(A) 2D 1H-15N HSQC spectra of 15N-labeled BOLA1 and BOLA3. (B) 15N R1, R2 relaxation rates and 15N(1H) NOE values per residue of BOLA1 and BOLA3 obtained at 500 MHz and 298 K. The proteins were in buffer N with 10% (v/v) D2O). The 1H-15N HSQC spectra of the apo forms of BOLA3 and BOLA1 showed well-dispersed resonances indicative of essentially folded proteins. The overall topology of the solution structure of human BOLA3 is α1β1β2η1α2η2β3 (h: 310-helix), in which β1 and β2 are antiparallel, and β3 is parallel to β2 (Figure 5). All α-helices are on the same side of the β-sheet and the 310-helix located between α2 and β3 contains the His96 residue that is invariant in all prokaryotic and eukaryotic BolA homologues (Li and Outten, 2012). A cysteine residue (Cys59), typically conserved in Bol3 homologues, is located close to the invariant His96 residue in an extended loop between β1 and β2. The two residues are not in the proper orientation to coordinate a [2Fe-2S] cluster (Sγ of Cys59 is at an average distance of ~8 Å from Nε2 of His96). The overall topology of the solution structure of BOLA1 is similar to that of BOLA3, with the exception of the presence, in BOLA1, of short helices in the loop regions and at the C-terminus (α1η1β1β2η2α2α3β3α4 topology) (Figure 5). In addition, at both termini BOLA1 has ca. 15 residues that are unstructured and whose backbone NH resonances are clustered in the central region of the 1H-15N HSQC spectrum (part B). The three His residues typically conserved in Bol1 homologues are located in two spatially close regions (Figure 5). Although they are close to each other, it is not possible to predict which ones may be involved in [2Fe-2S] cluster coordination. Information on backbone motions and on the protein oligomerization state of BOLA1 and BOLA3 were obtained through 15N R1, R2, heteronuclear 15N(1H)-NOEs NMR experiments. The 15N backbone relaxation properties showed that the N and C termini of BOLA1 are highly flexible, as about 15 to 20 residues on each terminus have negative 15N NOE values, while the folded domain is essentially rigid. BOLA3 is a rigid molecule, with the exception of the first detected residue at the N terminus, the last two residues at the C terminus and residues around invariant His96. All of them experience fast backbone motions in the ns-ps time scale, as indicated by their low or negative 15N NOE values.