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Purification and crystallization of yeast Ent1 ENTH domain.

ABSTRACT: Members of the Epsin protein family regulate the ubiquitin/clathrin-dependent trafficking of transmembrane proteins. The yeast Epsin-1 (ent1) gene was cloned and expressed in Escherichia coli. The protein product of a construct containing the ENTH-UIM modules was purified to homogeneity and subjected to crystallization screening using the sitting-drop vapour-diffusion method. Refined conditions containing polyethylene glycol 3350 and Tacsimate yielded thin rod-like crystals. X-ray analysis revealed that the crystallographic symmetry is primitive orthorhombic, space group P222, with unit-cell parameters a = 32.7, b = 35.5, c = 110.6?Å and a diffraction limit of 2.3?Å. Matthews coefficient calculations suggested that the crystal contained only the ENTH domain. This was corroborated by Coomassie Blue-stained SDS-PAGE analysis of dissolved crystals.

SUBMITTER: Tanner N

PROVIDER: S-EPMC3388931 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Purification and crystallization of yeast Ent1 ENTH domain.

Tanner Neta N Prag Gali G

Acta crystallographica. Section F, Structural biology and crystallization communications 20120628 Pt 7

Members of the Epsin protein family regulate the ubiquitin/clathrin-dependent trafficking of transmembrane proteins. The yeast Epsin-1 (ent1) gene was cloned and expressed in Escherichia coli. The protein product of a construct containing the ENTH-UIM modules was purified to homogeneity and subjected to crystallization screening using the sitting-drop vapour-diffusion method. Refined conditions containing polyethylene glycol 3350 and Tacsimate yielded thin rod-like crystals. X-ray analysis revea ...[more]

PMID: 22750874

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Project description:Epsins are eukaryotic, endocytic adaptor proteins primarily involved in the early steps of clathrin mediated endocytosis. Two epsins exist in Saccharomyces cerevisiae, Ent1 and Ent2, with single epsin knockouts being viable, while the double knockout is not. These proteins contain a highly conserved Epsin N-terminal homology (ENTH) domain that is essential for cell viability. In addition, overexpression of the ENTH domain of Ent2 (ENTH2) was shown to play a role in cell division by interacting with the septin organizing, Cdc42 GTPase activating protein, Bem3, leading to increased cytokinesis failure. In contrast, overexpression of the ENTH domain of Ent1 (ENTH1) does not affect cytokinesis, despite being 75% identical to ENTH2. An ENTH2(N112D, S114E, E118Q) mutant that switches residues in loop 7 to those found correspondingly in ENTH1 was incapable of inducing the cytokinesis phenotype. In order to better understand the role of loop 7 in the ENTH2-induced phenotype at a molecular level, X-ray crystallography was used to elucidate the structures of yeast ENTH2(WT) and ENTH2(DEQ). Our results indicate that mutations did not affect the conformation of loop 7, but rather introduce an increased negative charge on a potential interaction interface. Morphological analysis of cells overexpressing ENTH2 loop 7 mutants showed that the cytokinesis failure phenotype was abolished by the single mutants N112D, E118Q, and to a lesser extent by S114E. Taken together, our results indicate that the interaction surface that contains loop 7 and the specific nature of these residues are crucial for ENTH2 involvement in cytokinesis. This research provides insight into a molecular mechanism by which ENTH2, but not ENTH1, overexpression in yeast leads to cell division defects. Structural data of WT and mutant ENTH2 domains along with in vivo phenotypic analysis of ENTH2 overexpressing cells indicate that the biochemical nature of three loop 7 residues is crucial for its role in cytokinesis.

Dataset Information

Purification and crystallization of yeast Ent1 ENTH domain.

Publications

Purification and crystallization of yeast Ent1 ENTH domain.

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