Observation of substrate diffusion and ligand binding in enzyme crystals using high-repetition-rate mix-and-inject serial crystallography

Suraj Pandey; George Calvey; Andrea M Katz; Tek Narsingh Malla; Faisal H M Koua; Jose M Martin-Garcia; Ishwor Poudyal; Jay-How Yang; Mohammad Vakili; Oleksandr Yefanov; Kara A Zielinski; Sasa Bajt; Salah Awel; Katarina Doerner; Matthias Frank; Luca Gelisio; Rebecca Jernigan; Henry Kirkwood; Marco Kloos; Jayanath Koliyadu; Valerio Mariani; Mitchell D Miller; Grant Mills; Garrett Nelson; Jose L Olmos Jr; Alireza Sadri; Tokushi Sato; Alexandra Tolstikova; Weijun Xu; Abbas Ourmazd; John C H Spence; Peter Schwander; Anton Barty; Henry N Chapman; Petra Fromme; Adrian P Mancuso; George N Phillips Jr; Richard Bean; Lois Pollack; Marius Schmidt

doi:10.1107/S2052252521008125

Observation of substrate diffusion and ligand binding in enzyme crystals using high-repetition-rate mix-and-inject serial crystallography

IUCrJ. 2021 Sep 9;8(Pt 6):878-895. doi: 10.1107/S2052252521008125. eCollection 2021 Nov 1.

Authors

Suraj Pandey¹, George Calvey², Andrea M Katz², Tek Narsingh Malla¹, Faisal H M Koua³, Jose M Martin-Garcia^{4

5}, Ishwor Poudyal¹, Jay-How Yang⁴, Mohammad Vakili⁶, Oleksandr Yefanov³, Kara A Zielinski², Sasa Bajt^{7

8}, Salah Awel³, Katarina Doerner⁶, Matthias Frank⁹, Luca Gelisio³, Rebecca Jernigan⁴, Henry Kirkwood⁶, Marco Kloos⁶, Jayanath Koliyadu⁶, Valerio Mariani^{3

10}, Mitchell D Miller¹¹, Grant Mills⁶, Garrett Nelson¹², Jose L Olmos Jr^{11

13}, Alireza Sadri³, Tokushi Sato⁶, Alexandra Tolstikova³, Weijun Xu¹¹, Abbas Ourmazd¹, John C H Spence¹², Peter Schwander¹, Anton Barty⁷, Henry N Chapman^{3

8

14}, Petra Fromme⁴, Adrian P Mancuso^{6

15}, George N Phillips Jr^{11

16}, Richard Bean⁶, Lois Pollack², Marius Schmidt¹

Affiliations

¹ Physics Department, University of Wisconsin-Milwaukee, 3135 North Maryland Avenue, Milwaukee, WI 53211, USA.
² School of Applied and Engineering Physics, Cornell University, 254 Clark Hall, Ithaca, NY 14853, USA.
³ Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, Notkestrasse 85, 22607 Hamburg, Germany.
⁴ School of Molecular Sciences and Biodesign Center for Applied Structural Discovery, Arizona State University, Tempe, AZ 85287-1604, USA.
⁵ Institute of Physical Chemistry Rocasolano, Spanish National Research Council, Calle de Serrano 119, 28006 Madrid, Spain.
⁶ European XFEL, Holzkoppel 4, 22869 Schenefeld, Germany.
⁷ Deutsches Elektronen-Synchrotron DESY, Notkestrasse 85, 22607 Hamburg, Germany.
⁸ The Hamburg Centre for Ultrafast Imaging, Luruper Chaussee 149, 22761 Hamburg, Germany.
⁹ Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, CA 94550, USA.
¹⁰ SLAC National Accelerator Laboratory, 2575 Sand Hill Rd, Menlo Park, California 94025, USA.
¹¹ Department of BioSciences, Rice University, 6100 Main Street, Houston, TX 77005, USA.
¹² Department of Physics, Arizona State University, Tempe, AZ 85287, USA.
¹³ Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, CA 94158, USA.
¹⁴ Department of Physics, Universität Hamburg, Luruper Chaussee 149, 22761 Hamburg, Germany.
¹⁵ Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia.
¹⁶ Department of Chemistry, Rice University, 6100 Main Street, Houston, TX 77005, USA.

Abstract

Here, we illustrate what happens inside the catalytic cleft of an enzyme when substrate or ligand binds on single-millisecond timescales. The initial phase of the enzymatic cycle is observed with near-atomic resolution using the most advanced X-ray source currently available: the European XFEL (EuXFEL). The high repetition rate of the EuXFEL combined with our mix-and-inject technology enables the initial phase of ceftriaxone binding to the Mycobacterium tuberculosis β-lactamase to be followed using time-resolved crystallography in real time. It is shown how a diffusion coefficient in enzyme crystals can be derived directly from the X-ray data, enabling the determination of ligand and enzyme-ligand concentrations at any position in the crystal volume as a function of time. In addition, the structure of the irreversible inhibitor sulbactam bound to the enzyme at a 66 ms time delay after mixing is described. This demonstrates that the EuXFEL can be used as an important tool for biomedically relevant research.

Keywords: European X-ray Free-Electron Laser; X-ray crystallography; antibiotic resistance; ceftriaxone; drug discovery; enzyme kinetics; enzyme mechanisms; irreversible inhibition; megahertz pulse-repetition rate; mix-and-inject serial crystallography; protein structure determination; serial femtosecond crystallography; substrate diffusion in crystals; sulbactam; β-lactamases.

Abstract

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