Local structural preferences of calpastatin, the intrinsically unstructured protein inhibitor of calpain.

TitleLocal structural preferences of calpastatin, the intrinsically unstructured protein inhibitor of calpain.
Publication TypeJournal Article
Year of Publication2008
AuthorsKiss, R., D. Kovacs, P. Tompa, and A. Perczel
JournalBiochemistry
Volume47
Issue26
Pagination6936-45
Date Published2008 Jul 1
Type of Articleidp
ISSN1520-4995
KeywordsAmino Acid Sequence, Calcium-Binding Proteins, Calpain, Crystallography, X-Ray, Enzyme Inhibitors, Humans, Hydrogen-Ion Concentration, Models, Molecular, Molecular Sequence Data, Nuclear Magnetic Resonance, Biomolecular, Protein Structure, Secondary, Protein Structure, Tertiary, Sequence Alignment, Sequence Homology, Amino Acid, Temperature
Abstract

Calpain, the calcium-activated intracellular cysteine protease, is under the tight control of its intrinsically unstructured inhibitor, calpastatin. Understanding how potent inhibition by calpastatin can be reconciled with its unstructured nature provides deeper insight into calpain function and a more general understanding of how proteins devoid of a well-defined structure carry out their function. To this end, we performed a full NMR assignment of hCSD1 to characterize it in its solution state. Secondary chemical shift values and NMR relaxation data, R 1, R 2, and hetero-NOE, as well as spectral density function analysis have shown that conserved regions of calpastatin, subdomains A and C, which are responsible for calcium-dependent anchoring of the inhibitor to the enzyme, preferentially sample partially helical backbone conformations of a reduced flexibility. Moreover, the linker regions between subdomains are more flexible with no structural preference. The primary determinant of calpain inhibition, subdomain B, also has a non-fully random conformational preference, resembling a beta-turn structure also ascertained by prior studies of a 27-residue peptide encompassing the inhibitory region. This local structural preference is also confirmed by a deviation in chemical shift values between full-length calpastatin domain 1 and a truncated construct cut in the middle of subdomain B. At the C-terminal end of the molecule, a nascent helical region was found, which in contrast to the overall structural properties of the molecule may indicate a previously unknown functional region. Overall, these observations provide further evidence that supports previous suggestions that intrinsically unstructured proteins use preformed structural elements in efficient partner recognition.

DOI10.1021/bi800201a
Alternate JournalBiochemistry
PubMed ID18537264
Grant List067595 / / Wellcome Trust / United Kingdom