Faculty Bibliography

G-protein-coupled receptors (GPCRs) are critically regulated by beta-arrestins, which not only desensitize G-protein signalling but also initiate a G-protein-independent wave of signalling. A recent surge of structural data on a number of GPCRs, including the beta2 adrenergic receptor (beta2AR)-G-protein complex, has provided novel insights into the structural basis of receptor activation. However, complementary information has been lacking on the recruitment of beta-arrestins to activated GPCRs, primarily owing to challenges in obtaining stable receptor-beta-arrestin complexes for structural studies. Here we devised a strategy for forming and purifying a functional human beta2AR-beta-arrestin-1 complex that allowed us to visualize its architecture by single-particle negative-stain electron microscopy and to characterize the interactions between beta2AR and beta-arrestin 1 using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and chemical crosslinking. Electron microscopy two-dimensional averages and three-dimensional reconstructions reveal bimodal binding of beta-arrestin 1 to the beta2AR, involving two separate sets of interactions, one with the phosphorylated carboxy terminus of the receptor and the other with its seven-transmembrane core. Areas of reduced HDX together with identification of crosslinked residues suggest engagement of the finger loop of beta-arrestin 1 with the seven-transmembrane core of the receptor. In contrast, focal areas of raised HDX levels indicate regions of increased dynamics in both the N and C domains of beta-arrestin 1 when coupled to the beta2AR. A molecular model of the beta2AR-beta-arrestin signalling complex was made by docking activated beta-arrestin 1 and beta2AR crystal structures into the electron microscopy map densities with constraints provided by HDX-MS and crosslinking, allowing us to obtain valuable insights into the overall architecture of a receptor-arrestin complex. The dynamic and structural information presented here provides a framework for better understanding the basis of GPCR regulation by arrestins.

Cell signaling depends on dynamic protein-protein interaction (PPI) networks, often assembled through modular domains each interacting with multiple peptide motifs. This complexity raises a conceptual challenge, namely to define whether a particular cellular response requires assembly of the complete PPI network of interest or can be driven by a specific interaction. To address this issue, we designed variants of the Grb2 SH2 domain ("pY-clamps") whose specificity is highly biased toward a single phosphotyrosine (pY) motif among many potential pYXNX Grb2-binding sites. Surprisingly, directing Grb2 predominantly to a single pY site of the Ptpn11/Shp2 phosphatase, but not other sites tested, was sufficient for differentiation of the essential primitive endoderm lineage from embryonic stem cells. Our data suggest that discrete connections within complex PPI networks can underpin regulation of particular biological events. We propose that this directed wiring approach will be of general utility in functionally annotating specific PPIs.

Variability in the quality of antibodies to histone post-translational modifications (PTMs) is a widely recognized hindrance in epigenetics research. Here, we produced recombinant antibodies to the trimethylated lysine residues of histone H3 with high specificity and affinity and no lot-to-lot variation. These recombinant antibodies performed well in common epigenetics applications, and enabled us to identify positive and negative correlations among histone PTMs.

The dysregulated tyrosine kinase BCR-ABL causes chronic myelogenous leukemia in humans and forms a large multiprotein complex that includes the Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2). The expression of SHP2 is necessary for BCR-ABL-dependent oncogenic transformation, but the precise signaling mechanisms of SHP2 are not well understood. We have developed binding proteins, termed monobodies, for the N- and C-terminal SH2 domains of SHP2. Intracellular expression followed by interactome analysis showed that the monobodies are essentially monospecific to SHP2. Two crystal structures revealed that the monobodies occupy the phosphopeptide-binding sites of the SH2 domains and thus can serve as competitors of SH2-phosphotyrosine interactions. Surprisingly, the segments of both monobodies that bind to the peptide-binding grooves run in the opposite direction to that of canonical phosphotyrosine peptides, which may contribute to their exquisite specificity. When expressed in cells, monobodies targeting the N-SH2 domain disrupted the interaction of SHP2 with its upstream activator, the Grb2-associated binder 2 adaptor protein, suggesting decoupling of SHP2 from the BCR-ABL protein complex. Inhibition of either N-SH2 or C-SH2 was sufficient to inhibit two tyrosine phosphorylation events that are critical for SHP2 catalytic activity and to block ERK activation. In contrast, targeting the N-SH2 or C-SH2 revealed distinct roles of the two SH2 domains in downstream signaling, such as the phosphorylation of paxillin and signal transducer and activator of transcription 5. Our results delineate a hierarchy of function for the SH2 domains of SHP2 and validate monobodies as potent and specific antagonists of protein-protein interactions in cancer cells.

Insulin-degrading enzyme (IDE) selectively degrades the monomer of amyloidogenic peptides and contributes to clearance of amyloid beta (Abeta). Thus, IDE retards the progression of Alzheimer's disease. IDE possesses an enclosed catalytic chamber that engulfs and degrades its peptide substrates; however, the molecular mechanism of IDE function, including substrate access to the chamber and recognition, remains elusive. Here, we captured a unique IDE conformation by using a synthetic antibody fragment as a crystallization chaperone. An unexpected displacement of a door subdomain creates an ~18-A opening to the chamber. This swinging-door mechanism permits the entry of short peptides into the catalytic chamber and disrupts the catalytic site within IDE door subdomain. Given the propensity of amyloidogenic peptides to convert into beta-strands for their polymerization into amyloid fibrils, they also use such beta-strands to stabilize the disrupted catalytic site resided at IDE door subdomain for their degradation by IDE. Thus, action of the swinging door allows IDE to recognize amyloidogenicity by substrate-induced stabilization of the IDE catalytic cleft. Small angle X-ray scattering (SAXS) analysis revealed that IDE exists as a mixture of closed and open states. These open states, which are distinct from the swinging door state, permit entry of larger substrates (e.g., Abeta, insulin) to the chamber and are preferred in solution. Mutational studies confirmed the critical roles of the door subdomain and hinge loop joining the N- and C-terminal halves of IDE for catalysis. Together, our data provide insights into the conformational changes of IDE that govern the selective destruction of amyloidogenic peptides.

The functions of G-protein-coupled receptors (GPCRs) are primarily mediated and modulated by three families of proteins: the heterotrimeric G proteins, the G-protein-coupled receptor kinases (GRKs) and the arrestins. G proteins mediate activation of second-messenger-generating enzymes and other effectors, GRKs phosphorylate activated receptors, and arrestins subsequently bind phosphorylated receptors and cause receptor desensitization. Arrestins activated by interaction with phosphorylated receptors can also mediate G-protein-independent signalling by serving as adaptors to link receptors to numerous signalling pathways. Despite their central role in regulation and signalling of GPCRs, a structural understanding of beta-arrestin activation and interaction with GPCRs is still lacking. Here we report the crystal structure of beta-arrestin-1 (also called arrestin-2) in complex with a fully phosphorylated 29-amino-acid carboxy-terminal peptide derived from the human V2 vasopressin receptor (V2Rpp). This peptide has previously been shown to functionally and conformationally activate beta-arrestin-1 (ref. 5). To capture this active conformation, we used a conformationally selective synthetic antibody fragment (Fab30) that recognizes the phosphopeptide-activated state of beta-arrestin-1. The structure of the beta-arrestin-1-V2Rpp-Fab30 complex shows marked conformational differences in beta-arrestin-1 compared to its inactive conformation. These include rotation of the amino- and carboxy-terminal domains relative to each other, and a major reorientation of the 'lariat loop' implicated in maintaining the inactive state of beta-arrestin-1. These results reveal, at high resolution, a receptor-interacting interface on beta-arrestin, and they indicate a potentially general molecular mechanism for activation of these multifunctional signalling and regulatory proteins.

A set of phage display sorting strategies and validation methodologies are presented that are capable of producing high performance synthetic antibodies (sABs) with customized properties. Exquisite control of antigen and conditions during the phage display selection process can yield sABs that: (1) recognize conformational states, (2) target specific regions of the surface of a protein, (3) induce conformational changes, and (4) capture and stabilize multi-protein complexes. These unique capabilities open myriad opportunities to study complex macromolecular processes inaccessible to traditional affinity reagent technology. We present detailed protocols for de novo isolation of binders, as well as examples of downstream biophysical characterization. The methods described are generalizable and can be adapted to other in vitro direct evolution approaches based on yeast or mRNA display.

A synthetic phage-displayed antibody repertoire was constructed with equivalent chemical diversity in the third complementarity-determining regions of the heavy (CDR-H3) and light (CDR-L3) chains, which contrasts with natural antibodies in which CDR-H3 is much more diverse than CDR-L3 due to the genetic mechanisms that generate antibody encoding genes. Surprisingly, the synthetic repertoire yielded numerous functional antibodies that contained mutated CDR-L3 sequences but a fixed CDR-H3 sequence. Alanine-scanning analysis of antibodies that recognized 10 different antigens but contained a common CDR-H3 loop showed that, in most cases, the fixed CDR-H3 sequence was able to contribute favorably to antigen recognition, but in some cases, the loop was functionally inert. Structural analysis of one such antibody in complex with antigen showed that the inert CDR-H3 loop was nonetheless highly buried at the antibody-antigen interface. Taken together, these results show that CDR-H3 diversity is not necessarily required for the generation of antibodies that recognize diverse protein antigens with high affinity and specificity, and if given the chance, CDR-L3 readily assumes the dominant role for antigen recognition. These results contrast with the commonly accepted view of antigen recognition derived from the analysis of natural antibodies, in which CDR-H3 is presumed to be dominant and CDR-L3 is presumed to play an auxiliary role. Furthermore, the results show that natural antibody function is genetically constrained, and it should be possible to develop more functional synthetic antibody libraries by expanding the diversity of CDR-L3 beyond what is observed in nature.