BIOPHYSICAL THEORY AND MODELING |
The 
-helical propensity of the cytoplasmic
domain of phospholamban. A molecular dynamics study of
the effect of phosphorylation and mutation
M. Germana Paterlini 1* and David D. Thomas 2
1 Certusoft Inc.
2 University of Minnesota Medical School
* To whom correspondence should be addressed. E-mail: germana{at}certusoft.com.
Submitted on October 19, 2004
Revised on November 22, 2004
Accepted on 18 February 2005
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Abstract |
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We have used molecular dynamics simulations to investigate the effect of phosphorylation and mutation on the cytoplasmic domain of phospholamban, a 52-residue protein that regulates the calcium pump in cardiac muscle. Simulations were carried out in explicit water systems at 300K for three peptides spanning the first 25 residues of PLB: wild type (PLB1-25), PLB1-25 phosphorylated at Ser16 and PLB1-25 with the R9C mutation, which is known to cause human heart disease. The unphosphorylated peptide maintains a helical conformation from 3 to 15 throughout a 26 ns simulation, in agreement with spectroscopic data. Comparison with simulations of a fourth peptide truncated at Pro21 showed the importance of the region from 17 to 21 in preventing local unfolding of the helix. The results suggest that residues 11 to 16 are more likely to unfold when specific capping motifs are not present. It is proposed that protein kinase A exploits the intrinsic flexibility of the 11 to 21 region when binding PLB. In agreement with available CD and NMR data, the simulations show a decrease in the helical content upon phosphorylation. The phosphorylated peptide is characterized by helix spanning residues 3 to 11, followed by a turn that optimizes the salt-bridge interaction between the side chains of the phosphorylated Ser16 and Arg13. Replacing Arg9 with Cys results in unfolding of the helix from C9 and an overall decrease of the helical conformation. The simulations show that initiation of unfolding is due to increased solvent accessibility of the backbone atoms near the smaller Cys. It is proposed that the loss of inhibitory potency upon Ser16 phosphorylation or R9C mutation of PLB is due to a similar mechanism, in which the partial unfolding of the cytoplasmic helix of PLB results in a conformation that interacts with the cytoplasmic domain of the calcium pump to relieve its inhibition.
Key Words:
a-helix, EPR, NMR, PLB, SERCA, heart disease
