Design of a leucine zipper coiled coil stabilized 1.4 kcal mol-1 by phosphorylation of a serine in the e position.
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 (delta delta GP = -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 (delta delta GP = -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 delta delta GP = +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.
Szilák
L
L
Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Moitra
J
J
Vinson
C
C
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D030161:Avian Proteins; D050976:Basic-Leucine Zipper Transcription Factors; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002500:Centrifugation, Isopycnic; D002942:Circular Dichroism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D015342:DNA Probes / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051790:G-Box Binding Factors; D017930:Genes, Reporter; D018698:Glutamic Acid; D005998:Glycine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D016350:Leucine Zippers / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011489:Protein Denaturation; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013912:Threonine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection
D030161:Avian Proteins; D050976:Basic-Leucine Zipper Transcription Factors; D002352:Carrier Proteins; D015342:DNA Probes; D004268:DNA-Binding Proteins; D051790:G-Box Binding Factors; D010750:Phosphoproteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D013912:Threonine; D018698:Glutamic Acid; D012694:Serine; D001120:Arginine; D017868:Cyclic AMP-Dependent Protein Kinases; D005998:Glycine