PNNL

Abstract

The genome of the diurnal cyanobacterium Cyanothece sp. PCC 51142 has recently been sequenced and observed to contain 35 pentapeptide repeat proteins (PRPs). These proteins, while present throughout the prokaryotic and eukaryotic kingdoms, are most abundant in cyanobacteria. The sheer number of PRPs in cyanobacteria coupled with their predicted location in all the cyanobacteria cellular compartments argues for important, yet unknown, physiological and biochemical functions. To gain insights into the biochemical function of PRPs in cyanobacteria, the first crystal structure of a PRP from Cyanothece has been determined at 2.1 Å resolution. The native protein, annotated Rfr32 for repeated five-residue, is a 167-residue protein with an N-terminal 29-residue signal peptide. The signal peptide was replaced with a 43-residue tag that was invisible in the electron density maps of two different crystal forms from which essentially identical structures were solved. The structure is dominated by 21 tandem pentapeptide repeats that fold into a right-handed quadrilateral ß-helix, or Rfr-fold, reminiscent of a “square” tower with four distinct faces. Four consecutive pentapeptide repeats define a “floor” of the tower with a single repeat occupying a face. The Rfr-fold contains five complete, stacked, ascending floors (coils) that complete a revolution every 20 residues with a ~4.8 Å rise along the helix axis. The main chain backbone of the floors are held together with a narrow parallel ß-sheet on one face and stacked parallel The main chain backbone of the floors are held together with a narrow parallel ß-sheet on one face and stacked parallel ß-bridges (single-residue ß-sheets) on the other three faces. The regular shape of the tower is maintained by two distinct types of four-residue turns labeled pseudo type II and pseudo type IV ß-turns. The interior of the Rfr-fold is primarily hydrophobic, with all side chains of the i and i-2 residues inserted into the center of the ß-helix to form aligned, stacked, columns of hydrophobic side chains. The i-1, i+1, and i+2 residues are generally polar or charged and these side chains all point away from the Rfr-core to give the ß-helix a predominately hydrophilic surface. Two short, anti-parallel ß-helices, bridged with a disulfide bond, sit atop of the C-terminus of the Rfr-fold perhaps preventing edge-to-edge aggregation at the C-terminus of the Rfr-fold. The circular dichroism spectra of Rfr-32 is dominated by ß-turn and parallel ß-sheet features. The structure of Rfr32 is compared with the only other PRP structure, the mycobacterial fluoroquinoline resistance protein MfpA from Mycobacterium tuberculosis, and the general features of the amino acid sequences of the 35 Cyanothece PRPs are discussed.