The heat stable inhibitor of cAMP-dependent protein
kinase (PKIα) contains both a nuclear export signal
(NES) and a high affinity inhibitory region that is essential
for inhibition of the catalytic subunit of the kinase.
These functions are sequentially independent. Two-dimensional
NMR spectroscopy was performed on uniformly [15N]-labeled
PKIα to examine its structure free in solution. Seventy
out of 75 residues were identified, and examination of
the CαH chemical shifts revealed two regions of upfield
chemical shifts characteristic of α-helices. When PKIα
was fragmented into two functionally distinct peptides
for study at higher concentrations, no significant alterations
in chemical shifts or secondary structure were observed.
The first ordered region, identified in PKIα (1–25),
contains an α-helix from residues 1–13. This
helix extends by one turn the helix observed in the crystal
structure of a PKIα (5–24) peptide bound to the
catalytic subunit. The second region of well-defined secondary
structure, residues 35–47, overlaps with the nuclear
export signal in the PKIα (26–75) fragment. This
secondary structure consists of a helix with a hydrophobic
face comprised of Leu37, Leu41, and Leu44, followed by
a flexible turn containing Ile46. These four residues are
critical for nuclear export function. The remainder of
the protein in solution appears relatively unstructured,
and this lack of structure surrounding a few essential
and well-defined signaling elements may be characteristic
of a growing family of small regulatory proteins that interact
with protein kinases.