Site-specific phosphorylation of Lys-Ser-Pro repeat peptides from neurofilament H by cyclin-dependent kinase 5: structural basis for substrate recognition.
Recent work has shown that high molecular weight neurofilament (NF) proteins are phosphorylated in their carboxy-terminal tail portion by the enzyme cyclin-dependent kinase 5 (CDK-5). The tail domain of neurofilaments contains 52 tripeptide repeats, viz. Lys-Ser-Pro, which mainly exist as KSPXK and KSPXXX motifs (X = amino acid). CDK-5 specifically phosphorylates the serine residues within the KSPXK sites. We probed the structural basis for this type of substrate selectivity by studying the conformation of synthetic peptides containing either KSPXK or KSPXXX repeats designed from native neurofilament sequences. Synthetic peptides with KSPXK repeats were phosphorylated on serine with a recombinant CDK-5/p25 complex whereas those with KSPXXX repeats were unreactive in this system. Circular dichroism (CD) studies in 50% TFE/H2O revealed a predominantly helical conformation for the KSPXXX-containing peptides, whereas the CD spectra for KSPXK-containing peptides indicated the presence of a high population of extended structures in water and 50% TFE solutions. However, detailed NMR analysis of one such peptide which included two such KSPXK repeats suggested a turn-like conformation encompassing the first KSPXK repeat. Restrained molecular dynamics calculations yielded an unusually stable, folded structure with a double "S"-like bend incorporating the central residues of the peptide. The data suggest that a transient reverse turn or loop-type structure may be a requirement for CDK-5-promoted phosphate transfer to neurofilament-specific peptide segments.
Sharma
P
P
Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA.
Barchi
J J
JJ
Huang
X
X
Amin
N D
ND
Jaffe
H
H
Pant
H C
HC
D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008969:Molecular Sequence Data; D016900:Neurofilament Proteins / Q000378:metabolism*; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
D016900:Neurofilament Proteins; D009842:Oligopeptides; C051774:neurofilament protein H; D051360:Cyclin-Dependent Kinase 5; D017346:Protein Serine-Threonine Kinases; C495905:CDK5 protein, human; C495906:Cdk5 protein, mouse; D018844:Cyclin-Dependent Kinases