Insights into the structural variation between pentapeptide repeat proteins—Crystal structure of Rfr23 from Cyanothece 51142

Cyanothece sp. PCC 51142 contains 35 pentapeptide repeat proteins (PRPs), proteins that contain a minimum of eight tandem repeated five-residues (Rfr) of the general consensus sequence A[N/D]LXX. Published crystal structures of PRPs show that the tandem pentapeptide repeats adopt a type of right-handed quadrilateral β-helix called an Rfr-fold. To characterize how structural features of Rfr-folds might vary with different amino acid sequences, the crystal structure of Cyanothece Rfr23 (174 residues) was determined at 2.4 Å resolution. The structure is dominated by an Rfr-fold capped at the N-terminus with a nine-residue α-helix (M26∗–E34). The Rfr-fold of Rfr23 contains four structural features previously unobserved in Rfr-folds. First, Rfr23 is composed entirely of type II β-turns. Second, the pentapeptide repeats are not consecutive in the primary amino acid sequence. Instead, Rfr23 contains 24-residues protruding outside one corner of the first complete N-terminal coil of the Rfr-fold (L56–P79) (24-residue insertion). Third, a disulfide bond between C39 and C42 bridges the β-turn between the first and second pentapeptide repeats in the first coil (disulfide bracket). NMR spectroscopy indicates that the reduction of the disulfide bracket with the addition of DTT destroys the entire Rfr-fold. Fourth, a single-residue perturbs the Rfr-fold slightly in the last coil between the C-terminal two pentapeptide repeats (single-residue bulge).