Specific Conformational States Research Articles

Structural Refinement of Proteins by Restrained Molecular Dynamics Simulations with Non-interacting Molecular Fragments

The knowledge of multiple conformational states is a prerequisite to understand the function of membrane transport proteins. Unfortunately, the determination of detailed atomic structures for all these functionally important conformational states with conventional high-resolution approaches is often difficult and unsuccessful. In some cases, biophysical and biochemical approaches can provide important complementary structural information that can be exploited with the help of advanced computational methods to derive structural models of specific conformational states. In particular, functional and spectroscopic measurements in combination with site-directed mutations constitute one important source of information to obtain these mixed-resolution structural models. A very common problem with this strategy, however, is the difficulty to simultaneously integrate all the information from multiple independent experiments involving different mutations or chemical labels to derive a unique structural model consistent with the data. To resolve this issue, a novel restrained molecular dynamics structural refinement method is developed to simultaneously incorporate multiple experimentally determined constraints (e.g., engineered metal bridges or spin-labels), each treated as an individual molecular fragment with all atomic details. The internal structure of each of the molecular fragments is treated realistically, while there is no interaction between different molecular fragments to avoid unphysical steric clashes. The information from all the molecular fragments is exploited simultaneously to constrain the backbone to refine a three-dimensional model of the conformational state of the protein. The method is illustrated by refining the structure of the voltage-sensing domain (VSD) of the Kv1.2 potassium channel in the resting state and by exploring the distance histograms between spin-labels attached to T4 lysozyme. The resulting VSD structures are in good agreement with the consensus model of the resting state VSD and the spin-spin distance histograms from ESR/DEER experiments on T4 lysozyme are accurately reproduced.

Novel perspectives in cancer therapy: Targeting ion channels.

By controlling ion fluxes at multiple time scales, ion channels shape rapid cell signals, such as action potential and synaptic transmission, as well as much slower processes, such as mitosis and cell migration. As is currently increasingly recognized, a variety of channel types are involved in cancer hallmarks, and regulate specific stages of neoplastic progression. Long-term in vitro work has established that inhibition of these ion channels impairs the growth of cancer cells. Recently, these studies have been followed up in vivo, hence revealing that ion channels constitute promising pharmacological targets in oncology. The channel proteins can be often accessed from the extracellular milieu, which allows use of lower drug doses and decrease untoward toxicity. However, because of the central physiological roles exerted by ion channels in excitable cells, other types of side effects may arise, the gravest of which is cardiac arrhythmia. A paradigmatic case is offered by Kv11.1 (hERG1) channels. HERG1 blockers attenuate the progression of both hematologic malignancies and solid tumors, but may also lead to the lengthening of the electrocardiographic QT interval, thus predisposing the patient to ventricular arrhythmias. These side effects can be avoided by specifically inhibiting the channel isoforms which are highly expressed in certain tumors, such as Kv11.1B and the neonatal forms of voltage-gated Na(+) channels. Preclinical studies are also being explored in breast and prostate cancer (targeting voltage-gated Na(+) channels), and gliomas (targeting CLC-3). Overall, the possible approaches to improve the efficacy and safety of ion channel targeting in oncology include: (1) the development of specific inhibitors for the channel subtypes expressed in specific tumors; (2) drug delivery into the tumor by using antibodies or nanotechnology-based approaches; (3) combination regimen therapy and (4) blocking specific conformational states of the ion channel. We believe that expanding this relatively neglected field of oncology research might lead to unforeseen therapeutic benefits for cancer patients.

Computational study of correlated domain motions in the AcrB efflux transporter.

As active part of the major efflux system in E. coli bacteria, AcrB is responsible for the uptake and pumping of toxic substrates from the periplasm toward the extracellular space. In combination with the channel protein TolC and membrane fusion protein AcrA, this efflux pump is able to help the bacterium to survive different kinds of noxious compounds. With the present study we intend to enhance the understanding of the interactions between the domains and monomers, for example, the transduction of mechanical energy from the transmembrane domain into the porter domain, correlated motions of different subdomains within monomers, and cooperative effects between monomers. To this end, targeted molecular dynamics simulations have been employed either steering the whole protein complex or specific parts thereof. By forcing only parts of the complex towards specific conformational states, the risk for transient artificial conformations during the simulations is reduced. Distinct cooperative effects between the monomers in AcrB have been observed. Possible allosteric couplings have been identified providing microscopic insights that might be exploited to design more efficient inhibitors of efflux systems.

Conformational changes in human Norovirus polymerase

Human Noroviruses (NV) belong in the Caliciviridae family and are a major cause of gastroenteritis outbreaks throughout the world. Crystal structures of the RNA-dependent RNA polymerase from the human Norovirus have been determined in over ten different crystal forms in the presence and absence of divalent metal cations, nucleoside triphosphates, inhibitors and primer-template duplex RNA. These structures show how the polymerase enzyme can adopt a range of conformations in which the thumb, fingers and palm domains change orientations depending on the step of the enzymatic cycle trapped in different crystal forms. We discuss how the evidence from crystallographic and biochemical experiments combine to better understand how viral RNA polymerase enzymes from human Norovirus and related positive-strand RNA viruses can adopt a range of conformational states to facilitate RNA binding, NTP binding, catalysis, RNA translocation and pyrophosphate release. The detailed structural and mechanistic understanding of these conformational changes is important for providing a sound basis for understanding viral replication in general, as well as for the design of novel inhibitors capable of trapping the enzyme in specific conformational states.

The minimal pharmacophore for silent agonism of the α7 nicotinic acetylcholine receptor.

The minimum pharmacophore for activation of the human α7 nicotinic acetylcholine receptor (nAChR) is the tetramethylammonium cation. Previous work demonstrated that larger quaternary ammonium compounds, such as diethyldimethylammonium or 1-methyl quinuclidine, were α7-selective partial agonists, but additional increase in the size of the ammonium cation or the quinuclidine N-alkyl group by a single carbon to an N-ethyl group led to a loss of efficacy for ion channel activation. We report that although such compounds are ineffective at inducing the normal channel open state, they nonetheless regulate the induction of specific conformational states normally considered downstream of channel activation. We synthesized several panels of quaternary ammonium nAChR ligands that systematically varied the size of the substituents bonded to the central positively charged nitrogen atom. In these molecular series, we found a correlation between the molecular volume of the ligand and/or charge density, and the receptor's preferred distribution among conformational states including the closed state, the active state, a nonconducting state that could be converted to an activated state by a positive allosteric modulator (PAM), and a PAM-insensitive nonconducting state. We hypothesize that the changes of molecular volume of an agonist's cationic core subtly impact interactions at the subunit interface constituting the orthosteric binding site in such a way as to regulate the probability of conversions among the conformational states. We define a new minimal pharmacophore for the class of compounds we have termed "silent agonists," which are able to induce allosteric modulator-dependent activation but not the normal activated state.

Conformational Dynamics During Spliceosome Assembly Investigated by Single-Pair FRET

The ribonucleoprotein (RNP) machinery of the spliceosome is composed of several subunits, which assemble stepwise during the process of splicing. U2 auxiliary factor (U2AF), a heterodimer comprised of a large (65 kDa) and a small (35 kDa) subunit, is involved in the early recognition of the intron and stabilization during splicing reactions. U2AF65 specifically recognizes the polypyrimidine tract in pre-mRNA introns and additionally contacts further splicing factors, such as mBBP/SF1 and SAP155 [1,2]. U2AF65 contains three RNA recognition motifs (RRM), where RRM1 and RRM2 mediate RNA binding while RRM3 is a U2AF homology motif (UHM) that mediates the interaction with U2AF35 [3].A nuclear magnetic resonance spectroscopy study of recombinant human U2AF65 (RRM1-RRM2) showed a closed conformational state for the splicing factor in absence of RNA, while an open conformation is induced upon binding to polypyrimidine stretches [4].Here, conformational subpopulations of the protein were investigated using single-pair FRET in solution with multiparameter fluorescence detection and pulsed interleaved excitation [5]. Information on FRET efficiency, stoichiometry, and lifetime revealed different conformational states dependent on substrate recognition and provided clues for dynamic motions of the splicing factor. These were further analyzed on a total internal reflection microscope using molecules immobilized in lipid vesicles. Changes in FRET efficiency over time showed a highly flexible U2AF65 protein with stabilization of specific conformational states upon RNA binding. Single-pair FRET measurements provide detailed insights into the mechanistic action of polypyrimidine tract recognition.

Identification of the specific, shutter-like conformational reorientation in a chiroptical switching polycarbodiimide by VCD spectroscopy.

The specific conformational states responsible for the unique, reversible temperature- and solvent-driven chiroptical switching process experienced by poly(N-1-naphthyl-N'-octadecyl-carbodiimide) (PNOC) have been identified using VCD spectroscopy and DFT calculations. The distinct VCD spectra of PNOC corresponding to the two specific conformations were obtained for the polymer dissolved in DCM-d2 (state A) and CDCl3 (predominantly state B). To specifically assign the structures of both conformations, two simplified 7mer models were constructed and optimized using DFT calculations. The theoretical spectra associated with these model conformations show a high level of agreement when compared to the experimental VCD spectra. The two states consist of the naphthyl pendant groups aligned directionally opposing the helical rotation (model A) and aligned with the helicity of the backbone (model B). This pendant reorientation causes very large OR and ECD Cotton effect inversions upon modification to the temperature or solvent composition of dilute (+)-PNOC solutions in specific solvents. In addition, the pendant group equilibrium from state A to B causes a contraction of the helical pitch from the more expanded 5/1 pitch to the more contracted 7/2 pitch resulting in increased disorder of the solvation sphere surrounding the polymer chain.

Binding of Dopamine to Alpha-Synuclein is Mediated by Specific Conformational States

Parkinson’s disease is the second most common neurodegenerative disorder, in which both alpha-synuclein (α-syn) and dopamine (DA) have a critical role. α-Syn is known to be natively unstructured in equilibrium with subpopulations of more compact structures. It is these compact structures that are thought to be linked to amyloid formation. In the presence of DA, α-syn yields a diverse range of SDS-resistant, non-amyloid oligomers, however the precursor state conformation has not been established. Here, three DA molecules have been observed to bind per α-syn monomer by electrospray-ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS). Each of these DA molecules binds exclusively to the extended conformation of α-syn, and binding is not observed in the compact state of the protein. Measurements of collisional cross sectional areas show that the incremental uptake of DA pushes the protein towards a highly extended population, becoming fully populated upon the binding of three DA ligands. Tyrosine (Tyr) as a closely related structural analog, exhibited limited binding to the protein as compared with DA, with a maximum of two ligands being observed. Those Tyr ligands that do bind were observed as adducts to the extended conformation akin to DA. These findings suggest DA is able to modulate α-syn self-assembly by inducing the population of a highly extended state. Figureᅟ Electronic supplementary materialThe online version of this article (doi:10.1007/s13361-013-0676-z) contains supplementary material, which is available to authorized users.

Helix 69: A Multitasking RNA Motif as a Novel Drug Target

AbstractHigh‐resolution structures have shown that helix 69 (H69) of the large ribosomal subunit can assume variable conformational states during translation. Solution studies on small model RNAs, isolated subunits, and complete ribosomes also revealed a variety of H69 conformations. Specific nucleotides of H69 that undergo changes in their relative orientations within the ribosome structure play important roles in both translation and ribosome rescue. Furthermore, the presence of multiple pseudouridines influences the global conformational states of H69, and these highly conserved modifications play a role in regulating the positioning of key functional residues. Helix 69 has recently been identified as a novel antibiotic target site. Small molecules such as aminoglycosides bind to specific conformational states of H69 in bacterial ribosomes and affect the translation process. A variety of techniques have been employed to study H69, ranging from chemical synthesis to X‐ray crystallography, highlighting the importance of a detailed evaluation of the underlying principles of RNA conformational dynamics and drug targeting.

Specific Conformational States of Ras GTPase upon Effector Binding

To uncover the structural and dynamical determinants involved in the highly specific binding of Ras GTPase to its effectors, the conformational states of Ras in uncomplexed form and complexed to the downstream effectors Byr2, PI3Kγ, PLCε, and RalGDS were investigated using molecular dynamics and cross-comparison of the trajectories. The subtle changes in the dynamics and conformations of Ras upon effector binding require an analysis that targets local changes independent of global motions. Using a structural alphabet, a computational procedure is proposed to quantify local conformational changes. Positions detected by this approach were characterized as either specific for a particular effector, specific for an effector domain type, or as effector unspecific. A set of nine structurally connected residues (Ras residues 5–8, 32–35, 39–42, 55–59, 73–78, and 161–165), which link the effector binding site to the distant C-terminus, changed dynamics upon effector binding, indicating a potential effector-unspecific signaling route within the Ras structure. Additional conformational changes were detected along the N-terminus of the central β-sheet. Besides the Ras residues at the effector interface (e.g., D33, E37, D38, and Y40), which adopt effector-specific local conformations, the binding signal propagates from the interface to distant hot-spot residues, in particular to Y5 and D57. The results of this study reveal possible conformational mechanisms for the stabilization of the active state of Ras upon downstream effector binding and for the structural determinants responsible for effector specificity.

Abstract SY06-01: Intracellular antibody therapy: Specific delivery of synthetic antibodies to cytoplasmic targets in cancer cells

Abstract Synthetic Antigen Binders (sABs) are a new class of antibody-based reagents engineered using novel phage display libraries and selection strategies. These attributes provide for the ability to generate sABs that are customized to: 1) target specific regions on the surface of the protein, 2) recognize specific conformational or oligomeric states, 3) induce conformational changes, and 4) capture and stabilize multi-protein complexes. As such, they have the potential to outperform traditional monoclonal antibodies as therapeutic modalities for biomedical applications. However, a critical limitation is that antibodies can only be directed at extracellular targets because they cannot penetrate the cell membrane. This eliminates a rich set of potential cytoplasmic targets that function as crucial components of signaling pathways or structural networks. To overcome this limitation we have developed receptor-mediated delivery systems that have proven effective in internalizing a novel class of synthetic antigen binders in their fully functional form. Further, this delivery system inherently seeks out certain types of cancer cells, while being inert towards cells on normal tissue. As a demonstration of the power of the approach, we have generated a set of sABs that have been tailored to induce conformational changes in F-actin filaments that substantially alter actin cytoskeletal structure by mechanisms involving depolymerizing, severing, bundling and capping of actin filaments. We introduce these sABs into cancer cells using receptor-mediated delivery and show the properties selected for in the in vitro phage display sorting are exactly recapitulated in the live cells. The effects of actin filament bundling are shown to be particularly detrimental to the cancer cells suggesting that sABs performing this function may be useful therapeutic agents. This approach is being expanded to make agents that target nuclear components to alter cell function at the DNA level. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY06-01. doi:1538-7445.AM2012-SY06-01

Symmetric C–C Stretching Mode Splitting versus CH2-Chain Conformation Order in Sodium Montmorillonite Modified by Cetyltrimethylammonium Bromide

Exploiting Raman spectroscopy and computational modeling, for the first time, we report and explain an interesting phenomenon in clay modified by cetyltrimethylammonium bromide. A splitting of the CH(2)-chain's symmetric C-C stretching Raman mode found at ~1128 cm(-1) in cetyltrimethylammonium bromide into two bands at 1128 and 1139 cm(-1) in clay modified by cetyltrimethylammonium bromide is observed. We demonstrate that this splitting appears if two types of trans-segments with nonequivalent lengths and terminal groups coexist in the CH(2)-chain of the alkylammonium ion embedded into the clay interlayer space. We report Raman experimental evidence for a CH(2)-chain bending within the clay galleries, resulting in the symmetric C-C stretching band splitting, as was also suggested by computational modeling. Noteworthy, we postulate that this unique behavior based on CH(2)-chain bending provides a general understanding of conformation reorganization and switching within long CH(2)-chain molecules confined within modified clay interlayer galleries. For all modifier concentrations, we show that the intercalated cetyltrimethylammonium ions exist in a liquid-like state, consisting mainly of trans conformations (~86%) of two types in approximately equal proportions. Moreover, we demonstrate that the integral Raman intensity ratio I(1295)(CH(2))/I(705)(clay) provides a rapid nondestructive quantification of the relative content of alkylammonium ions in modified clays. These results demonstrate that a simple direct monitoring of specific modifier-dependent interlayer conformational states is possible, which is of great importance for a tunable fabrication of modified clays-based nanocomposites with desired properties.

Characterisation of a multimeric protein complex associated with ERp57 within the nucleus in paclitaxel-sensitive and -resistant epithelial ovarian cancer cells: The involvement of specific conformational states of β-actin

Ovarian cancer is the second most frequently diagnosed malignancy of the reproductive system and is the leading cause of gynecological cancer mortality. Although the majority of advanced ovarian carcinomas initially respond successfully to taxane-based chemotherapy, resistance to chemotherapy remains the primary factor accounting for the low 5-year survival in this patient population. Recent data obtained by our group demonstrate that the disulphide isomerase ERp57 is strongly modulated in paclitaxel resistance suggesting that it may represent a chemoresistance biomarker in ovarian cancer. In the present study, we characterise a nuclear multimeric complex where ERp57 is associated with protein species involved in cell division and gene expression, as Nucleolin, Nucleophosmin, Vimentin, Aurora kinase C and beta-actin. In particular, we show that the occurrence of the interaction of nuclear ERp57 with beta-actin is associated with paclitaxel resistance and that specific actin conformations modulate this complex. We propose the involvement of the nuclear ERp57 complex in mechanisms associated with chromosome segregation in which specific conformational states of actin play a role in the pathway involved in paclitaxel resistance.

Protein Aggregation as a Phase Transition Process

In this model we attempt to mimic the aggregation pattern found in real proteins using a modified Ising model. Similar to a Blume-Campel model, we restrict the proteins to occupy discrete lattice points and define a pairwise interaction depending on the configuration of the protein (as in the Potts model). We define three specific conformational states: native, intermediate and unfolded and consider the aggregation process as a series of second-order phase transitions. In this framework we can analyze the zeros of the partition function analytically continued onto the complex plane, specifically the Yang-Lee and Fisher zeros. Using combinatorial arguments we can simplify the partition function and predict the occurrences of phase transitions as a function of the interaction potential and protein density.

Conformational states and association mechanism of Yersinia pestis Caf1 subunits

Bacterial infectivity often relies on efficient attachment to the host cells through adhesive extensions. Unveiling the structural basis of the formation of these organelles is of paramount importance for both academic and applicative implications. Computational approaches may fruitfully complement experimental studies by providing information on specific conformational states whose characterization is difficult. Here, we report molecular dynamics characterizations of Yersinia pestis Caf1 subunit in its monomeric-unbound and dimeric states. Data on the monomeric form indicate that it is highly reactive and evolves toward compact states, which likely hamper subunit–subunit association. In line with recent experimental reports, this finding implies that chaperone release and subunit–subunit association must be simultaneous. MD analysis on Caf1 dimer lead to the formation of a novel assembly endowed with a significant stability in the simulation timescale. Using these data, an end-to-end model of the fiber, which well agrees with available experimental data, was also generated.

Test of the nonequilibrium kinetic focusing of voltage-gated ion channels

It has been shown numerically (Millonas and Chialvo 1996 Phys. Rev. Lett. 76 550) that voltage-gated ion channels can be focused into specific conformational states by the application of fluctuating voltages, such as dichotomous noise. We present results of an experimental test performed on Shaker K+ channels. We applied dichotomous noise with properties mimicking the numerical simulations as closely as physiologically feasible. We observed that the probability for intermediate states can be increased as much as 50% above the maximal value for any static voltages.

Computationally Efficient Strategy for Modeling the Effect of Ion Current Modifiers

Electrophysiological studies often seek to relate changes in ion current properties caused by a chemical modifier to changes in cellular properties. Therefore, quantifying concentration-dependent effects of modifiers on ion currents is a topic of importance. In this paper, we sought a mathematical method for using ion current data to predict the effect of several theoretical ion current modifiers on cellular and tissue properties that is computationally efficient without compromising predictive power. We focused on the K+ current I(K,r) as an example case due to its link to long QT syndrome and arrhythmias, but these methods should be generally applicable to other electrophysiological studies. We compared predictions using a Markov model with mass action binding of the modifiers to specific conformational states of the channel to predictions generated by two simplified models. We investigated scaling I(K,r) conductance, and found that although this method produced predictions that agreed qualitatively with the more complicated model, it did not generate quantitatively consistent predictions for all modifiers tested. Our simulations showed that a more computationally efficient Hodgkin-Huxley model that incorporates the effect of modifiers through functional changes in the current produced quantitatively consistent predictions of concentration-dependent changes in cell and tissue properties for all modifiers tested.

Solitonic Effects of the Local Electromagnetic Field on Neuronal Microtubules

Current wisdom in classical neuroscience suggests that the only direct action of the electric field in neurons is upon voltage-gated ion channels which open and close their gates during the passage of ions. The intraneuronal biochemical activities are thought to be modulated indirectly either by entering into the cytoplasmic ions that act as second messengers, or via linkage to the ion channels enzymes. In this paper we present a novel possibility for the subneuronal processing of information by cytoskeletal microtubule tubulin tails and we show that the local electromagnetic field supports information that could be converted into specific protein tubulin tail conformational states. Long-range collective coherent behavior of the tubulin tails could be modelled in the form of solitary waves such as sine-Gordon kinks, antikinks or breathers that propagate along the microtubule outer surface, and the tubulin tail soliton collisions could serve as elementary computational gates that control cytoskeletal processes. The biological importance of the presented model is due to the unique biological enzymatic energase action of the tubulin tails, which is experimentally verified for controlling the sites of microtubule-associated protein attachment and the kinesin transport of post-Golgi vesicles.

Influenza A H5N1 hemagglutinin cleavable signal sequence substitutions

Eleven influenza A H5N1 hemagglutinin N-terminal cleavable signal sequences, coded by single nucleotide substitutions relative to reference A/Viet Nam/1203/2004, were identified by BLASTN search of GenBank and were characterized by molecular modeling. The signal sequences statistically segregated into two classes of states. Members of one class were uncharged and conformationally compact while members of the second class each carried a 2+ electric charge and were conformationally extended. Virtual signal sequences, not found on GenBank and based upon hypothetical transversions in the third codon, had molecular characteristics intermediate to those of the two classes of actual signal sequences. The high incidence of non-synonymous substitutions (63.6%), the high transition/transversion ratio (10/1) and the results of molecular modeling all suggest that the N-terminal cleavable signal sequence is mutationally evolving more rapidly than proteins which must assume specific conformational states in the mature influenza virion.

Receptor-specific Ubiquitination of β-Arrestin Directs Assembly and Targeting of Seven-transmembrane Receptor Signalosomes

Angiotensin II type 1a (AT1a), vasopressin V2, and neurokinin 1 (NK1) receptors are seven-transmembrane receptors (7TMRs) that bind and co-internalize with the multifunctional adaptor protein, β-arrestin. These receptors also lead to robust and persistent activation of extracellular-signal regulated kinase 1/2 (ERK1/2) localized on endosomes. Recently, the co-trafficking of receptor-β-arrestin complexes to endosomes was demonstrated to require stable β-arrestin ubiquitination (Shenoy, S. K., and Lefkowitz, R. J. (2003) <i>J. Biol. Chem.</i> 278, 14498–14506). We now report that lysines at positions 11 and 12 in β-arrestin2 are specific and required sites for its AngII-mediated sustained ubiquitination. Thus, upon AngII stimulation the mutant β-arrestin2^K11,12R is only transiently ubiquitinated, does not form stable endocytic complexes with the AT1aR, and is impaired in scaffolding-activated ERK1/2. Fusion of a ubiquitin moiety in-frame to β-arrestin2^K11,12R restores AngII-mediated trafficking and signaling. Wild type β-arrestin2 and β-arrestin2^K11R,K12R-Ub, but not β-arrestin2^K11R,K12R, prevent nuclear translocation of pERK. These findings imply that sustained β-arrestin ubiquitination not only directs co-trafficking of receptor-β-arrestin complexes but also orchestrates the targeting of "7TMR signalosomes" to microcompartments within the cell. Surprisingly, binding of β-arrestin2^K11R,K12R to V2R and NK1R is indistinguishable from that of wild type β-arrestin2. Moreover, ubiquitination patterns and ERK scaffolding of β-arrestin2^K11,12R are unimpaired with respect to V2R stimulation. In contrast, a quintuple lysine mutant (β-arrestin2_{K18R,K107R,K108R,K207R,K296R}) is impaired in endosomal trafficking in response to V2R but not AT1aR stimulation. Our findings delineate a novel regulatory mechanism for 7TMR signaling, dictated by the ubiquitination of β-arrestin on specific lysines that become accessible for modification due to the specific receptor-bound conformational states of β-arrestin2.