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Phosphorylation and mutation of phospholamban alter physical interactions with the sarcoplasmic reticulum calcium pump.

Abstract：

:Phospholamban physically interacts with the sarcoplasmic reticulum calcium pump (SERCA) and regulates contractility of the heart in response to adrenergic stimuli. We studied this interaction using electron microscopy of 2D crystals of SERCA in complex with phospholamban. In earlier studies, phospholamban oligomers were found interspersed between SERCA dimer ribbons and a 3D model was constructed to show interactions with SERCA. In this study, we examined the oligomeric state of phospholamban and the effects of phosphorylation and mutation of phospholamban on the interaction with SERCA in the 2D crystals. On the basis of projection maps from negatively stained and frozen-hydrated crystals, phosphorylation of Ser16 selectively disordered the cytoplasmic domain of wild type phospholamban. This was not the case for a pentameric gain-of-function mutant (Lys27Ala), which retained inhibitory activity and remained ordered in the phosphorylated state. A partial loss-of-function mutation that altered the charge state of phospholamban (Arg14Ala) retained an ordered state, while a complete loss-of-function mutation (Asn34Ala) was also disordered. The functional state of phospholamban was correlated with an order-to-disorder transition of the phospholamban cytoplasmic domain in the 2D co-crystals. Furthermore, co-crystals of the gain-of-function mutant (Lys27Ala) facilitated data collection from frozen-hydrated crystals. An improved projection map was calculated to a resolution of 8 Å, which supports the pentamer as the oligomeric state of phospholamban in the crystals. The 2D co-crystals with SERCA require a functional pentameric form of phospholamban, which physically interacts with SERCA at an accessory site distinct from that used by the phospholamban monomer for the inhibitory association.

journal_name

J Mol Biol

journal_title

Journal of molecular biology

authors

Glaves JP,Trieber CA,Ceholski DK,Stokes DL,Young HS

doi

10.1016/j.jmb.2010.11.014

subject

Has Abstract

pub_date

2011-01-21 00:00:00

pages

707-23

issue

eissn

0022-2836

issn

1089-8638

pii

S0022-2836(10)01228-3

journal_volume

405

pub_type

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Phosphorylation and mutation of phospholamban alter physical interactions with the sarcoplasmic reticulum calcium pump.