TPR domain of NrfG mediates complex formation between heme lyase and formate-dependent nitrite reductase in Escherichia coli O157:H7.

Escherichia coli synthesize C-type cytochromes only during anaerobic growth in media supplemented with nitrate and nitrite. The reduction of nitrate to ammonium in the periplasm of Escherichia coli involves two separate periplasmic enzymes, nitrate reductase and nitrite reductase. The nitrite reductase involved, NrfA, contains cytochrome C and is synthesized coordinately with a membrane-associated cytochrome C, NrfB, during growth in the presence of nitrite or in limiting nitrate concentrations. The genes NrfE, NrfF, and NrfG are required for the formate-dependent nitrite reduction pathway, which involves at least two C-type cytochrome proteins, NrfA and NrfB. The NrfE, NrfF, and NrfG genes (heme lyase complex) are involved in the maturation of a special C-type cytochrome, apocytochrome C (apoNrfA), to cytochrome C (NrfA) by transferring a heme to the unusual heme binding motif of the Cys-Trp-Ser-Cys-Lys sequence in apoNrfA protein. Thus, in order to further investigate the roles of NrfG in the formation of heme lyase complex (NrfEFG) and in the interaction between heme lyase complex and formate-dependent nitrite reductase (NrfA), we determined the crystal structure of NrfG at 2.05 A. The structure of NrfG showed that the contact between heme lyase complex (NrfEFG) and NrfA is accomplished via a TPR domain in NrfG which serves as a binding site for the C-terminal motif of NrfA. The portion of NrfA that binds to TPR domain of NrfG has a unique secondary motif, a helix followed by about a six-residue C-terminal loop (the so called "hook conformation"). This study allows us to better understand the mechanism of special C-type cytochrome assembly during the maturation of formate-dependent nitrite reductase, and also adds a new TPR binding conformation to the list of TPR-mediated protein-protein interactions.

Crystal structure of TTC0263, a thermophilic TPR protein from Thermus thermophilus HB27.

The hypothetical protein TTC0263 of Thermus thermophilus HB27 is a thermophilic tetratricopeptide repeat (TPR)-containing protein. In the present study, the TPR region (residues 26-230) was resolved at 2.5 A with R-factors of R/Rfree = 23.6%/28.6%. TTC0263 consists of 11 helices that form five TPR units. Uniquely, it contains one atypical "extended" TPR (eTPR) unit. This comprises extended helical residues near the loop region of TTC0263, such that the helical length of eTPR is longer than that of the canonical TPR sequence. In addition, the hybrid TPR domain of TTC0263 possesses oligomer-forming characteristics. TPR domains are generally involved in forming multi-subunit complexes by interacting with each other or with other subunit proteins. The dynamic structure of TTC0263 described here goes some way to explaining how TPR domains mediate the formation of multi-subunit complexes.

Crystal structure of YrrB: a TPR protein with an unusual peptide-binding site.

YrrB is a hypothetical protein containing a tetratricopeptide repeat (TPR) domain from a Gram-positive bacterium, Bacillus subtilis. We determined YrrB structure in the C2 space group to 2.5A resolution, which is the first TPR structure of the Gram-positive bacterium B. subtilis. In contrast to other known TPR structures, the concave surface of the YrrB TPR domain is composed of the putative peptide-binding pocket lined with positively-charged residues. This unique charge distribution reveals that YrrB can interact with partner proteins via an unusual TPR-mediated interaction mode, compared to that of other TPR-containing structures. Functional annotation using genomics analysis suggested that YrrB may be an interacting mediator in the complex formation among RNA sulfuration components. No proteins containing a TPR domain have been identified in the biosynthesis of sulfur-containing biomolecules. Thus, YrrB could play a new role as a connecting module among those proteins in the conserved gene cluster for RNA sulfuration.

Crystal structure of PilF: functional implication in the type 4 pilus biogenesis in Pseudomonas aeruginosa.

PilF is a requisite protein involved in the type 4 pilus biogenesis system from the Gram-negative human pathogenic bacteria, Pseudomonas aeruginosa. We determined the PilF structure at a 2.2A resolution; this includes six tandem tetratrico peptide repeat (TPR) units forming right-handed superhelix. PilF structure was similar to the heat shock protein organizing protein, which interacts with the C-terminal peptide of Hsp90 and Hsp70 via a concave Asn ladder in the inner groove of TPR superhelix. After simulated screening, the C-terminal pentapeptides of PilG, PilU, PilY, and PilZ proved to be a likely candidate binding to PilF, which are ones of 25 necessary components involved in the type 4 pilus biogenesis system. We proposed that PilF would be critical as a bridgehead in protein-protein interaction and thereby, PilF may bind a necessary molecule in type 4 pilus biogenesis system such as PilG, PilU, PilY, and PilZ.