Investigating the position of the hairpin loop in New Delhi metallo-β-lactamase, NDM-1, during catalysis and inhibitor binding

• We used double electron electron resonance (DEER) spectroscopy to study metallo-β-lactamase.
• We used rapid-freeze DEER studies to probe loop motions during catalysis.
• Loop motions in New Delhi metallo-β-lactamase, NDM-1, were evaluated.
• One reaction was freeze quenched at 500 μs.
• Product binding does not affect the position of the loop.

In an effort to examine the relative position of a hairpin loop in New Delhi metallo-β-lactamase, NDM-1, during catalysis, rapid freeze quench double electron electron resonance (RFQ-DEER) spectroscopy was used. A doubly-labeled mutant of NDM-1, which had one spin label on the invariant loop at position 69 and another label at position 235, was prepared and characterized. The reaction of the doubly spin labeled mutant with chromacef was freeze quenched at 500 μs and 10 ms. DEER results showed that the average distance between labels decreased by 4 Å in the 500 μs quenched sample and by 2 Å in the 10 ms quenched sample, as compared to the distance in the unreacted enzyme, although the peaks corresponding to distance distributions were very broad. DEER spectra with the doubly spin labeled enzyme with two inhibitors showed that the distance between the loop residue at position 69 and the spin label at position 235 does not change upon inhibitor binding. This study suggests that the hairpin loop in NDM-1 moves over the metal ion during the catalysis and then moves back to its original position after hydrolysis, which is consistent with a previous hypothesis based on NMR solution studies on a related metallo-β-lactamase. This study also demonstrates that this loop motion occurs in the millisecond time domain.

Rapid-freeze quench double electron electron resonance spectroscopy (RFQ-DEER) was used to probe the position of an invariant loop in New Delhi metallo-β-lactamase, NDM-1, during catalysis and inhibitor binding. The results show that the loop moves over the active site during catalysis but stays in the same position during inhibitor binding.Figure optionsDownload as PowerPoint slide