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Biochemistry. 2005 Mar 22;44(11):4386-96.

Serine 16 phosphorylation induces an order-to-disorder transition in monomeric phospholamban.

Author information

1
Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, USA.

Abstract

Phospholamban (PLB) is a 52 amino acid membrane-endogenous regulator of the sarco(endo)plasmic calcium adenosinetriphosphatase (SERCA) in cardiac muscle. PLB's phosphorylation and dephosphorylation at S16 modulate its regulatory effect on SERCA by an undetermined mechanism. In this paper, we use multidimensional (1)H/(15)N solution NMR methods to establish the structural and dynamics basis for PLB's control of SERCA upon S16 phosphorylation. For our studies, we use a monomeric, fully active mutant of PLB, where C36, C41, and C46 have been mutated to A36, F41, and A46, respectively. Our data show that phosphorylation disrupts the "L-shaped" structure of monomeric PLB, causing significant unwinding of both the cytoplasmic helix (domain Ia) and the short loop (residues 17-21) connecting this domain to the transmembrane helix (domains Ib and II). Concomitant with this conformational transition, we also find pronounced changes in both the pico- to nanosecond and the micro- to millisecond time scale dynamics. The (1)H/(15)N heteronuclear NOE values for residues 1-25 are significantly lower than those of unphosphorylated PLB, with slightly lower NOE values in the transmembrane domain, reflecting less restricted motion throughout the whole protein. These data are supported by the faster spin-lattice relaxation rates (R(1)) present in both the cytoplasmic and loop regions and by the enhanced spin-spin transverse relaxation rates (R(2)) observed in the transmembrane domain. These results demonstrate that while S16 phosphorylation induces a localized structural transition, changes in PLB's backbone dynamics are propagated throughout the protein backbone. We propose that the regulatory mechanism of PLB phosphorylation involves an order-to-disorder transition, resulting in a decrease in the PLB inhibition of SERCA.

PMID:
15766268
DOI:
10.1021/bi047571e
[Indexed for MEDLINE]

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