Precipitant–ligand exchange technique reveals the ADP binding mode inMycobacterium tuberculosisdethiobiotin synthetase

Abstract

Dethiobiotin synthetase from Mycobacterium tuberculosis (MtDTBS) is a promising antituberculosis drug target. Small‐molecule inhibitors that target MtDTBS provide a route towards new therapeutics for the treatment of antibiotic‐resistant tuberculosis. Adenosine diphosphate (ADP) is an inhibitor of MtDTBS; however, structural studies into its mechanism of inhibition have been unsuccessful owing to competitive binding to the enzyme by crystallographic precipitants such as citrate and sulfate. Here, a crystallographic technique termed precipitant–ligand exchange has been developed to exchange protein‐bound precipitants with ligands of interest. Proof of concept for the exchange method was demonstrated using cytidine triphosphate (CTP), which adopted the same binding mechanism as that obtained with traditional crystal‐soaking techniques. Precipitant–ligand exchange also yielded the previously intractable structure of MtDTBS in complex with ADP solved to 2.4 Å resolution. This result demonstrates the utility of precipitant–ligand exchange, which may be widely applicable to protein crystallography.

Mycobacterium tuberculosis Dethiobiotin Synthetase Facilitates Nucleoside Triphosphate Promiscuity through Alternate Binding Modes

“…However, this was not observed for the other weaker binding NTPs, even at concentrations greater than 100 mM. P-loop-bound precipitants were competitively displaced with NTPs using our precipitant-ligand exchange technique, 16 whereby crystals grown in ammonium sulfate were transferred to a solution in which the ammonium sulfate precipitant was replaced by high concentrations of NTPs.…”

“…To overcome this, a crystallographic technique was developed to displace the precipitant with the target ligandsan approach we termed precipitant−ligand exchange. 16 Here, crystals were initially grown to approximately 100−400 μm in conditions containing ammonium sulfate. They were then transferred into a soaking solution where the precipitant was replaced with NTP at high concentrations (200−300 mM).…”

“…Proof of concept that a protein bound precipitant could be displaced by a competitive ligand was established using CTP. The crystal structure obtained using our new crystal soaking approach demonstrated that CTP adopted the same mode of binding described above . Here, we use this technique to investigate the atomic interactions between NTPs and Mt DTBS.…”

“…Repeated efforts to crystallize the enzyme with alternate NTPs proved unsuccessful, presumably due to the presence of crystallographic precipitants competing with ligands for binding to the MtDTBS active site. We recently developed a crystallographic technique termed precipitantligand exchange, 16 which allowed us to overcome this technical hurdle. Proof of concept that a protein bound precipitant could be displaced by a competitive ligand was established using CTP.…”

“…The crystal structure obtained using our new crystal soaking approach demonstrated that CTP adopted the same mode of binding described above. 16 Here, we use this technique to investigate the atomic interactions between NTPs and MtDTBS.…”