Snapshots of actin and tubulin folding inside the TRiC chaperonin.

The integrity of a cell's proteome depends on correct folding of polypeptides by chaperonins. The chaperonin TCP-1 ring complex (TRiC) acts as obligate folder for >10% of cytosolic proteins, including he cytoskeletal proteins actin and tubulin. Although its architecture and how it recognizes folding substrates are emerging from structural studies, the subsequent fate of substrates inside the TRiC chamber is not defined. We trapped endogenous human TRiC with substrates (actin, tubulin) and cochaperone (PhLP2A) at different folding stages, for structure determination by cryo-EM. The already-folded regions of client proteins are anchored at the chamber wall, positioning unstructured regions toward the central space to achieve their native fold. Substrates engage with different sections of the chamber during the folding cycle, coupled to TRiC open-and-close transitions. Further, the cochaperone PhLP2A modulates folding, acting as a molecular strut between substrate and TRiC chamber. Our structural snapshots piece together an emerging model of client protein folding within TRiC.

PF-07059013: A Noncovalent Modulator of Hemoglobin for Treatment of Sickle Cell Disease.

Sickle cell disease (SCD) is a genetic disorder caused by a single point mutation (ß6 Glu → Val) on the ß-chain of adult hemoglobin (HbA) that results in sickled hemoglobin (HbS). In the deoxygenated state, polymerization of HbS leads to sickling of red blood cells (RBC). Several downstream consequences of polymerization and RBC sickling include vaso-occlusion, hemolytic anemia, and stroke. We report the design of a noncovalent modulator of HbS, clinical candidate PF-07059013 (23). The seminal hit molecule was discovered by virtual screening and confirmed through a series of biochemical and biophysical studies. After a significant optimization effort, we arrived at 23, a compound that specifically binds to Hb with nanomolar affinity and displays strong partitioning into RBCs. In a 2-week multiple dose study using Townes SCD mice, 23 showed a 37.8% (±9.0%) reduction in sickling compared to vehicle treated mice. 23 (PF-07059013) has advanced to phase 1 clinical trials.

Decay of an active GPCR: Conformational dynamics govern agonist rebinding and persistence of an active, yet empty, receptor state.

Here, we describe two insights into the role of receptor conformational dynamics during agonist release (all-trans retinal, ATR) from the visual G protein-coupled receptor (GPCR) rhodopsin. First, we show that, after light activation, ATR can continually release and rebind to any receptor remaining in an active-like conformation. As with other GPCRs, we observe that this equilibrium can be shifted by either promoting the active-like population or increasing the agonist concentration. Second, we find that during decay of the signaling state an active-like, yet empty, receptor conformation can transiently persist after retinal release, before the receptor ultimately collapses into an inactive conformation. The latter conclusion is based on time-resolved, site-directed fluorescence labeling experiments that show a small, but reproducible, lag between the retinal leaving the protein and return of transmembrane helix 6 (TM6) to the inactive conformation, as determined from tryptophan-induced quenching studies. Accelerating Schiff base hydrolysis and subsequent ATR dissociation, either by addition of hydroxylamine or introduction of mutations, further increased the time lag between ATR release and TM6 movement. These observations show that rhodopsin can bind its agonist in equilibrium like a traditional GPCR, provide evidence that an active GPCR conformation can persist even after agonist release, and raise the possibility of targeting this key photoreceptor protein by traditional pharmaceutical-based treatments.

Development and characterization of pepducins as Gs-biased allosteric agonists.

The ß2-adrenergic receptor (ß2AR) is a prototypical G protein-coupled receptor that mediates many hormonal responses, including cardiovascular and pulmonary function. ß-Agonists used to combat hypercontractility in airway smooth muscle stimulate ß2AR-dependent cAMP production that ultimately promotes airway relaxation. Chronic stimulation of the ß2AR by long acting ß-agonists used in the treatment of asthma can promote attenuated responsiveness to agonists and an increased frequency of fatal asthmatic attacks. ß2AR desensitization to ß-agonists is primarily mediated by G protein-coupled receptor kinases and ß-arrestins that attenuate receptor-Gs coupling and promote ß2AR internalization and degradation. A biased agonist that can selectively stimulate Gs signaling without promoting receptor interaction with G protein-coupled receptor kinases and ß-arrestins should serve as an advantageous asthma therapeutic. To identify such molecules, we screened ∼50 lipidated peptides derived from the intracellular loops of the ß2AR, known as pepducins. This screen revealed two classes of Gs-biased pepducins, receptor-independent and receptor-dependent, as well as several ß-arrestin-biased pepducins. The receptor-independent Gs-biased pepducins operate by directly stimulating G protein activation. In contrast, receptor-dependent Gs-biased pepducins appear to stabilize a Gs-biased conformation of the ß2AR that couples to Gs but does not undergo G protein-coupled receptor kinase-mediated phosphorylation or ß-arrestin-mediated internalization. Functional studies in primary human airway smooth muscle cells demonstrate that Gs-biased pepducins are not subject to conventional desensitization and thus may be good candidates for the development of next generation asthma therapeutics. Our study reports the first Gs-biased activator of the ß2AR and provides valuable tools for the study of ß2AR function.

Inter-domain communication of human cystathionine ß-synthase: structural basis of S-adenosyl-L-methionine activation.

Cystathionine ß-synthase (CBS) is a key enzyme in sulfur metabolism, and its inherited deficiency causes homocystinuria. Mammalian CBS is modulated by the binding of S-adenosyl-l-methionine (AdoMet) to its regulatory domain, which activates its catalytic domain. To investigate the underlying mechanism, we performed x-ray crystallography, mutagenesis, and mass spectrometry (MS) on human CBS. The 1.7 Å structure of a AdoMet-bound CBS regulatory domain shows one AdoMet molecule per monomer, at the interface between two constituent modules (CBS-1, CBS-2). AdoMet binding is accompanied by a reorientation between the two modules, relative to the AdoMet-free basal state, to form interactions with AdoMet via residues verified by mutagenesis to be important for AdoMet binding (Phe(443), Asp(444), Gln(445), and Asp(538)) and for AdoMet-driven inter-domain communication (Phe(443), Asp(538)). The observed structural change is further supported by ion mobility MS, showing that as-purified CBS exists in two conformational populations, which converged to one in the presence of AdoMet. We therefore propose that AdoMet-induced conformational change alters the interface and arrangement between the catalytic and regulatory domains within the CBS oligomer, thereby increasing the accessibility of the enzyme active site for catalysis.

Pepducin targeting the C-X-C chemokine receptor type 4 acts as a biased agonist favoring activation of the inhibitory G protein.

Short lipidated peptide sequences derived from various intracellular loop regions of G protein-coupled receptors (GPCRs) are named pepducins and act as allosteric modulators of a number of GPCRs. Recently, a pepducin selectively targeting the C-X-C chemokine receptor type 4 (CXCR4) was found to be an allosteric agonist, active in both cell-based assays and in vivo. However, the precise mechanism of action of this class of ligands remains poorly understood. In particular, given the diversity of signaling effectors that can be engaged by a given receptor, it is not clear whether pepducins can show biased signaling leading to functional selectivity. To explore the ligand-biased potential of pepducins, we assessed the effect of the CXCR4 selective pepducin, ATI-2341, on the ability of the receptor to engage the inhibitory G proteins (Gi1, Gi2 and Gi3), G13, and ß-arrestins. Using bioluminescence resonance energy transfer-based biosensors, we found that, in contrast to the natural CXCR4 ligand, stromal cell-derived factor-1α, which promotes the engagement of the three Gi subtypes, G13 and the two ß-arrestins, ATI-2341 leads to the engagement of the Gi subtypes but not G13 or the ß-arrestins. Calculation of the transduction ratio for each pathway revealed a strong negative bias of ATI-2341 toward G13 and ß-arrestins, revealing functional selectivity for the Gi pathways. The negative bias toward ß-arrestins results from the reduced ability of the pepducin to promote GPCR kinase-mediated phosphorylation of the receptor. In addition to revealing ligand-biased signaling of pepducins, these findings shed some light on the mechanism of action of a unique class of allosteric regulators.

Direct interaction between an allosteric agonist pepducin and the chemokine receptor CXCR4.

Cell surface heptahelical G protein-coupled receptors (GPCRs) mediate critical cellular signaling pathways and are important pharmaceutical drug targets. (1) In addition to traditional small-molecule approaches, lipopeptide-based GPCR-derived pepducins have emerged as a new class of pharmaceutical agents. (2, 3) To better understand how pepducins interact with targeted receptors, we developed a cell-based photo-cross-linking approach to study the interaction between the pepducin agonist ATI-2341 and its target receptor, chemokine C-X-C-type receptor 4 (CXCR4). A pepducin analogue, ATI-2766, formed a specific UV-light-dependent cross-link to CXCR4 and to mutants with truncations of the N-terminus, the known chemokine docking site. These results demonstrate that CXCR4 is the direct binding target of ATI-2341 and suggest a new mechanism for allosteric modulation of GPCR activity. Adaptation and application of our findings should prove useful in further understanding pepducin modulation of GPCRs as well as enable new experimental approaches to better understand GPCR signal transduction.

G protein-coupled receptor modulation with pepducins: moving closer to the clinic.

At the 2nd Pepducin Science Symposium held in Cambridge, Massachusetts, on November 4-5, 2010, investigators working in G protein-coupled receptor (GPCR) research convened to discuss progress since last year's inaugural conference. This year's symposium focused on increasing knowledge of the structure and function of this ubiquitous superfamily of membrane receptors and their potential modulation for disease treatment. Presentations also focused on how GPCR mechanisms might be exploited to treat diseases with pepducins, novel synthetic lipopeptide pharmacophores that modulate heptahelical GPCR activity. While the multiple roles of GPCRs in physiological and pathophysiological processes offer significant opportunities for novel drug development, the global nature of their activity challenges drug-specific and validated target identification. This year's conference highlighted advances in understanding of GPCR agonist and antagonist ligand-binding motifs, their ligand-independent functions, structure-activity relationships (SARs), and evolving unique methods to probe GPCR structure and function. Study results summarized at the meeting also provided evidence for evolving views of how signaling mechanisms work through these receptors.

Discovery of a CXCR4 agonist pepducin that mobilizes bone marrow hematopoietic cells.

The G protein-coupled receptor (GPCR), chemokine CXC-type receptor 4 (CXCR4), and its ligand, CXCL12, mediate the retention of polymorphonuclear neutrophils (PMNs) and hematopoietic stem and progenitor cells (HSPCs) in the bone marrow. Agents that disrupt CXCL12-mediated chemoattraction of CXCR4-expressing cells mobilize PMNs and HSPCs into the peripheral circulation and are therapeutically useful for HSPC collection before autologous bone marrow transplantation (ABMT). Our aim was to develop unique CXCR4-targeted therapeutics using lipopeptide GPCR modulators called pepducins. A pepducin is a synthetic molecule composed of a peptide derived from the amino acid sequence of one of the intracellular (IC) loops of a target GPCR coupled to a lipid tether. We prepared and screened a small CXCR4-targeted pepducin library and identified several pepducins with in vitro agonist activity, including ATI-2341, whose peptide sequence derives from the first IC loop. ATI-2341 induced CXCR4- and G protein-dependent signaling, receptor internalization, and chemotaxis in CXCR4-expressing cells. It also induced dose-dependent peritoneal recruitment of PMNs when administered i.p. to mice. However, when administered systemically by i.v. bolus, ATI-2341 acted as a functional antagonist and dose-dependently mediated release of PMNs from the bone marrow of both mice and cynomolgus monkeys. ATI-2341-mediated release of granulocyte/macrophage progenitor cells from the bone marrow was confirmed by colony-forming assays. We conclude that ATI-2341 is a potent and efficacious mobilizer of bone marrow PMNs and HSPCs and could represent a previously undescribed therapeutic approach for the recruitment of HSPCs before ABMT.

A novel interaction between atrophin-interacting protein 4 and beta-p21-activated kinase-interactive exchange factor is mediated by an SH3 domain.

Cross-talk between G protein-coupled receptors and receptor tyrosine kinase signaling pathways is crucial to the efficient relay and integration of cellular information. Here we identify and define the novel binding interaction of the E3 ubiquitin ligase atrophin-interacting protein 4 (AIP4) with the GTP exchange factor beta-p21-activated kinase-interactive exchange factor (beta PIX). We demonstrate that this interaction is mediated in part by the beta PIX-SH3 domain binding to a proline-rich stretch of AIP4. Analysis of the interaction by isothermal calorimetry is consistent with a heterotrimeric complex with one AIP4-derived peptide binding to two beta PIX-SH3 domains. We determined the crystal structure of the beta PIX-SH3.AIP4 complex to 2.0-A resolution. In contrast to the calorimetry results, the crystal structure shows a monomeric complex in which AIP4 peptide binds the beta PIX-SH3 domain as a canonical Class I ligand with an additional type II polyproline helix that makes extensive contacts with another face of beta PIX. Taken together, the novel interaction between AIP4 and beta PIX represents a new regulatory node for G protein-coupled receptor and receptor tyrosine kinase signal integration. Our structure of the beta PIX-SH3.AIP4 complex provides important insight into the mechanistic basis for beta PIX scaffolding of signaling components, especially those involved in cross-talk.

Crystal structure of the SH3 domain of betaPIX in complex with a high affinity peptide from PAK2.

The p21-activated kinases (PAKs) are important effector proteins of the small GTPases Cdc42 and Rac and control cytoskeletal rearrangements and cell proliferation. The direct interaction of PAKs with guanine nucleotide exchange factors from the PIX/Cool family, which is responsible for the localization of PAK kinases to focal complexes in the cell, is mediated by a 24-residue peptide segment in PAKs and an N-terminal src homology 3 (SH3) domain in PIX/Cool. The SH3-binding segment of PAK contains the atypical consensus-binding motif PxxxPR, which is required for unusually high affinity binding. In order to understand the structural basis for the high affinity and specificity of the PIX-PAK interaction, we solved crystal structures for the N-terminal SH3 domain of betaPIX and for the complex of the atypical binding segment of PAK2 with the N-terminal SH3 domain of betaPIX at 0.92 A and 1.3A resolution, respectively. The asymmetric unit of the crystal contains two SH3 domains and two peptide ligands. The bound peptide adopts a conformation that allows for intimate contacts with three grooves on the surface of the SH3 domain that lie between the n-Src and RT-loops. Most notably, the arginine residue of the PxxxPR motif forms a salt-bridge and is tightly coordinated by a number of residues in the SH3 domain. This arginine-specific interaction appears to be the key determinant for the high affinity binding of PAK peptides. Furthermore, C-terminal residues of the peptide engage in additional interactions with the surface of the RT-loop, which significantly increases binding specificity. Compared to a recent NMR structure of a similar complex, our crystal structure reveals an alternate binding mode. Finally, we compare our crystal structure with the recently published betaPIX/Cbl-b complex structure, and suggest the existence of a molecular switch.

Role of the retinal hydrogen bond network in rhodopsin Schiff base stability and hydrolysis.

Little is known about the molecular mechanism of Schiff base hydrolysis in rhodopsin. We report here our investigation into this process focusing on the role of amino acids involved in a hydrogen bond network around the retinal Schiff base. We find conservative mutations in this network (T94I, E113Q, S186A, E181Q, Y192F, and Y268F) increase the activation energy (E(a)) and abolish the concave Arrhenius plot normally seen for Schiff base hydrolysis in dark state rhodopsin. Interestingly, two mutants (T94I and E113Q) show dramatically faster rates of Schiff base hydrolysis in dark state rhodopsin, yet slower hydrolysis rates in the active MII form. We find deuterium affects the hydrolysis process in wild-type rhodopsin, exhibiting a specific isotope effect of approximately 2.5, and proton inventory studies indicate that multiple proton transfer events occur during the process of Schiff base hydrolysis for both dark state and MII forms. Taken together, our study demonstrates the importance of the retinal hydrogen bond network both in maintaining Schiff base integrity in dark state rhodopsin, as well as in catalyzing the hydrolysis and release of retinal from the MII form. Finally, we note that the dramatic alteration of Schiff base stability caused by mutation T94I may play a causative role in congenital night blindness as has been suggested by the Oprian and Garriga laboratories.

Rhodopsin activation exposes a key hydrophobic binding site for the transducin alpha-subunit C terminus.

Conformational changes enable the photoreceptor rhodopsin to couple with and activate the G-protein transducin. Here we demonstrate a key interaction between these proteins occurs between the C terminus of the transducin alpha-subunit (G(Talpha)) and a hydrophobic cleft in the rhodopsin cytoplasmic face exposed during receptor activation. We mapped this interaction by labeling rhodopsin mutants with the fluorescent probe bimane and then assessed how binding of a peptide analogue of the G(Talpha) C terminus (containing a tryptophan quenching group) affected their fluorescence. From these and other assays, we conclude that the G(Talpha) C-terminal tail binds to the inner face of helix 6 in a retinal-linked manner. Further, we find that a "hydrophobic patch" comprising key residues in the exposed cleft is required for transducin binding/activation because it enhances the binding affinity for the G(Talpha) C-terminal tail, contributing up to 3 kcal/mol for this interaction. We speculate the hydrophobic interactions identified here may be important in other GPCR signaling systems, and our Trp/bimane fluorescence methodology may be generally useful for mapping sites of protein-protein interaction.

Assessing structural elements that influence Schiff base stability: mutants E113Q and D190N destabilize rhodopsin through different mechanisms.

The stability of the retinal chromophore attachment varies between different visual pigments and may factor in some retinal disease states. Opsin appears to stabilize this Schiff base linkage by: (i) affecting the hydrolysis chemistry, (ii) shielding the retinal linkage from solvent, or (iii) acting as a kinetic trap to slow retinal release. Here we describe methods to determine Schiff base stability in rhodopsin, present examples of dark state and MII rhodopsin stability differences, and show that studies of mutants E113Q and D190N demonstrate different parts of rhodopsin influence Schiff base stability in different ways.

Stability of dark state rhodopsin is mediated by a conserved ion pair in intradiscal loop E-2.

The rhodopsin crystal structure reveals that intradiscal loop E-2 covers the 11-cis-retinal, creating a "retinal plug." Recently, we noticed the ends of loop E-2 are linked by an ion pair between residues Arg-177 and Asp-190, near the highly conserved disulfide bond. This ion pair appears biologically significant; it is conserved in almost all vertebrate opsins and may occur in other G-protein-coupled receptors. We report here that the Arg-177/Asp-190 ion pair is critical for the folding and stability of dark state rhodopsin. We find ion pair mutants that regenerate with retinal are functionally and spectrally wild-type-like yet thermally unstable in their dark state because of rapid hydrolysis of the retinal Schiff base linkage. Surprisingly, Arrhenius analysis indicates that the activation energies for the hydrolysis process are similar between the ion pair mutants and wild-type rhodopsin. Furthermore, the ion pair mutants do not show increased reactivity toward hydroxylamine, suggesting that their instability is not caused by an increased exposure to bulk solvent. Our results indicate that the loop E-2 ion pair is important for rhodopsin stability and thus suggest that retinitis pigmentosa observed in patients with Asp-190 mutations may in part be the result of thermally unstable rhodopsin proteins.