Using selenomethionyl derivatives to assign sequence in low-resolution structures of the AP2 clathrin adaptor.


Change log
Authors
Kelly, Bernard T 
Graham, Stephen C 
Owen, David J 
Abstract

Selenomethionine incorporation is a powerful technique for assigning sequence to regions of electron density at low resolution. Genetic introduction of methionine point mutations and the subsequent preparation and crystallization of selenomethionyl derivatives permits unambiguous sequence assignment by enabling the placement of the anomalous scatterers (Se atoms) thus introduced. Here, the use of this approach in the assignment of sequence in a part of the AP2 clathrin adaptor complex that is responsible for clathrin binding is described. AP2 plays a pivotal role in clathrin-mediated endocytosis, a tightly regulated process in which cell-surface transmembrane proteins are internalized from the plasma membrane by incorporation into lipid-enclosed transport vesicles. AP2 binds cargo destined for internalization and recruits clathrin, a large trimeric protein that helps to deform the membrane to produce the transport vesicle. By selenomethionine labelling of point mutants, it was shown that the clathrin-binding site is buried within a deep cleft of the AP2 complex. A membrane-stimulated conformational change in AP2 releases the clathrin-binding site from autoinhibition, thereby linking clathrin recruitment to membrane localization.

Description
Keywords
AP2 clathrin adaptor complex, low resolution, selenomethionyl derivatives, sequence assignment, Adaptor Protein Complex 2, Animals, Binding Sites, Clathrin, Crystallization, Crystallography, X-Ray, Endocytosis, Humans, Mice, Models, Molecular, Protein Binding, Protein Conformation, Rats, Selenomethionine
Journal Title
Acta Crystallogr D Struct Biol
Conference Name
Journal ISSN
2059-7983
2059-7983
Volume Title
72
Publisher
International Union of Crystallography (IUCr)
Sponsorship
Wellcome Trust (090909/Z/09/Z)
Wellcome Trust (098406/Z/12/Z)
Wellcome Trust (079895/Z/06/B)
We would like to thank the I02, I03 and I04-1 beamline staff at the Diamond Light Source (mx6641) and Chris Oubridge for advice and assistance with SeMet mapping of the hinge residues. D.J.O. and B.T.K. are supported by Wellcome Trust Principal Research Fellowship (090909/Z/09/Z). S.C.G. is supported by a Sir Henry Dale Fellowship from the Wellcome Trust and the Royal Society (098406/Z/12/Z). CIMR is supported by a Wellcome Trust Strategic Award (079895).