Purification and crystallization of yeast Ent1 ENTH domain

Tanner, Neta; Prag, Gali

doi:10.1107/s1744309112022488

Cited by 3 publications

(5 citation statements)

References 14 publications

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“…Many of the proteins involved in CME have been well characterized structurally. For instance, the structure and function of the clathrin coat has been described extensively, and certain adaptor proteins have domains with solved structures, such as the EFC/F‐BAR of the yeast Syp1 3 or the ENTH domain of the yeast epsins Ent1/2 . However, other protein components are only understood genetically or biochemically, due to an inability to solve their structures using crystallography, caused in part by the presence of intrinsically disordered regions in these proteins.…”

Section: Introductionmentioning

confidence: 99%

Pan1 is an intrinsically disordered protein with homotypic interactions

2013

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The yeast scaffold protein Pan1 contains two EH domains at its N-terminus, a predicted coiled-coil central region, and a C-terminal proline-rich domain. Pan1 is also predicted to contain regions of intrinsic disorder, characteristic of proteins that have many binding partners. In vitro biochemical data suggest that Pan1 exists as a dimer, and we have identified amino acids 705–848 as critical for this homotypic interaction. Tryptophan fluorescence was used to further characterize Pan1 conformational states. Pan1 contains four endogenous tryptophans, each in a distinct region of the protein: Trp312 and Trp642 are each in an EH domain, Trp957 is in the central region, and Trp1280 is a critical residue in the Arp2/3 activation domain. To examine the local environment of each of these tryptophans, three of the four tryptophans were mutagenized to phenylalanine to create four proteins, each with only one tryptophan residue. When quenched with acrylamide, these single tryptophan mutants appeared to undergo collisional quenching exclusively and were moderately accessible to the acrylamide molecule. Quenching with iodide or cesium, however, revealed different Stern-Volmer constants due to unique electrostatic environments of the tryptophan residues. Time-resolved fluorescence anisotropy data confirmed structural and disorder predictions of Pan1. Further experimentation to fully develop a model of Pan1 conformational dynamics will assist in a deeper understanding of the mechanisms of endocytosis.

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Section: Introductionmentioning

confidence: 99%

Pan1 is an intrinsically disordered protein with homotypic interactions

2013

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“…However, in ENTH 1-152 , a construct that lacks the UIMs and consequently possesses only a single UBD, K103 and K148 were found to be the sole ubiquitylation sites. Based on our structures of apo yeast and zebrafish ENTH domains (PDB accession codes 5LOZ and 5LP0, respectively), and their homology to the human STAM1–VHS domain complex with Ub (PDB accession code 3LDZ), we constructed a structural model ENTH:Ub of non-covalent complexes [18,19]. The yeast ENTH:Ub complex provides a molecular explanation for the ubiquitylation sites found in the ENTH–UIMs construct, which better represent the natural structure of full-length Ent1 (Supplementary Figure S2).…”

Section: Resultsmentioning

confidence: 99%

“…Positive residues at the hydrophilic face of H 0 interact with the PIP 2 lipid head-group (Figure 3). We determined the crystal structure of the yeast Ent1–ENTH domain that lacks the H 0 [18,19]. Given the high homology between the yeast and the rat epsin proteins, we constructed a structural model for H 0 (purple helix in Figure 3b).…”

Section: Resultsmentioning

confidence: 99%

“…To test this hypothesis biochemically, we conducted two independent, in vitro competition experiments for the binding of apo Ent1 to PIP 2 versus ubiquitylated Ent1. Ent1 apo and ubiquitylated protein were purified as previously described [7,19]. First, we assessed the binding of the purified proteins to an immobilized PIP array (Figure 4).…”

Section: Resultsmentioning

confidence: 99%

“…The crystal structure of Ent1–ENTH domain a.a. 17-150 (PDB 5LOZ), determined by Tanner, was the basis for our model [18,19]. We used Protein Homology/analogY Recognition Engine V 2.0 (Phyre 2 ) to model the first 16 a.a. [20].…”

Section: Methodsmentioning

confidence: 99%

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Remodeling Membrane Binding by Mono-Ubiquitylation

et al. 2019

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Ubiquitin (Ub) receptors respond to ubiquitylation signals. They bind ubiquitylated substrates and exert their activity in situ. Intriguingly, Ub receptors themselves undergo rapid ubiquitylation and deubiquitylation. Here we asked what is the function of ubiquitylation of Ub receptors? We focused on yeast epsin, a Ub receptor that decodes the ubiquitylation signal of plasma membrane proteins into an endocytosis response. Using mass spectrometry, we identified lysine-3 as the major ubiquitylation site in the epsin plasma membrane binding domain. By projecting this ubiquitylation site onto our crystal structure, we hypothesized that this modification would compete with phosphatidylinositol-4,5-bisphosphate (PIP2) binding and dissociate epsin from the membrane. Using an E. coli-based expression of an authentic ubiquitylation apparatus, we purified ubiquitylated epsin. We demonstrated in vitro that in contrast to apo epsin, the ubiquitylated epsin does not bind to either immobilized PIPs or PIP2-enriched liposomes. To test this hypothesis in vivo, we mimicked ubiquitylation by the fusion of Ub at the ubiquitylation site. Live cell imaging demonstrated that the mimicked ubiquitylated epsin dissociates from the membrane. Our findings suggest that ubiquitylation of the Ub receptors dissociates them from their products to allow binding to a new ubiquitylated substrates, consequently promoting cyclic activity of the Ub receptors.

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A bacterial genetic selection system for ubiquitylation cascade discovery

et al. 2016

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About one-third of the eukaryotic proteome undergoes ubiquitylation, but the enzymatic cascades leading to substrate modification are largely unknown. We present a genetic selection tool that utilizes Escherichia coli, which lack deubiquitylases, to identify interactions along ubiquitylation cascades. Coexpression of split antibiotic resistance protein tethered to ubiquitin and ubiquitylation target together with a functional ubiquitylation apparatus results in a covalent assembly of the resistance protein, giving rise to bacterial growth on selective media. We applied the selection system to uncover an E3 ligase from the pathogenic bacteria EHEC and to identify the epsin ENTH domain as an ultraweak ubiquitin-binding domain. The latter was complemented with a structure-function analysis of the ENTH-ubiquitin interface. We also constructed and screened a yeast fusion library, discovering Sem1 as a novel ubiquitylation substrate of Rsp5 E3 ligase. Collectively, our selection system provides a robust high-throughput approach for genetic studies of ubiquitylation cascades and for small-molecule modulator screening.

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Purification and crystallization of yeast Ent1 ENTH domain

Cited by 3 publications

References 14 publications

Pan1 is an intrinsically disordered protein with homotypic interactions

Pan1 is an intrinsically disordered protein with homotypic interactions

Remodeling Membrane Binding by Mono-Ubiquitylation

A bacterial genetic selection system for ubiquitylation cascade discovery

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