Capturing Protein-Protein Interactions with Acidic Amino Acids Reactive Cross-Linkers.

Acidic residues (Asp and Glu) have a high prevalence on protein surfaces, but cross-linking reactions targeting these residues are limited. Existing methods either require high-concentration coupling reagents or have low structural compatibility. Here a previously reported "plant-and-cast" strategy is extended to develop heterobifunctional cross-linkers. These cross-linkers first react rapidly with Lys sidechains and then react with Asp and Glu sidechains, in a proximity-enhanced fashion. The cross-linking reaction proceeds at neutral pH and room temperature without coupling reagents. The efficiency and robustness of cross-linking using model proteins, ranging from small monomeric proteins to large protein complexes are demonstrated. Importantly, it is shown that this type of cross-linkers are efficient at identifying protein-protein interactions involving acidic domains. The Cross-linking mass spectrometry (XL-MS) study with p53 identified 87 putative binders of the C-terminal domain of p53. Among them, SARNP, ZRAB2, and WBP11 are shown to regulate the expression and alternative splicing of p53 target genes. Thus, these carboxylate-reactive cross-linkers will further expand the power of XL-MS in the analysis of protein structures and protein-protein interactions.

Ready for the sheet: ß-strand folding of phosphorylation clusters guides GPCR binding to arrestin.

The molecular dynamics of arrestin binding to G protein-coupled receptors (GPCRs) are still poorly understood. In this issue of Structure, Guillien et al. show that negative charges in GPCR key phosphorylation clusters induce the formation of a transient ß-strand that participates in an intermolecular ß-sheet in the associated complex.

Genetically encoded crosslinkers to address protein-protein interactions.

Noncanonical amino acids (ncAAs) for photo- and chemical crosslinking are powerful biochemical tools for studying and manipulating interactions between proteins both in vitro and in intact cells. Since the first crosslinking ncAAs were genetically encoded about 20 years ago, the technology has now ripened beyond the proof-of-principle demonstrations and is contributing to the study of relevant biological questions in the frame of modern integrative approaches. Here, we provide an overview of available photo-activatable ncAAs for photo-crosslinking and electrophilic ncAAs for genetically encoded chemical crosslinking (GECX), with a major focus on the most recent entries such as ncAAs for SuFEx click chemistry and photo-activatable ncAAs for chemical crosslinking. We present recent examples of the application of genetically encoded crosslinkers to capture protein-protein interactions and identify interaction partners in live cells, to investigate molecular mechanisms of protein function, to stabilize protein complexes for structural studies, to derive structural information about protein complexes from the physiological cell environment, up to perspective applications of GECX-ncAAs for the development of covalent drugs.

Sparse pseudocontact shift NMR data obtained from a non-canonical amino acid-linked lanthanide tag improves integral membrane protein structure prediction.

A single experimental method alone often fails to provide the resolution, accuracy, and coverage needed to model integral membrane proteins (IMPs). Integrating computation with experimental data is a powerful approach to supplement missing structural information with atomic detail. We combine RosettaNMR with experimentally-derived paramagnetic NMR restraints to guide membrane protein structure prediction. We demonstrate this approach using the disulfide bond formation protein B (DsbB), an α-helical IMP. Here, we attached a cyclen-based paramagnetic lanthanide tag to an engineered non-canonical amino acid (ncAA) using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry reaction. Using this tagging strategy, we collected 203 backbone HN pseudocontact shifts (PCSs) for three different labeling sites and used these as input to guide de novo membrane protein structure prediction protocols in Rosetta. We find that this sparse PCS dataset combined with 44 long-range NOEs as restraints in our calculations improves structure prediction of DsbB by enhancements in model accuracy, sampling, and scoring. The inclusion of this PCS dataset improved the Cα-RMSD transmembrane segment values of the best-scoring and best-RMSD models from 9.57 Å and 3.06 Å (no NMR data) to 5.73 Å and 2.18 Å, respectively.

Structural details of a Class B GPCR-arrestin complex revealed by genetically encoded crosslinkers in living cells.

Understanding the molecular basis of arrestin-mediated regulation of GPCRs is critical for deciphering signaling mechanisms and designing functional selectivity. However, structural studies of GPCR-arrestin complexes are hampered by their highly dynamic nature. Here, we dissect the interaction of arrestin-2 (arr2) with the secretin-like parathyroid hormone 1 receptor PTH1R using genetically encoded crosslinking amino acids in live cells. We identify 136 intermolecular proximity points that guide the construction of energy-optimized molecular models for the PTH1R-arr2 complex. Our data reveal flexible receptor elements missing in existing structures, including intracellular loop 3 and the proximal C-tail, and suggest a functional role of a hitherto overlooked positively charged region at the arrestin N-edge. Unbiased MD simulations highlight the stability and dynamic nature of the complex. Our integrative approach yields structural insights into protein-protein complexes in a biologically relevant live-cell environment and provides information inaccessible to classical structural methods, while also revealing the dynamics of the system.

Detecting Active Deconjugating Enzymes with Genetically Encoded Activity-Based Ubiquitin and Ubiquitin-like Protein Probes.

Post-translational modification of proteins by Ubiquitin (Ub) and Ubiquitin-like proteins (Ubls) can be reversed by deconjugating enzymes, which have been implicated in different pathways and associated with various human diseases. To understand the activity and dynamics of deconjugating enzymes, multiple synthetic and semi-synthetic Ub/Ubl probes have been developed, and some of them have been applied to screen inhibitors of deconjugating enzymes. Since these Ub/Ubl probes are generally not cell-permeable, different strategies have been developed to deliver Ub/Ubl probes to live cells. However, till now, no Ub/Ubl probes can be expressed in live cells to directly report on the activities of deconjugating enzymes in the most relevant cellular environment. Here, we genetically encoded cross-linkable Ub/Ubl probes in live E. coli and HEK293T cells. These probes can cross-link with deconjugating enzymes in vitro and in vivo. Using these Ub probes combined with mass spectrometry, we have successfully identified endogenous deconjugating enzymes in live cells. We believe that these genetically encoded Ub/Ubl probes are valuable for investigating biological functions of deconjugating enzymes in physiological environments.

Binding of Natural Peptide Ligands to the Neuropeptide Y5 Receptor.

The binding mode of natural peptide ligands to the Y5 G protein-coupled receptor (Y5 R), an attractive therapeutic target for the treatment of obesity, is largely unknown. Here, we apply complementary biochemical and computational approaches, including scanning of the receptor surface with a genetically encoded crosslinker, Ala-scanning of the ligand and double-cycle mutagenesis, to map interactions in the ligand-receptor interface and build a structural model of the NPY-Y5 R complex guided by the experimental data. In the model, the carboxyl (C)-terminus of bound NPY is placed close to the extracellular loop (ECL) 3, whereas the characteristic α-helical segment of the ligand drapes over ECL1 and is tethered towards ECL2 by a hydrophobic cluster. We further show that the other two natural ligands of Y5 R, peptide YY (PYY) and pancreatic polypeptide (PP) dock to the receptor in a similar pose.

Biochemical insights into structure and function of arrestins.

Arrestins (arr) are multifunctional cytosolic adaptors that bind to active and phosphorylated G protein-coupled receptors (GPCRs) via a highly versatile interface. Arrestins stop G protein signaling and trigger other signaling pathways. Recently, 3D structures of arr-GPCR complexes have been solved, which provide a bulk of structural information for understanding the mechanism of arr recruitment and activation. However, many questions about the functional consequences of structural details and the dynamics of the arr-GPCR interaction remain open. A wealth of information about key determinants for the arr-GPCR interaction and their functional relevance, and dynamic insights into the process of arr binding and the functional outcomes of different binding modes have been provided by a series of biochemical methods which we review here. Importantly, most of these methods provide information from the live cell, which is a necessary validation and complement for structural data. With the main focus on the most recent research, we will highlight major findings about arr structure, function, and dynamics derived from mutagenesis studies, cross-linking studies, conformational probes, and sensors, and we summarize available systems to detect arr recruitment. Furthermore, we discuss recent findings and directions of in silico investigations in arr-GPCR complexes.

Determination of G-protein-coupled receptor oligomerization by molecular brightness analyses in single cells.

Oligomerization of membrane proteins has received intense research interest because of their importance in cellular signaling and the large pharmacological and clinical potential this offers. Fluorescence imaging methods are emerging as a valid tool to quantify membrane protein oligomerization at high spatial and temporal resolution. Here, we provide a detailed protocol for an image-based method to determine the number and oligomerization state of fluorescently labeled prototypical G-protein-coupled receptors (GPCRs) on the basis of small out-of-equilibrium fluctuations in fluorescence (i.e., molecular brightness) in single cells. The protocol provides a step-by-step procedure that includes instructions for (i) a flexible labeling strategy for the protein of interest (using fluorescent proteins, small self-labeling tags or bio-orthogonal labeling) and the appropriate controls, (ii) performing temporal and spatial brightness image acquisition on a confocal microscope and (iii) analyzing and interpreting the data, excluding clusters and intensity hot-spots commonly observed in receptor distributions. Although specifically tailored for GPCRs, this protocol can be applied to diverse classes of membrane proteins of interest. The complete protocol can be implemented in 1 month.

Capturing Peptide-GPCR Interactions and Their Dynamics.

Many biological functions of peptides are mediated through G protein-coupled receptors (GPCRs). Upon ligand binding, GPCRs undergo conformational changes that facilitate the binding and activation of multiple effectors. GPCRs regulate nearly all physiological processes and are a favorite pharmacological target. In particular, drugs are sought after that elicit the recruitment of selected effectors only (biased ligands). Understanding how ligands bind to GPCRs and which conformational changes they induce is a fundamental step toward the development of more efficient and specific drugs. Moreover, it is emerging that the dynamic of the ligand-receptor interaction contributes to the specificity of both ligand recognition and effector recruitment, an aspect that is missing in structural snapshots from crystallography. We describe here biochemical and biophysical techniques to address ligand-receptor interactions in their structural and dynamic aspects, which include mutagenesis, crosslinking, spectroscopic techniques, and mass-spectrometry profiling. With a main focus on peptide receptors, we present methods to unveil the ligand-receptor contact interface and methods that address conformational changes both in the ligand and the GPCR. The presented studies highlight a wide structural heterogeneity among peptide receptors, reveal distinct structural changes occurring during ligand binding and a surprisingly high dynamics of the ligand-GPCR complexes.

Exploring GPCR-arrestin interfaces with genetically encoded crosslinkers.

ß-arrestins (ßarr1 and ßarr2) are ubiquitous regulators of G protein-coupled receptor (GPCR) signaling. Available data suggest that ß-arrestins dock to different receptors in different ways. However, the structural characterization of GPCR-arrestin complexes is challenging and alternative approaches to study GPCR-arrestin complexes are needed. Here, starting from the finger loop as a major site for the interaction of arrestins with GPCRs, we genetically incorporate non-canonical amino acids for photo- and chemical crosslinking into ßarr1 and ßarr2 and explore binding topologies to GPCRs forming either stable or transient complexes with arrestins: the vasopressin receptor 2 (rhodopsin-like), the corticotropin-releasing factor receptor 1, and the parathyroid hormone receptor 1 (both secretin-like). We show that each receptor leaves a unique footprint on arrestins, whereas the two ß-arrestins yield quite similar crosslinking patterns. Furthermore, we show that the method allows defining the orientation of arrestin with respect to the GPCR. Finally, we provide direct evidence for the formation of arrestin oligomers in the cell.

Allosteric interactions in the parathyroid hormone GPCR-arrestin complex formation.

Peptide ligands of class B G-protein-coupled receptors act via a two-step binding process, but the essential mechanisms that link their extracellular binding to intracellular receptor-arrestin interactions are not fully understood. Using NMR, crosslinking coupled to mass spectrometry, signaling experiments and computational approaches on the parathyroid hormone (PTH) type 1 receptor (PTHR), we show that initial binding of the PTH C-terminal part constrains the conformation of the flexible PTH N-terminal signaling epitope before a second binding event occurs. A 'hot-spot' PTH residue, His9, that inserts into the PTHR transmembrane domain at this second step allosterically engages receptor-arrestin coupling. A conformational change in PTHR intracellular loop 3 permits favorable interactions with ß-arrestin's finger loop. These results unveil structural determinants for PTHR-arrestin complex formation and reveal that the two-step binding mechanism proceeds via cooperative fluctuations between ligand and receptor, which extend to other class B G-protein-coupled receptors.

Quantitative Single-Residue Bioorthogonal Labeling of G Protein-Coupled Receptors in Live Cells.

High-end microscopy studies of G protein-coupled receptors (GPCRs) require installing onto the receptors bright and photostable dyes. Labeling must occur in quantitative yields, to allow stoichiometric data analysis, and in a minimally invasive fashion, to avoid perturbing GPCR function. We demonstrate here that the genetic incorporation of trans-cyclooct-2-ene lysine (TCO*) allows achieving quantitative single-residue labeling of the extracellular loops of the ß2-adrenergic and the muscarinic M2 class A GPCRs, as well as of the corticotropin releasing factor class B GPCR. Labeling occurs within a few minutes by reaction with dye-tetrazine conjugates on the surface of live cells and preserves the functionality of the receptors. To precisely quantify the labeling yields, we devise a method based on fluorescence fluctuation microscopy that extracts the number of labeling sites at the single-cell level. Further, we show that single-residue labeling is better suited for studies of GPCR diffusion than fluorescent-protein tags, since the latter can affect the mobility of the receptor. Finally, by performing dual-color competitive labeling on a single TCO* site, we devise a method to estimate the oligomerization state of a GPCR without the need for a biological monomeric reference, which facilitates the application of fluorescence methods to oligomerization studies. As TCO* and the dye-tetrazines used in this study are commercially available and the described microscopy techniques can be performed on a commercial microscope, we expect our approach to be widely applicable to fluorescence microscopy studies of membrane proteins in general.

Exploring Pairwise Chemical Crosslinking To Study Peptide-Receptor Interactions.

Pairwise crosslinking is a powerful technique to characterize interactions between G protein coupled receptors and their ligands in the live cell. In this work, the "thiol trapping" method, which exploits the proximity-enhanced reaction between haloacetamides and cysteine, is examined to identify intermolecular pairs of vicinal positions. By incorporating cysteine into the corticotropin-releasing factor receptor and either α-chloro- or α-bromoacetamide groups into its ligands, it is shown that thiol trapping provides highly reproducible signals and a low background, and represents a valid alternative to classical "disulfide trapping". The method is advantageous if reducing agents are required during sample analysis. Moreover, it can provide partially distinct spatial constraints, thus giving access to a wider dataset for molecular modeling. Finally, by applying recombinant mini-Gs, GTPγS, and Gαs-depleted HEK293 cells to modulate Gs coupling, it is shown that yields of crosslinking increase in the presence of elevated levels of Gs.

Application of non-canonical crosslinking amino acids to study protein-protein interactions in live cells.

The genetic incorporation of non-canonical amino acids (ncAAs) equipped with photo-crosslinking and chemical crosslinking moieties has found broad application in the study of protein-protein interactions from a unique perspective in live cells. We highlight here applications of photo-activatable ncAAs to map protein interaction surfaces and to capture protein-protein interactions, and we describe recent efforts to efficiently couple photo-crosslinking with mass spectrometric analysis. In addition, we describe recent advances in the development and application of ncAAs for chemical crosslinking, including protein stapling, photo-control of protein conformation, two-dimensional crosslinking, and stabilization of transient and low-affinity protein-protein interactions. We expect that the field will keep growing in the near future and enable the tackling of ambitious biological questions.

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells.

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

Mapping of Protein Interfaces in Live Cells Using Genetically Encoded Crosslinkers.

Understanding the topology of protein-protein interactions is a matter of fundamental importance in the biomedical field. Biophysical approaches such as X-ray crystallography and nuclear magnetic resonance can investigate in detail only isolated protein complexes that are reconstituted in an artificial environment. Alternative methods are needed to investigate protein interactions in a physiological context, as well as to characterize protein complexes that elude the direct structural characterization. We describe here a general strategy to investigate protein interactions at the molecular level directly in the live mammalian cell, which is based on the genetic incorporation of photo- and chemical crosslinking noncanonical amino acids. First a photo-crosslinking amino acid is used to map putative interaction surfaces and determine which positions of a protein come into proximity of an associated partner. In a second step, the subset of residues that belong to the binding interface are substituted with a chemical crosslinker that reacts selectively with proximal cysteines strategically placed in the interaction partner. This allows determining inter-molecular spatial constraints that provide the basis for building accurate molecular models. In this chapter, we illustrate the detailed application of this experimental strategy to unravel the binding modus of the 40-mer neuropeptide hormone Urocortin1 to its class B G-protein coupled receptor, the corticotropin releasing factor receptor type 1. The approach is in principle applicable to any protein complex independent of protein type and size, employs established techniques of noncanonical amino acid mutagenesis, and is feasible in any molecular biology laboratory.

Designer tRNAs for efficient incorporation of non-canonical amino acids by the pyrrolysine system in mammalian cells.

The pyrrolysyl-tRNA synthetase/tRNAPyl pair is the most versatile and widespread system for the incorporation of non-canonical amino acids (ncAAs) into proteins in mammalian cells. However, low yields of ncAA incorporation severely limit its applicability to relevant biological targets. Here, we generate two tRNAPyl variants that significantly boost the performance of the pyrrolysine system. Compared to the original tRNAPyl, the engineered tRNAs feature a canonical hinge between D- and T-loop, show higher intracellular concentrations and bear partially distinct post-transcriptional modifications. Using the new tRNAs, we demonstrate efficient ncAA incorporation into a G-protein coupled receptor (GPCR) and simultaneous ncAA incorporation at two GPCR sites. Moreover, by incorporating last-generation ncAAs for bioorthogonal chemistry, we achieve GPCR labeling with small organic fluorophores on the live cell and visualize stimulus-induced GPCR internalization. Such a robust system for incorporation of single or multiple ncAAs will facilitate the application of a wide pool of chemical tools for structural and functional studies of challenging biological targets in live mammalian cells.

Structural insight into the activation of a class B G-protein-coupled receptor by peptide hormones in live human cells.

The activation mechanism of class B G-protein-coupled receptors (GPCRs) remains largely unknown. To characterize conformational changes induced by peptide hormones, we investigated interactions of the class B corticotropin-releasing factor receptor type 1 (CRF1R) with two peptide agonists and three peptide antagonists obtained by N-truncation of the agonists. Surface mapping with genetically encoded photo-crosslinkers and pair-wise crosslinking revealed distinct footprints of agonists and antagonists on the transmembrane domain (TMD) of CRF1R and identified numerous ligand-receptor contact sites, directly from the intact receptor in live human cells. The data enabled generating atomistic models of CRF- and CRF(12-41)-bound CRF1R, further explored by molecular dynamics simulations. We show that bound agonist and antagonist adopt different folds and stabilize distinct TMD conformations, which involves bending of helices VI and VII around flexible glycine hinges. Conservation of these glycine hinges among all class B GPCRs suggests their general role in activation of these receptors.

Genetically encoded chemical probes in cells reveal the binding path of urocortin-I to CRF class B GPCR.

Molecular determinants regulating the activation of class B G-protein-coupled receptors (GPCRs) by native peptide agonists are largely unknown. We have investigated here the interaction between the corticotropin releasing factor receptor type 1 (CRF1R) and its native 40-mer peptide ligand Urocortin-I directly in mammalian cells. By incorporating unnatural amino acid photochemical and new click-chemical probes into the intact receptor expressed in the native membrane of live cells, 44 intermolecular spatial constraints have been derived for the ligand-receptor interaction. The data were analyzed in the context of the recently resolved crystal structure of CRF1R transmembrane domain and existing extracellular domain structures, yielding a complete conformational model for the peptide-receptor complex. Structural features of the receptor-ligand complex yield molecular insights on the mechanism of receptor activation and the basis for discrimination between agonist and antagonist function.