Matthew P Pond1, Rebecca Eells2, Bradley W Treece2, Frank Heinrich3, Mathias Lösche4, Benoît Roux5. 1. Department of Biochemistry and Molecular Biology, Gordon Center for Integrative Science, University of Chicago, Chicago, IL, 60637, USA. 2. Department of Physics, Carnegie Mellon University, Pittsburgh, PA, 15213, USA. 3. Department of Physics, Carnegie Mellon University, Pittsburgh, PA, 15213, USA; Center for Neutron Research, NIST, Gaithersburg, MD, 20899, USA. 4. Department of Physics, Carnegie Mellon University, Pittsburgh, PA, 15213, USA; Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA, 15213, USA; Center for Neutron Research, NIST, Gaithersburg, MD, 20899, USA. 5. Department of Biochemistry and Molecular Biology, Gordon Center for Integrative Science, University of Chicago, Chicago, IL, 60637, USA. Electronic address: roux@uchicago.edu.
Abstract
Src family kinases (SFKs) are a group of nonreceptor tyrosine kinases that are characterized by their involvement in critical signal transduction pathways. SFKs are often found attached to membranes, but little is known about the conformation of the protein in this environment. Here, solution nuclear magnetic resonance (NMR), neutron reflectometry (NR), and molecular dynamics (MD) simulations were employed to study the membrane interactions of the intrinsically disordered SH4 and Unique domains of the Src family kinase Hck. Through development of a procedure to combine the information from the different techniques, we were able produce a first-of-its-kind atomically detailed structural ensemble of a membrane-bound intrinsically disordered protein. Evaluation of the model demonstrated its consistency with previous work and provided insight into how SFK Unique domains act to differentiate the family members from one another. Fortuitously, the position of the ensemble on the membrane allowed the model to be combined with configurations of the multidomain Hck kinase previously determined from small-angle solution X-ray scattering to produce full-length models of membrane-anchored Hck. The resulting models allowed us to estimate that the kinase active site is positioned about 65 ± 35 Å away from the membrane surface, offering the first estimations of the length scale associated with the concept of SFK subcellular localization.
Src family kinases (SFKs) are a group of nonreceptor pan class="Chemical">tyrosine kinases that are characterized by their involvement in critical signal transduction pathways. SFKs are often found attached to membranes, but little is known about the conformation of the protein in this environment. Here, solution nuclear magnetic resonance (NMR), neutron reflectometry (NR), and molecular dynamics (MD) simulations were employed to study the membrane interactions of the intrinsically disordered SH4 and Unique domains of the Src family kinase Hck. Through development of a procedure to combine the information from the different techniques, we were able produce a first-of-its-kind atomically detailed structural ensemble of a membrane-bound intrinsically disordered protein. Evaluation of the model demonstrated its consistency with previous work and provided insight into how SFK Unique domains act to differentiate the family members from one another. Fortuitously, the position of the ensemble on the membrane allowed the model to be combined with configurations of the multidomain Hck kinase previously determined from small-angle solution X-ray scattering to produce full-length models of membrane-anchored Hck. The resulting models allowed us to estimate that the kinase active site is positioned about 65 ± 35 Å away from the membrane surface, offering the first estimations of the length scale associated with the concept of SFK subcellular localization.
Authors: Woo Sung Son; Sang Ho Park; Henry J Nothnagel; George J Lu; Yan Wang; Hua Zhang; Gabriel A Cook; Stanley C Howell; Stanley J Opella Journal: J Magn Reson Date: 2011-10-25 Impact factor: 2.229
Authors: Jagabandhu Das; Ping Chen; Derek Norris; Ramesh Padmanabha; James Lin; Robert V Moquin; Zhongqi Shen; Lynda S Cook; Arthur M Doweyko; Sidney Pitt; Suhong Pang; Ding Ren Shen; Qiong Fang; Henry F de Fex; Kim W McIntyre; David J Shuster; Kathleen M Gillooly; Kamelia Behnia; Gary L Schieven; John Wityak; Joel C Barrish Journal: J Med Chem Date: 2006-11-16 Impact factor: 7.446