**3. The DnaB Helicase and the ABC Transporter BmrA**

The usefulness of particular ATP mimics for structural studies strongly depends on the nature of the protein of interest, as shown in Figure 2B for the example of DNA helicases and ABC transporters. The two proteins were subject to studies in the last years in our laboratories: the bacterial helicase DnaB from *Helicobacter pylori* [38,80–88] and the ABC transporter BmrA from *Bacillus subtilis* [86,89–92]. In the presence of double-stranded DNA, the DnaB from *Helicobacter pylori* is a double-homo hexamer of

59 kDa monomers with each hexamer moving along its single DNA strand, whereas BmrA from *Bacillus subtilis* is a dimeric membrane protein of 65 kDa monomers. The two proteins are well-characterized ATP-fuelled proteins. In both proteins, the chemical energy released during ATP hydrolysis in the nucleotide-binding domain (NBD) is converted into mechanical work, which, e.g., enables the movement of DnaB along a double-stranded DNA and its unzipping, as well as the transportation of molecules across the membrane by ABC transporters. Both proteins belong to the vast family of P-loop fold NTPases, one of the largest protein superfamilies. In any genome 10–20% of proteins code for P-loop fold domains [93–95]. P-loop fold NTPases are characterized by their signature GxxxxGK [S/T] sequence motif, also known as the Walker A motif [96]. This motif is responsible for binding the triphosphate chain and is often called the P-loop (phosphate-binding loop) motif [97]. In the P-loop fold, the conserved Lys residue forms hydrogen bonds with the β- and γ-phosphate groups of ATP or GTP. Another conserved motif, known as the Walker B motif, is composed of four hydrophobic residues ended by an aspartate residue. The conserved Asp residue stabilizes the metal ion cofactor Mg2<sup>+</sup> [96].

The *C*-terminal NBD of DnaB belongs to the superfamily 4 (SF4) of helicases, which in turn belongs to the class "RecA and F1/FO-related ATPases" (hereafter abbreviated as RecA/F1-related ATPases) of P-loop old NTPases. The ABC transporter BmrA belongs to a separate class of ABC transporters [93–95]. Both the RecA/F1-related ATPases and ABC transporters belong to the ASCE division of P-loop fold NTPases. The members of this division are characterized by an additional β-strand in the P-loop fold and a catalytic glutamate (E) residue next to the attacking water molecule [94,95,98]. The glutamate residue stabilizes the catalytic water molecule and, perhaps, operates as a catalytic base for ATP hydrolysis [99].

To avoid a futile NTP hydrolysis, P-loop fold NTPases are initiated before each turnover by activating moieties provided either by other proteins or by domains of the same protein [100–104]. The activating moiety interacts with the triphosphate chain and triggers the hydrolysis. The ATP hydrolysis in DnaB is induced by an interaction with an arginine residue that is provided by the neighbouring subunit of the same oligomer [105,106]. In ABC transporters, one of the NBDs is believed to activate hydrolysis within the active site in the other NBD by providing a signature LSGGQ motif [64,106].

Two analogues were mainly used in structural studies of helicases and ABC transporters (Figure 2): AMPPNP and ATP-γ-S, which both mimic the pre-hydrolytic state. The transition state is mainly mimicked by ADP:AlF<sup>x</sup> for the helicases, and ADP:Vi for the ABC transporters. Regarding the literature, this state is underrepresented compared to the pre-hydrolytic state.

We here gather information from published experiments, as well as present complementary original data, in order to give a compilation of ATP analogues and their mimicking power for the two proteins DnaB and BmrA, as assessed by magnetic-resonance methods, namely NMR and EPR.
