Design of a leucine zipper coiled coil stabilized 1.4 kcal mol-1 by phosphorylation of a serine in the e position

L. Szilák, Jaideep Moitra, Charles Vinson

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

Using a dimeric bZIP protein, we have designed a leucine zipper that becomes more stable after a serine in the e position is phosphorylated by protein kinase A (ΔΔG(P) = -1.4 kcal mol-1 dimer-1 or -0.7 kcal mol- 1 residue-1). Mutagenesis studies indicate that three arginines form a network of inter-helical (i,i' + 5; i, i' + 2) and intra-helical (i,i +4) attractive interactions with the phosphorylated serine. When the arginines are replaced with lysines, the stabilizing effect of serine phosphorylation is reduced (ΔΔG(P) = -0.5 kcal mol-1 dimer-1). The hydrophobic interface of the leucine zipper needs a glycine in the d position to obtain an increase in stability after phosphorylation. The phosphorylated protein binds DNA with a 15-fold higher affinity. Using a transient transfection assay, we document a PKA dependent four-fold activation of a reporter gene. Phosphorylation of a threonine in the same e position decreases the stability by ΔΔG(P) = +1.2 kcal mol-1 dimer-1. We present circular dichroism (CD) thermal denaturations of 15 bZIP proteins before and after phosphorylation. These data provide insights into the structural determinants that result in stabilization of a coiled coil by phosphorylation.

Original languageEnglish
Pages (from-to)1273-1283
Number of pages11
JournalProtein Science
Volume6
Issue number6
Publication statusPublished - Jun 1997

Fingerprint

Leucine Zippers
Phosphorylation
Serine
Basic-Leucine Zipper Transcription Factors
Dimers
Arginine
Cyclic GMP-Dependent Protein Kinases
Mutagenesis
Denaturation
Threonine
Circular Dichroism
Cyclic AMP-Dependent Protein Kinases
Reporter Genes
Glycine
Lysine
Transfection
Assays
Stabilization
Hot Temperature
Genes

Keywords

  • α-Helix
  • Coiled coil
  • Leucine zipper
  • Phosphorylation
  • PKA
  • Protein stability
  • Serine

ASJC Scopus subject areas

  • Biochemistry

Cite this

Design of a leucine zipper coiled coil stabilized 1.4 kcal mol-1 by phosphorylation of a serine in the e position. / Szilák, L.; Moitra, Jaideep; Vinson, Charles.

In: Protein Science, Vol. 6, No. 6, 06.1997, p. 1273-1283.

Research output: Contribution to journalArticle

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