Methionine acts as a “magnet” in photoaffinity crosslinking experiments

Wittelsberger, Angela; Thomas, Beena; Mierke, Dale F.; Rosenblatt, Michael

doi:10.1016/j.febslet.2006.02.050

Cited by 102 publications

(107 citation statements)

References 21 publications

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“…4A). Moreover, the photoreactive benzophenone (pBpF) group does not react avidly with all amino acids but shows enhanced reactivity with Met residues (35). Because no cross-linking with SdhA had been observed with SdhE-8pBpF, the SdhA-E186 and SdhA-T187 residues were substituted with Met.…”

Section: Resultsmentioning

confidence: 99%

Binding of the Covalent Flavin Assembly Factor to the Flavoprotein Subunit of Complex II

Maklashina

Rajagukguk

Starbird³

et al. 2016

Journal of Biological Chemistry

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Escherichia coli harbors two highly conserved homologs of the essential mitochondrial respiratory complex II (succinate: ubiquinone oxidoreductase). Aerobically the bacterium synthesizes succinate:quinone reductase as part of its respiratory chain, whereas under microaerophilic conditions, the quinol: fumarate reductase can be utilized. All complex II enzymes harbor a covalently bound FAD co-factor that is essential for their ability to oxidize succinate. In eukaryotes and many bacteria, assembly of the covalent flavin linkage is facilitated by a small protein assembly factor, termed SdhE in E. coli. How SdhE assists with formation of the covalent flavin bond and how it binds the flavoprotein subunit of complex II remain unknown. Using photo-cross-linking, we report the interaction site between the flavoprotein of complex II and the SdhE assembly factor. These data indicate that SdhE binds to the flavoprotein between two independently folded domains and that this binding mode likely influences the interdomain orientation. In so doing, SdhE likely orients amino acid residues near the dicarboxylate and FAD binding site, which facilitates formation of the covalent flavin linkage. These studies identify how the conserved SdhE assembly factor and its homologs participate in complex II maturation.

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Section: Resultsmentioning

confidence: 99%

Binding of the Covalent Flavin Assembly Factor to the Flavoprotein Subunit of Complex II

Maklashina

Rajagukguk

Starbird³

et al. 2016

Journal of Biological Chemistry

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“…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.…”

Section: Discussionmentioning

confidence: 99%

“…The results from the disulfide-trapping experiments were utilized as distance restraints in a molecular dynamics simulation, using the NAMD simulation package, following published procedures (14). In these simulations, a restraint of 6.0 Å was placed between the Cβ atoms of the Ser1 of PTH and the appropriate residue of the receptor: L368, Y421, F424, and M425.…”

Section: Molecular Modelingmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2008

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Efforts to elucidate the nature of the bimolecular interaction of parathyroid hormone (PTH) with its cognate receptor, the PTH receptor type 1 (PTHR1), have relied heavily on benzoylphenylalanine-(Bpa-) based photoaffinity cross-linking. However, given the flexibility, size, and shape of Bpa, the resolution at the PTH-PTHR1 interface appears to be reaching the limit of this technique. Here we employ a disulfide-trapping approach developed by others primarily for use in screening compound libraries to identify novel ligands. In this method, cysteine substitutions are introduced into a specific site within the ligand and a region in the receptor predicted to interact with each other. Upon ligand binding, if these cysteines are in close proximity, they form a disulfide bond. Since the geometry governing disulfide bond formation is more constrained than Bpa cross-linking, this novel approach can be employed to generate a more refined molecular model of the PTH-PTHR1 complex. Using a PTH analogue containing a cysteine at position 1, we probed 24 sites and identified 4 in PTHR1 to which cross-linking occurred. Importantly, previous photoaffinity cross-linking studies using a PTH analogue with Bpa at position 1 only identified a single interaction site. The new sites identified by the disulfide-trapping procedure were used as constraints in molecular dynamics simulations to generate an updated model of the PTH-PTHR1 complex. Mapping by disulfide trapping extends and complements photoaffinity cross-linking. It is applicable to other peptide-receptor interfaces and should yield insights about yet unknown sites of ligand-receptor interactions, allowing for generation of more refined models.Class II G protein-coupled receptors (GPCRs) 1 interact with physiologically important peptides. There is intense study of how these peptides associate with their cognate receptors since elucidation of these interactions should provide important insights for the rational design of ligands with enhanced pharmacological properties for use in treating an array of diseases (1,2). Over the past decade, photoaffinity cross-linking has been employed to study the interaction of a number of peptide-GPCR interactions (3). This technique involves systematically probing the receptor for regions of interaction using a peptide that incorporates a photoreactive moiety that irreversibly cross-links to the receptor. We and others have used this approach extensively to study the interaction of parathyroid hormone (PTH) with its cognate receptor, the PTH receptor type 1 (PTHR1) (4-12). This hormone-receptor system plays an integral role in calcium metabolism and bone biology. † This work was supported in part by Grants DK47490 (to M.R.) and GM54082 (to D.F.M.) from the National Institutes of Health.* Address correspondence to this author. Phone: (617) Our research is focused on studying the bimolecular interface of the PTH-PTHR1 complex in order to gain insights that will aid the design of ligands of PTHR1 for the treatment of osteoporosis and o...

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“…Signaling via the cAMP/PKA pathway was assessed using HEK-293-derived cell lines that stably express the Glosensor cAMP reporter (Promega Corp.) (32), along with either the WT rat PTHR1 (GR-35 cells) or the PD rat PTHR1 (GPD-4 cells). cAMP responses were also assessed using HEK-293 cells transiently transfected to express a cAMP-response-element/ luciferase (Cre-Luc) reporter (33), and either the WT or PD rat PTHR1. Intracellular calcium (iCa ++ ) signaling was assessed in transiently PTHR1-transfected HEK-293 cells using Fura2-AM (Invitrogen, Life Tech.)…”

Section: Methodsmentioning

confidence: 99%

Critical role of parathyroid hormone (PTH) receptor-1 phosphorylation in regulating acute responses to PTH

Maeda

Okazaki

Baron

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Agonist-induced phosphorylation of the parathyroid hormone (PTH) receptor 1 (PTHR1) regulates receptor signaling in vitro, but the role of this phosphorylation in vivo is uncertain. We investigated this role by injecting "knock-in" mice expressing a phosphorylation-deficient (PD) PTHR1 with PTH ligands and assessing acute biologic responses. Following injection with PTH (1-34), or with a unique, long-acting PTH analog, PD mice, compared with WT mice, exhibited enhanced increases in cAMP levels in the blood, as well as enhanced cAMP production and gene expression responses in bone and kidney tissue. Surprisingly, however, the hallmark hypercalcemic and hypophosphatemic responses were markedly absent in the PD mice, such that paradoxical hypocalcemic and hyperphosphatemic responses were observed, quite strikingly with the long-acting PTH analog. Spot urine analyses revealed a marked defect in the capacity of the PD mice to excrete phosphate, as well as cAMP, into the urine in response to PTH injection. This defect in renal excretion was associated with a severe, PTH-induced impairment in glomerular filtration, as assessed by the rate of FITC-inulin clearance from the blood, which, in turn, was explainable by an overly exuberant systemic hypotensive response. The overall findings demonstrate the importance in vivo of PTH-induced phosphorylation of the PTHR1 in regulating acute ligand responses, and they serve to focus attention on mechanisms that underlie the acute calcemic response to PTH and factors, such as blood phosphate levels, that influence it.calcium homeostasis | family B GPCR | phosphate homeostasis | receptor phosphorylation P arathyroid hormone (PTH) plays a critical role in regulating blood concentrations of ionized calcium by acting via its receptor, the PTH receptor 1 (PTHR1), expressed by target cells of bone and kidney. In response to decreases in blood Ca 2+ levels, PTH thus acts to promote the release of calcium from bone, and the reabsorption of calcium from the glomerular filtrate. PTH also acts to lower blood inorganic phosphate (Pi) by promoting the renal excretion of Pi into the urine. PTH acts in concert with several other calcium-and phosphate-regulating factors, including 1,25(OH) 2 -vitamin-D 3 and FGF23, as part of a tightly regulated homeostatic system of mineral ion control (1). Of note, PTH peptides injected daily at low dose can stimulate new bone formation and are thus in use to treat osteoporosis (2). Because of their calcemic actions, PTH peptides also hold promise as treatments for hypoparathyroidism (3).The PTHR1 is a class B G protein-coupled receptor (GPCR) that signals via the Gαs/cAMP/PKA and Gαq/phopholipase C (PLC)/intracellular calcium (iCa)/PKC pathways (4). In cultured cells, agonist activation of the PTHR1 induces rapid receptor internalization and signal desensitization. As for most GPCRs, the PTHR1 is phosphorylated on its C-terminal tail following agonist activation, and this phosphorylation promotes the internalization and desensitization responses, in large part...

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Methionine acts as a “magnet” in photoaffinity crosslinking experiments

Cited by 102 publications

References 21 publications

Binding of the Covalent Flavin Assembly Factor to the Flavoprotein Subunit of Complex II

Binding of the Covalent Flavin Assembly Factor to the Flavoprotein Subunit of Complex II

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

Critical role of parathyroid hormone (PTH) receptor-1 phosphorylation in regulating acute responses to PTH

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