The cryo-EM structure of gastric H+,K+-ATPase with bound BYK99, a high-affinity member of K+-competitive, imidazo[1,2-a]pyridine inhibitors

Kazuhiro Abe,Jun Shimokawa,Mao Naito,George Sachs,Kazutoshi Tani,Keith Munson,Hiroshi Suzuki,Olga Vagin,Yoshinori Fujiyoshi

doi:10.1038/s41598-017-06698-8

Abstract

The gastric proton pump H+,K+-ATPase acidifies the gastric lumen, and thus its inhibitors, including the imidazo[1,2-a]pyridine class of K+-competitive acid blockers (P-CABs), have potential application as acid-suppressing drugs. We determined the electron crystallographic structure of H+,K+-ATPase at 6.5 Å resolution in the E2P state with bound BYK99, a potent P-CAB with a restricted ring structure. The BYK99 bound structure has an almost identical profile to that of a previously determined structure with bound SCH28080, the original P-CAB prototype, but is significantly different from the previously reported P-CAB-free form, illustrating a common conformational change is required for P-CAB binding. The shared conformational changes include a distinct movement of transmembrane helix 2 (M2), from its position in the previously reported P-CAB-free form, to a location proximal to the P-CAB binding site in the present BYK99-bound structure. Site-specific mutagenesis within M2 revealed that D137 and N138, which face the P-CAB binding site in our model, significantly affect the inhibition constant (Ki) of P-CABs. We also found that A335 is likely to be near the bridging nitrogen at the restricted ring structure of the BYK99 inhibitor. These provide clues to elucidate the binding site parameters and mechanism of P-CAB inhibition of gastric acid secretion.

Highlights

The H+,K+-ATPase enzyme utilises ATP hydrolysis to drive electroneutral exchange of cytoplasmic protons for luminal K+, acidifying the gastric juice[1, 2]
We previously reported a cryo-electron microscopy structure for H+,K+-ATPase with bound SCH28080 at 7 Å resolution and identified its binding location in a cavity facing the gastric lumen[10]
The quality of the density map of the BYK99-bound form analysed at 6.5 Å resolution was significantly improved over that of the 7 Å structure with bound SCH28080 [(SCH)E2BeF]10

Summary

Results and Discussion

Mutagenesis of the M2 helix showed that only mutations at D137 and N138 affected SCH28080 affinity (Table 1) This part of M2 is in close proximity to the inhibitor after rearrangement of the TM helices to give the P-CAB bound structures (Fig. 2E); the inhibitor binding site is bounded by M2 on one side, and by M4, M5, and M6 helices on the other. D137 is the only acidic amino acid in the M2 helix near the binding site in our model, and its accessibility to the solvent in the absence of the inhibitor is expected to give it a negative charge This led us to evaluate a possible charge effect at the position of D137 (Table 3). The Ki value of the charge-conserved mutant D137E could not be readily determined from global fitting due to its unusual kinetic parameters (see Materials and Methods), its affinity (Ki’) determined from a Km/Vmax plot indicated a smaller effect on the SCH28080 and BYK99 binding affinity

WT WT

Methods

Author Contributions

Additional Information