NrdH‐redoxin of Corynebacterium ammoniagenes forms a domain‐swapped dimer

Abstract

NrdH-redoxins constitute a family of small redox proteins, which contain a conserved CXXC sequence motif, and are characterized by a glutaredoxin-like amino acid sequence but a thioredoxin-like activity profile. Here we report the structure of Corynebacterium ammoniagenes NrdH at 2.7 A resolution, determined by molecular replacement using E. coli NrdH as model. The structure is the first example of a domain-swapped dimer from the thioredoxin family. The domain-swapped structure is formed by an inter-chain two-stranded anti-parallel beta-sheet and is stabilized by electrostatic interactions at the dimer interface. Size exclusion chromatography, and MALDI-ESI experiments revealed however, that the protein exists as a monomer in solution. Similar to E. coli NrdH-redoxin and thioredoxin, C. ammoniagenes NrdH-redoxin has a wide hydrophobic pocket at the surface that could be involved in binding to thioredoxin reductase. However, the loop between alpha2 and beta3, which is complementary to a crevice in the reductase in the thioredoxin-thioredoxin reductase complex, is the hinge for formation of the swapped dimer in C. ammoniagenes NrdH-redoxin. C. ammoniagenes NrdH-redoxin has the highly conserved sequence motif W61-S-G-F-R-P-[DE]67 which is unique to the NrdH-redoxins and which determines the orientation of helix alpha3. An extended hydrogen-bond network, similar to that in E. coli NrdH-redoxin, determines the conformation of the loop formed by the conserved motif.

NrdH-redoxin of Mycobacterium tuberculosis and Corynebacterium glutamicum Dimerizes at High Protein Concentration and Exclusively Receives Electrons from Thioredoxin Reductase

“…This crystal structure differs significantly from the structures of both Ec_NrdH-redoxin and Cg_NrdH-redoxin despite the high amino acid sequence identity with both proteins (41 and 75%, respectively). The Ca_NrdH-redoxin crystal structure appears as a domain-swapped dimer (9). However, the authors noted that Ca_NrdH-redoxin is a monomer in solution.…”

“…8). In all structures of NrdH-redoxins (Ec, Ca, and Cg), a central water molecule controls this hydrogen bond network (9,21). We included the Ca_NrdH-redoxin swapped dimer structure in this analysis because none of the amino acids of the hydrogen bond network are involved in domain swapping.…”

“…The hydrophobic amino acids of this pocket are identical in both NrdH-redoxins, and the hydrophobicity of these residues is conserved among all NrdH-redoxins (9,21). In the NrdH-redoxins of Mt and Cg, this pocket consists of Tyr-6, Ile-33, Val-43, Leu-49, Gln-50, Ala-51, and Val-53 (Fig.…”

“…Recently, Gustafsson et al (15) have shown that NrdH-redoxin from B. anthracis is not reduced by bacillithiol but accepts electrons from TrxR. Within the actinomycetes, it has been shown that the NrdH-redoxin of Corynebacterium ammoniagenes (Ca) accepts electrons from TrxR, although the possible reduction of NrdH-redoxin by MSH has not been examined (9).…”

“…The Cg_NrdH-redoxin structure was solved by molecular replacement using BALBES (20). The models designated by the PDB accession numbers 1R7H, 1H75, 3IC4, and 1ABA were used for molecular replacement (9,21,22). The solution of the Balbes run was submitted to the ARP/wARP server (23) for model building.…”