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Conformational dynamics of metallo-β-lactamase CcrA during catalysis investigated by using DEER spectroscopy

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Abstract

Previous crystallographic and mutagenesis studies have implicated the role of a position-conserved hairpin loop in the metallo-β-lactamases in substrate binding and catalysis. In an effort to probe the motion of that loop during catalysis, rapid-freeze-quench double electron–electron resonance (RFQ-DEER) spectroscopy was used to interrogate metallo-β-lactamase CcrA, which had a spin label at position 49 on the loop and spin labels (at positions 82, 126, or 233) 20–35 Å away from residue 49, during catalysis. At 10 ms after mixing, the DEER spectra show distance increases of 7, 10, and 13 Å between the spin label at position 49 and the spin labels at positions 82, 126, and 233, respectively. In contrast to previous hypotheses, these data suggest that the loop moves nearly 10 Å away from the metal center during catalysis and that the loop does not clamp down on the substrate during catalysis. This study demonstrates that loop motion during catalysis can be interrogated on the millisecond time scale.

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Abbreviations

CcrA*:

Spin-labeled site-directed variant CcrA:C155S

CcrA*(49):

Spin-labeled site-directed variant CcrA:C155S/W49C

CcrA*(49/82):

Spin-labeled site-directed variant CcrA:C155S/W49C/N82C

CcrA*(49/126):

Spin-labeled site-directed variant CcrA:C155S/W49C/D126C

CcrA*(49/233):

Spin-labeled site-directed variant CcrA:C155SW49C/E233C

DEER:

Double electron–electron resonance/pulsed electron–electron double resonance

EPR:

Electron paramagnetic (spin) resonance

MTSL:

(S-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate)

MβL:

Metallo-β-lactamase

RFQ:

Rapid freeze quench

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Acknowledgments

Funding and support from Miami University, National Science Foundation (CHE1151658 to MWC), and the National Institutes of Health (GM108026 to GAL) are gratefully acknowledged. Zahilyn Roche was an NSF-REU student in 2012. The National Biomedical EPR Center (James S. Hyde, Medical College of Wisconsin) is supported by a NIH P41 EB001980 Grant. The pulsed EPR spectrometer at Miami was purchased through NSF MRI-0722403 and the Ohio Board of Regents Grants. The authors thank Christian Altenbach, UCLA, for providing LongDistance and Candice S. Klug, Medical College of Wisconsin, for assistance with LongDistance and discussions.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, Miami University, 650 East High Street, Oxford, OH, 45056, USA

    Mahesh Aitha, Lindsay Moritz, Indra D. Sahu, Omar Sanyurah, Zahilyn Roche, Robert McCarrick, Gary A. Lorigan & Michael W. Crowder

  2. Physics Department, Marquette University, 540 N. 15th Street, Milwaukee, WI, 53233, USA

    Brian Bennett

  3. Department of Biophysics, Medical College of Wisconsin, 8701 W. Watertown Plank Road, Milwaukee, WI, 53226, USA

    Brian Bennett

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  1. Mahesh Aitha
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  2. Lindsay Moritz
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Correspondence to Brian Bennett or Michael W. Crowder.

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Aitha, M., Moritz, L., Sahu, I.D. et al. Conformational dynamics of metallo-β-lactamase CcrA during catalysis investigated by using DEER spectroscopy. J Biol Inorg Chem 20, 585–594 (2015). https://doi.org/10.1007/s00775-015-1244-8

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