study, we observed that phosphorylation of the peptide was necessary for binding and the formation of short helical segments in bound peptides was a common structural element. By comparing the binding of these different segments we conclude that while phosphorylation is necessary for affinity, the primary amino acid sequence of the peptides can affect the final bound conformation. The NOE data of the bound peptides and mutational studies of arrestin taken from the literature were used to predict models describing the interaction interface between arrestin and peptides using the docking program. This study will expand our knowledge of signal transduction and may provide avenues for novel methods of modulating GPCR function.

End-to-End Approach to Surfactant Selection, Risk Mitigation, and Control Strategies for Protein-Based Therapeutics.

A survey performed by the AAPS Drug Product Handling community revealed a general, mostly consensus, approach to the strategy for the selection of surfactant type and level for biopharmaceutical products. Discussing and building on the survey results, this article describes the common approach for surfactant selection and control strategy for protein-based therapeutics and focuses on key studies, common issues, mitigations, and rationale. Where relevant, each section is prefaced by survey responses from the 22 anonymized respondents. The article format consists of an overview of surfactant stabilization, followed by a strategy for the selection of surfactant level, and then discussions regarding risk identification, mitigation, and control strategy. Since surfactants that are commonly used in biologic formulations are known to undergo various forms of degradation, an effective control strategy for the chosen surfactant focuses on understanding and controlling the design space of the surfactant material attributes to ensure that the desired material quality is used consistently in DS/DP manufacturing. The material attributes of a surfactant added in the final DP formulation can influence DP performance (e.g., protein stability). Mitigation strategies are described that encompass risks from host cell proteins (HCP), DS/DP manufacturing processes, long-term storage, as well as during in-use conditions

The Influence of Charge Distribution on Self-association and Viscosity Behavior of Monoclonal Antibody Solutions

The present work investigates the influence of electrostatic surface potential distribution of monoclonal antibodies (MAbs) on intermolecular interactions and viscosity. Electrostatic models suggest MAb-1 has a less uniform surface charge distribution than MAb-2. The patches of positive and negative potential on MAb-1 are predicted to favor intermolecular attraction, even in the presence of a small net positive charge. Consistent with this expectation, MAb-1 exhibits a negative second virial coefficient (B22), an increase in static structure factor, S(q→0), and a decrease in hydrodynamic interaction parameter, H(q→0), with increase in MAb-1 concentration. Conversely, MAb-2 did not show such heterogeneous charge distribution as MAb-1 and hence favors intermolecular repulsion (positive B22), lower static structure factor, S(q→0), and repulsion induced increase in momentum transfer, H(q→0), to result in lower viscosity of MAb-2. Charge swap mutants of MAb-1, M-5 and M-7, showed a decrease in charge asymmetry and concomitantly a loss in self-associating behavior and lower viscosity than MAb-1. However, replacement of charge residues in the sequence of MAb-2, M-10, did not invoke charge distribution to the same extent as MAb-1 and hence exhibited a similar viscosity and self-association profile as MAb-2