Solution structures of a 30-residue amino-terminal domain of the carp granulin-1 protein and its amino-terminally truncated 3-30 subfragment: Implications for the conformational stability of the stack of two β-hairpins

Abstract

Carp granulins are members of an emerging class of proteins with a sequence motif encoding a parallel stack of two to four β-hairpins. The carp granulin-1 protein forms a stack of four β-hairpins, whereas its amino-terminal fragment appears to adopt a very stable stack of two β-hairpins in solution. Here we determined a refined three-dimensional structure of this peptide fragment to examine potential conformational changes compared with the full-length protein. The structures were calculated with both a traditional method and a fast semiautomated method using ambiguous NMR distance restraints. The resulting sets of structures are very similar and show that a well-defined stack of two β-hairpins is retained in the peptide. Conformational rearrangements compensating the loss of the carboxy-terminal subdomain of the native protein are restricted to the carboxy-terminal end of the peptide, the turn connecting the two β-hairpins, and the Tyr21 and Tyr25 aromatic side chains. Further removal of the Val1 and Ile2 residues, which are part of the first β-hairpin and components of two major hydrophobic clusters in the two β-hairpin structure, results in the loss of the first β-hairpin. The second β-hairpin, which is closely associated with the first, retains a similar but somewhat less stable conformation. The invariable presence of the second β-hairpin and the dependence of its stability on the first β-hairpin suggest that the stack of two β-hairpins may be an evolutionary conserved and autonomous folding unit. In addition, the high conformational stability makes the stack of two β-hairpins an attractive scaffold for the development of peptide-based drug candidates. Proteins 2002;47:14–24. © 2002 Wiley-Liss, Inc.