*4.5. Structural Considerations*

It seems worthy to compare the NMR data with structural information as available for proteins that are closely related to DnaB of *Helicobacter pylori* and BmrA of *Bacillus subtilis*, respectively.

<sup>−</sup> γ γ *BmrA and its structural counterpart.* The most suited for comparison appears to be the set of structures that shows the maltose ABC-transporter of *E. coli* (MBP-MalFGK2) in the outward-facing conformation, with two interacting NBDs and in the presence of AMPPNP (PDB 3RLF), ADP:BeF<sup>3</sup> (PDB 3PUX), ADP:Vi (PDB 3PUV), and ADP:AlF<sup>4</sup> - (PDB 3PUW) [64]. The collection of these different structures shapes the view of the transport cycle [122]. Chen and Oldham noticed that, despite the different ATP-analogues used, all residues within the NBD are essentially superimposable. However, structural differences between the pre-hydrolytic state (AMPPNP) and the transition state (ADP:Vi and ADP:AlF<sup>4</sup> <sup>−</sup>) are (i) the distance between the γ-phosphate or the mimicked γ-phosphate by the analogues and the bridging oxygen of

the β-phosphate and (ii) the presence of a water molecule, essential for the ATP hydrolysis, only in the transition state. Although the transmembrane part of the maltose transporter essentially differs from that of BmrA, the NBD homodimers of the two proteins are relatively similar (RMSD of 1.7 Å from the alignment of MBP-MalFGK2:AMPPNP, PDB 3RLF, with BmrAE504A:ATP, PDB 6R72). The two NBDs differ mainly in their ATPase activity. The ATPase activity of MBP-MalFGK2 is one order of magnitude lower than BmrA [124]. For BmrA, a major conformational transition between the open (inward-facing) and closed (outward-facing) conformation was for example experimentally demonstrated by hydrogen/deuterium exchange (HDX) coupled to mass spectrometry [125] and NMR spectroscopy [92]. It is believed that the protein adopts the closed conformation, with interacting NBDs, upon substrate binding. Generally, in membrane transporters, the energies of their sub-conformations should be close to each other and should essentially depend on the protein environment. The NMR spectra of BmrA in the presence of ADP:Vi and ADP:AlF<sup>4</sup> <sup>−</sup> might be taken as reporters of the enzyme transition state; in the presence of AlF<sup>4</sup> <sup>−</sup>, the crystal structure of the maltose transporter shows a classical picture with the catalytic water molecule in the apical attack position (Figure 11A). Even in the presence of AlF<sup>4</sup> - , the <sup>31</sup>P 1D CP spectra give two signals for the α- and β-phosphates (Figure 9D), respectively, which points to a certain nonequivalence of the two substrate-binding sites in the two similar NBDs. This finding might indicate that the two catalytic sites operate not simultaneously but sequentially.

*DnaB of Helicobacter pylori and its structural counterparts.* The conformation of DnaB, as could be judged from the 2D <sup>13</sup>C-13C DARR spectra, essentially depends on the nature of the analogue used, which matches the great structural variability reported for DnaB from other bacteria and their viral homologues [126–132]. Depending on the presence of substrate analogues and their nature, the SF4 helicase subunits can either form rings of distinct shapes [126–130] or arrange themselves as a hexameric ladder along a DNA strand [131,132]. The latter type of the structure was reported for DnaB from *Bacillus stearothermophilus* (currently *Geobacillus stearothermophilus*)*,* which was crystalized, in the presence of a DNA strand, with GDP:AlF<sup>4</sup> <sup>−</sup> in five of its six catalytic sites [131] (see Figure 11B). In this structure, each monomer of DnaB interacts in a similar way with two nucleotides of DNA; together, the subunits make a kind of a spiral ladder. It is noteworthy, that the position of AlF<sup>4</sup> <sup>−</sup> in the structure of *Geobacillus stearothermophilus* DnaB (Figure 11B) differs from that in other P-loop fold NTPases. No catalytic water molecule is present apically to the plane of AlF<sup>4</sup> <sup>−</sup> (see Figure 11A as a typical example), and the position of the AlF<sup>4</sup> <sup>−</sup> moiety does not correspond to that of the γ-phosphate group (see Figure 11C,F). Interestingly, the NMR data on DnaB from *Helicobacter pylori* discussed herein point to a full occupation of all six NBDs and a rather high symmetry in the oligomer [82] as it potentially could be achieved by more flat conformations of the helicase hexamer, as reported for several DnaB proteins, including the one from *Geobacillus stearothermophilus*, which were crystallized in the absence of AlF<sup>4</sup> <sup>−</sup> [127,128]. Whether the physiological shape of the DnaB ring is flat or spiral has to be established yet.

Figure 11D,E show the structures of the NBD of the ABC transporter MBP-MalFGK2 (Figure 11D) and the gene 4 helicase from bacteriophage T (Figure 11E) complexed with the pre-hydrolytic ATP analogue AMPPNP. The overlay of the structures (Figure 11F) shows a similar conformation of the bound phosphate chain of the ATP mimic. Although quite similar enzymes seem to bind ATP mimics in a similar way, they might behave differently to the huge number of ATP analogues available and solid-state NMR seems to be the method-of-choice to address such different behaviors.

α β γ **Figure 11.** Structural comparison of nucleotide-binding sites in SF4 helicases and ABC-transporters. Different protein subunits are colored in different shades of the same color. Mg2<sup>+</sup> or Ca2<sup>+</sup> ions are shown as green spheres; water molecules are shown as red spheres; hydrogen bonds and metal interactions involving α- and β-phosphates are shown as cyan dashes, interactions with γ-phosphate or its fluoroaluminate complex mimic shown as magenta dashes. Nucleotide analogue, P-loop motif residues and activating residues (Arg residue or LSGGQ motif) are shown as thick sticks, other interacting amino acid residues are shown as thin sticks. Enzymes complexed with NDP:AlF<sup>4</sup> − (**A**–**C**). Maltose/maltodextrin import ATP-binding protein MalK from *Escherichia coli K-12* (PDB ID 3PUW, chain B) (**A**), Replicative helicase DnaB from *Geobacillus stearothermophilus* (PDB ID 4ESV, chain E) (**B**), Structures 4ESV and 3PUW superimposed by phosphate chain and ribose atoms of NDP moieties (**C**); Enzymes complexed with the slowly hydrolyzable ATP analogue AMPPNP (**D**–**F**). Maltose/maltodextrin import ATP-binding protein MalK from *Escherichia coli K-12,* (PDB ID 3RLF, chain A) (**D**), Gene 4 Ring Helicase from Escherichia phage T7 (PDB ID 1E0J, chain A) (**E**), Structures 1E0J and 3RLF superimposed by phosphate chain (**F**).
