Monitoring ssDNA Binding to the DnaB Helicase from Helicobacter pylori by Solid‐State NMR Spectroscopy

DnaB helicases are bacterial, ATP‐driven enzymes that unwind double‐stranded DNA during DNA replication. Herein, we study the sequential binding of the “non‐hydrolysable” ATP analogue AMP‐PNP and of single‐stranded (ss) DNA to the dodecameric DnaB helicase from Helicobacter pylori using solid‐state NMR. Phosphorus cross‐polarization experiments monitor the binding of AMP‐PNP and DNA to the helicase. ¹³C chemical‐shift perturbations (CSPs) are used to detect conformational changes in the protein upon binding. The helicase switches upon AMP‐PNP addition into a conformation apt for ssDNA binding, and AMP‐PNP is hydrolyzed and released upon binding of ssDNA. Our study sheds light on the conformational changes which are triggered by the interaction with AMP‐PNP and are needed for ssDNA binding of H. pylori DnaB in vitro. They also demonstrate the level of detail solid‐state NMR can provide for the characterization of protein–DNA interactions and the interplay with ATP or its analogues.     

Paramagnetic Solid-State NMR to Localize the Metal-Ion Cofactor in an Oligomeric DnaB Helicase

Paramagnetic metal ions can be inserted into ATP-fueled motor proteins by exchanging the diamagnetic Mg²⁺ cofactor with Mn²⁺ or Co²⁺. Then, paramagnetic relaxation enhancement (PRE) or pseudo-contact shifts (PCSs) can be measured to report on the localization of the metal ion within the protein. We determine the metal position in the oligomeric bacterial DnaB helicase from Helicobacter pylori complexed with the transition-state ATP-analogue ADP:AlF₄⁻ and single-stranded DNA using solid-state NMR and a structure-calculation protocol employing CYANA. We discuss and compare the use of Mn²⁺ and Co²⁺ in localizing the ATP cofactor in large oligomeric protein assemblies. ³¹P PCSs induced in the Co²⁺-containing sample are then used to localize the DNA phosphate groups on the Co²⁺ PCS tensor surface enabling structural insights into DNA binding to the DnaB helicase.     

Poly­sulfonyl­amines. CXLV.

The prominent features in the molecular structure of the title compound (alternative name: 2‐diethyl­carbamoyl‐1,1,3,3‐tetraoxo‐1,3,2‐benzodi­thia­zole), C₁₁H₁₄N₂O₅S₂, arise in the urea moiety S₂N—C(O)—N′C₂: the sum of the angles at N is 332.3 (1)°, the N—C(O)—N′C₂ unit is planar, and distances N—C(O) = 1.494 (3) Å, N′—C(O) = 1.325 (2) Å and C—O = 1.215 (2) Å. The mol­ecules are associated via five C—H?O hydrogen bonds to form layers parallel to the yz plane. This compound and its di­methyl homologue, which were synthesized by treating the silver salt of o‐benzene­disulfon­imide with carbamoyl chlorides, are prone to rapid hydro­lysis at the weak N—C(O) bond. For both mol­ecules, the rotational barrier about the partial N′—C(O) double bond is ca 50 kJ mol^?1 at 250 K (from dynamic ¹H NMR experiments).