p-Benzoyl-L-phenylalanine, a new photoreactive amino acid. Photolabeling of calmodulin with a synthetic calmodulin-binding peptide.

Kauer, James C.; Erickson‐Viitanen, Susan; Wolfe, Henry R.; DeGrado, William F.

doi:10.1016/s0021-9258(18)67441-1

Cited by 209 publications

(103 citation statements)

References 23 publications

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“…We used a photo-cross-linking approach to identify additional C-terminal binding site(s) for bound peptide substrates. We designed an 11-mer cross-linking probe, designated DG023, with peptide sequence based on a known ERAP1 substrate 8 , the nonhydrolyzable phosphinic group replacing the first peptide bond, and the unnatural amino-acid p -benzoyl- l -phenylalanine (BPA)-amide at the C-terminus 33 (Fig. 3a ).…”

Section: Resultsmentioning

confidence: 99%

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Conformational dynamics linked to domain closure and substrate binding explain the ERAP1 allosteric regulation mechanism

et al. 2021

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The endoplasmic-reticulum aminopeptidase ERAP1 processes antigenic peptides for loading on MHC-I proteins and recognition by CD8 T cells as they survey the body for infection and malignancy. Crystal structures have revealed ERAP1 in either open or closed conformations, but whether these occur in solution and are involved in catalysis is not clear. Here, we assess ERAP1 conformational states in solution in the presence of substrates, allosteric activators, and inhibitors by small-angle X-ray scattering. We also characterize changes in protein conformation by X-ray crystallography, and we localize alternate C-terminal binding sites by chemical crosslinking. Structural and enzymatic data suggest that the structural reconfigurations of ERAP1 active site are physically linked to domain closure and are promoted by binding of long peptide substrates. These results clarify steps required for ERAP1 catalysis, demonstrate the importance of conformational dynamics within the catalytic cycle, and provide a mechanism for the observed allosteric regulation and Lys/Arg528 polymorphism disease association.

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Section: Resultsmentioning

confidence: 99%

“…3a ). UV irradiation (350 nm) of BPA generates a carbene that can form a covalent bond with nearby molecules 33 . We performed cross-linking reactions in this manner using DG023 and purified ERAP1 and identified three cross-linking sites by LC-MS/MS (Fig.…”

Section: Resultsmentioning

confidence: 99%

Conformational dynamics linked to domain closure and substrate binding explain the ERAP1 allosteric regulation mechanism

et al. 2021

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“…Affinity Labeling of CHO Cells with PI]-BPA Insulin and Internalization ofLabeled Receptors Benzoylphenylalamne (Bps) was synthesized as described by DeGrado and coworkers (Kauer et al ., 1986 ["5 I]iodine (1 mCi/10-cm dish) as previously described (Backer et al ., 1989) . The cells were washed five times in cold PBS and then incubated in the absence or presence of 100 nM insulin at 37°C .…”

Section: Analysis Ofinternalization Datamentioning

confidence: 99%

Insulin receptors internalize by a rapid, saturable pathway requiring receptor autophosphorylation and an intact juxtamembrane region.

Backer

Shoelson

Haring

et al. 1991

The Journal of Cell Biology

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Abstract. The effect of receptor occupancy on insulin receptor endocytosis was examined in CHO cells expressing normal human insulin receptors (CHO/IR), autophosphorylation-and internalization-deficient receptors (CHOARmo,8), and receptors which undergo autophosphorylation but lack a sequence required for internalization (CHO/IR,96o) . The rate of [ 121 1]insulin internalization in CHO/IR cells at 37°C was rapid at physiological concentrations, but decreased markedly in the presence of increasing unlabeled insulin (EDso = 1-3 nM insulin, or 75,000 occupied receptors/cell). In contrast, [125I]insulin internalization by CHO/IRA,o,s and CHO/1:Re96o cells was slow and was not inhibited by unlabeled insulin . At saturating insulin concentrations, the rate of internalization by wild-type and mutant receptors was similar. Moreover, depletion of intracellular potassium, which has been shown to disrupt coated pit formation, inhibited the rapid in-T HE uptake of a variety of hormones and nutrients by eukaryotic cells occurs by receptor-mediated endocytosis . Occupied cell surface receptors internalize via clathrin-coated vesicles and are transported to endosomes, where ligands dissociate during the gradual acidification of the endosomel lumen (Tycko et al., 1983). Recently, a number of these events have been reconstituted in vitro : the formation ofcoated pits onto isolated membranes, the endocytosis ofreceptors from coated pits to coated vesicles, and the fusion of endosomel vesicles have all been demonstrated in cell-free or broken-cell systems (Mahaffey et al., 1989 ;Smythe et al., 1989; Podbilewicz and Mellman,1990 ; Gruenberg and Howell, 1989;Braell, 1987; Diaz et al., 1989;Davey et al., 1985;Mullock et al., 1989;Ward et al., 1990). However, the mechanisms which govern the entry of receptors from the plasma membrane into the coated pit/coated vesicle/endosome continuum remain poorly defined .Cell surface receptors have been broadly divided into class I receptors, which are constitutively located in coated pits under basal conditions, and class II receptors, which move from non-coated to coated regions ofthe plasma membrane when stimulated by ligand These data suggest that the insulinstimulated entry of the insulin receptor into a rapid, coated pit-mediated internalization pathway is saturable and requires receptor autophosphorylation and an intact juxtamembrane region . Furthermore, CHO cells also contain a constitutive nonsaturable pathway which does not require receptor autophosphorylation or an intact juxtamembrane region ; this second pathway is unaffected by depletion of intracellular potassium, and therefore may be independent of coated pits. Our data suggest that the ligand-stimulated internalization of the insulin receptor may require specific saturable interactions between the receptor and components of the endocytic system.

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“…Further systems were developed that allow to genetically encode unnatural amino acids in proteins. The tyrosyl-tRNAsynthetase/tRNA CUA from Methanocaldococcus jannaschii was evolved to allow incorporation of the photoactivatable crosslinkers p-benzoyl-L-phenylalanine and p-azido-Lphenylalanine proteins (Kauer et al, 1986;Chin et al, 2002a,b). This enables to photo-crosslink interaction partners and to stabilize otherwise transient interactions.…”

Section: Synthetic Biology: Genetically Encoding Acetyl-l-lysine In Proteins Allows To Unravel the Real Consequences Of Lysine Acetylatiomentioning

confidence: 99%

Post-translational Lysine Ac(et)ylation in Bacteria: A Biochemical, Structural, and Synthetic Biological Perspective

Lammers

2021

Front. Microbiol.

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Ac(et)ylation is a post-translational modification present in all domains of life. First identified in mammals in histones to regulate RNA synthesis, today it is known that is regulates fundamental cellular processes also in bacteria: transcription, translation, metabolism, cell motility. Ac(et)ylation can occur at the ε-amino group of lysine side chains or at the α-amino group of a protein. Furthermore small molecules such as polyamines and antibiotics can be acetylated and deacetylated enzymatically at amino groups. While much research focused on N-(ε)-ac(et)ylation of lysine side chains, much less is known about the occurrence, the regulation and the physiological roles on N-(α)-ac(et)ylation of protein amino termini in bacteria. Lysine ac(et)ylation was shown to affect protein function by various mechanisms ranging from quenching of the positive charge, increasing the lysine side chains’ size affecting the protein surface complementarity, increasing the hydrophobicity and by interfering with other post-translational modifications. While N-(ε)-lysine ac(et)ylation was shown to be reversible, dynamically regulated by lysine acetyltransferases and lysine deacetylases, for N-(α)-ac(et)ylation only N-terminal acetyltransferases were identified and so far no deacetylases were discovered neither in bacteria nor in mammals. To this end, N-terminal ac(et)ylation is regarded as being irreversible. Besides enzymatic ac(et)ylation, recent data showed that ac(et)ylation of lysine side chains and of the proteins N-termini can also occur non-enzymatically by the high-energy molecules acetyl-coenzyme A and acetyl-phosphate. Acetyl-phosphate is supposed to be the key molecule that drives non-enzymatic ac(et)ylation in bacteria. Non-enzymatic ac(et)ylation can occur site-specifically with both, the protein primary sequence and the three dimensional structure affecting its efficiency. Ac(et)ylation is tightly controlled by the cellular metabolic state as acetyltransferases use ac(et)yl-CoA as donor molecule for the ac(et)ylation and sirtuin deacetylases use NAD+ as co-substrate for the deac(et)ylation. Moreover, the accumulation of ac(et)yl-CoA and acetyl-phosphate is dependent on the cellular metabolic state. This constitutes a feedback control mechanism as activities of many metabolic enzymes were shown to be regulated by lysine ac(et)ylation. Our knowledge on lysine ac(et)ylation significantly increased in the last decade predominantly due to the huge methodological advances that were made in fields such as mass-spectrometry, structural biology and synthetic biology. This also includes the identification of additional acylations occurring on lysine side chains with supposedly different regulatory potential. This review highlights recent advances in the research field. Our knowledge on enzymatic regulation of lysine ac(et)ylation will be summarized with a special focus on structural and mechanistic characterization of the enzymes, the mechanisms underlying non-enzymatic/chemical ac(et)ylation are explained, recent technological progress in the field are presented and selected examples highlighting the important physiological roles of lysine ac(et)ylation are summarized.

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p-Benzoyl-L-phenylalanine, a new photoreactive amino acid. Photolabeling of calmodulin with a synthetic calmodulin-binding peptide.

Cited by 209 publications

References 23 publications

Conformational dynamics linked to domain closure and substrate binding explain the ERAP1 allosteric regulation mechanism

Conformational dynamics linked to domain closure and substrate binding explain the ERAP1 allosteric regulation mechanism

Insulin receptors internalize by a rapid, saturable pathway requiring receptor autophosphorylation and an intact juxtamembrane region.

Post-translational Lysine Ac(et)ylation in Bacteria: A Biochemical, Structural, and Synthetic Biological Perspective

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