Mapping Ligand–receptor Interfaces: Approaching the Resolution Limit of Benzophenone‐based Photoaffinity Scanning

Abstract

Photoaffinity crosslinking has yielded important insights in the study of G protein-coupled receptors and the mode of ligand binding. The most widely used photolabile moiety is p-benzoylphenylalanine largely because of its reportedly high site specificity, reduced reactivity to water and light, photokinetics, and ease of incorporation into peptide ligands during synthesis. However, in the course of our studies directed at characterizing the binding of parathyroid hormone to its cognate G proteincoupled receptor, we find that inherent properties of p-benzoylphenylalanine, such as its size and conformational flexibility, limit the resulting resolution of the ligand-receptor structure. Here, we examine and define these limits. Keywords molecular modeling; parathyroid hormone receptor; p-benzoylphenylalanine; photoaffinity crosslinking; resolution limit G protein-coupled receptors represent the largest family of membrane proteins, with over 800 members identified in the human genome. They constitute one of the most important groups of targets for drug discovery, with approximately half of all drugs currently in clinical use targeted at G protein-coupled receptors (GPCRs). Yet, the nature of ligand binding to GPCRs and the mechanism of activation of GPCRs are poorly understood; the hydrophobic nature of the seven membrane-spanning helices and the conformational complexity of GPCRs pose a problem for direct structure determination by crystallography or NMR.To gain insight into ligand-GPCR interactions, photoaffinity scanning has been commonly applied as a means of mapping the points and zones of contact between hormones and their receptors. Using p-benzoylphenylalanine (Bpa)-based photoaffinity scanning, important advances in our understanding of the ligand-receptor interface have been obtained for a number of ligand-GPCR systems. For example, the 27 amino acid hormone secretin was found to adopt an extended conformation while bound to its receptor, a family B GPCR, with the N-terminal residues of the hormone reaching to the extracellular part of TM6 (1,2). Similarly, photoaffinity crosslinking reveals a binding groove for calcitonin on the calcitonin receptor, also a family B GPCR, extending from residues in the N-ECD to contacts in extracellular loop (ECL) 3, with the N-terminus of the ligand positioned in proximity to TM6 (3). In addition, the crosslinking sites for calcitonin agonists versus antagonists differ due to differential interaction of the analogs with active versus inactive receptor states, suggesting conformational changes involving the N-ECD upon receptor activation (4). The angiotensin-receptor system is an *Corresponding author: Angela Wittelsberger, angela.wittelsberger@tufts.edu. example from GPCR family A. Bpa-based crosslinking shows that the eight amino acid ligand adopts an extended conformation which inserts into the TM bundle with position 8 of the ligand contacting residues in TM3, TM6, and TM7 (5). Finally, for the parathyroid hormone (PTH)-receptor (PTHR1) system, molecular dynamic...

Mapping Peptide Hormone−Receptor Interactions Using a Disulfide-Trapping Approach

“…We believe that we are reaching the limit of the resolving power of this technique, making the generation of newer, more detailed models of the PTH-PTHR1 interaction increasingly difficult. Also, we have recently explored an important caveat associated with the commonly employed photoreactive Bpa moiety: we delineated the "methionine magnet effect" over a range of at least 11 amino acid residues in the PTHR1 (14,19). Although highly useful for surveying a hormone-receptor in order to derive a first-generation map of the bindecular interface, the properties of Bpa have important implications that compromise its utility in photoaffinity cross-linking to delineate contact sites for use in creating high-resolution models of the PTH-PTHR1 interaction and peptide-receptor interactions in general.…”

“…Using benzoylphenylalanine-(Bpa-) mediated photoaffinity cross-linking, we previously reported that an analogue of PTH incorporating a Bpa moiety at position 1 (Bpa1-PTH) crosslinks to M425 of the receptor, close to the top of transmembrane (TM) 6 (6). However, based on recent findings that Bpa exhibits a strong cross-linking preference for methionine, the precision of this contact site is not well-defined (14)(15)(16)(17)(18)(19). Given the methionine preference, coupled with the rotational freedom of the Bpa moiety (encompassing a large radius for crosslinking), further refinement of this PTH/PTHR1 region of interaction using a Bpa photoaffinity cross-linking approach is precluded.…”

Two distinct sites of client protein interaction with the chaperone cpSRP43

“…Crosslinking occurs when the ketone oxygen of the incorporated pBpa is within 3.1 Å of an interaction partner (36). To specifically examine the interactions between cpSRP43 and Lhcb 5 , we used a superactive mutant of cpSRP43 (intein-cpSRP43) that has been shown to mimic the conformation and activity of cpSRP54-activated cpSRP43 in both NMR spectroscopy and biochemical assays (22).…”

Design and characterization of a photo-activatable hedgehog probe that mimics the natural lipidated form

“…Our approach to identify interactions between Shh and potential binding partners and to circumvent the challenges of reconstituting what might be low affinity binding interactions, was to generate a photo-activatable version of Shh using benzophenone [45, 46] to target and cross-link these interactions. Benzophenone photophores are used extensively for photoaffinity-labeling studies as they are one of the most stable photoreactive groups and the wavelength for UV crosslinking (~350 nm) does not typically affect proteins [47]. Benzophenone-containing molecules undergo photo-activatable cross-linking to adjacent molecules with high specificity by efficient covalent modification to C–H bonds, even in aqueous buffers.…”