Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure

Abstract

We designed a single-chain variant of the Arc repressor homodimer in which the ␤ strands that contact operator DNA are connected by a hairpin turn and the ␣ helices that form the tetrahelical scaffold of the dimer are attached by a short linker. The designed protein represents a noncyclic permutation of secondary structural elements in another single-chain Arc molecule (Arc-L1-Arc), in which the two subunits are fused by a single linker. The permuted protein binds operator DNA with nanomolar affinity, refolds on the submillisecond time scale, and is as stable as Arc-L1-Arc. The crystal structure of the permuted protein reveals an essentially wild-type fold, demonstrating that crucial folding information is not encoded in the wild-type order of secondary structure. Noncyclic rearrangement of secondary structure may allow grouping of critical activesite residues in other proteins and could be a useful tool for protein design and minimization. circular permutation ͉ protein folding ͉ protein structure I magine a protein in which the ␣-helices and ␤-strands are disconnected but otherwise arranged properly in three dimensions, so that the hydrophobic core and most native interactions remain intact. There are probably many ways to connect the secondary-structure elements that are compatible with stable folding. For example, cyclic permutation of secondary structure (Fig. 1A) is tolerated in numerous proteins and occurs naturally in some protein families (1-10). After cyclic permutation, a wild-type block of N-terminal sequence is shifted to the C terminus of the permuted protein. Threedimensional domain swapping, in which a monomeric protein fold is reproduced with structural parts donated by different subunits in a multimer (Fig. 1B), provides another instance of rearranged structural elements (11,12). These examples suggest that the essential feature of a protein fold is the complementary packing of secondary structural elements and not the precise manner in which these elements are connected. If this model is correct, then many permutations of protein structural elements should be allowed. However, to our knowledge, no successful examples of noncyclic permutations of protein structural elements have been reported.Arc repressor from bacteriophage P22 is a small transcription factor that folds as a symmetric homodimer. Two Arc dimers bind cooperatively to adjacent subsites in arc operator DNA (13-16). Each Arc subunit consists of a ␤-strand and two ␣-helices (␣A and ␣B); the packing of these elements from both subunits of the dimer forms a single hydrophobic core. The ␤-strands pair to form an antiparallel ␤-sheet, which contacts the major groove of operator DNA. A f lexible N-terminal arm and residues near the beginning of helix B make other DNA contacts. Joining two Arc subunits with a 15-residue linker results in a single-chain molecule (Arc-L1-Arc) that is more stable than wild-type Arc and binds operator DNA at lower protein concentrations (17,18). The wild-type order of secondary structure is preserved in Arc-L...

The energy cost of polypeptide knot formation and its folding consequences

“…The heterogeneous behavior of Arc-L1-Arc was not detected when pulling a non-circular permutant of it, pARC. This molecule retains the architecture of Arc-L1-Arc without the linker L1, making it impossible to form a knot 32 (Supplementary Fig. 3 ).…”

Topology independent protein structural alignment

“…Although there has been previous work on the detection of non-cyclic permutations [ 14 - 16 , 26 ], compared to cyclic-permutations there has been relatively little research of noncyclic-permutations. As an example of this important class of topologically permuted proteins, Tabtiang et al (2004) were able to artificially create a noncyclic permutation of the Arc repressor that was thermodynamically stable, refolds on the sub-millisecond time scale, and binds operator DNA with nanomolar affinity [ 12 ]. This raises the question of whether or not these non-cyclic permutations can arise naturally.…”

“…Tabtiang et al . were able to create a thermodynamically stable and biologically functional non-cyclic permutation, indicating that non-cyclic permutations may be as important as circular permutations [ 12 ]. In this study, we present a novel method that detects spatially similar structures that can identify structures related by circular and more complex non-cyclic permutations.…”

Topology Independent Protein Structural Alignment

“…Although there has been previous work on the detection of non-cyclic permutations [14][15][16]26], compared to cyclic-permutations there has been relatively little research of noncyclic-permutations. As an example of this important class of topologically permuted proteins, Tabtiang et al (2004) were able to artificially create a noncyclic permutation of the Arc repressor that was thermodynamically stable, refolds on the sub-millisecond time scale, and binds operator DNA with nanomolar affinity [12]. This raises the question of whether or not these noncyclic permutations can arise naturally.…”

“…We refer to these as non-cyclic permutations from now on. Tabtiang et al were able to create a thermodynamically stable and biologically functional non-cyclic permutation, indicating that non-cyclic permutations may be as important as circular permutations [12]. In this study, we present a novel method that detects spatially similar structures that can identify structures related by circular and more complex non-cyclic permutations.…”