IUCrJ (Nov 2021)
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
Affiliations
- Suraj Pandey
- Physics Department, University of Wisconsin-Milwaukee, 3135 North Maryland Avenue, Milwaukee, WI 53211, USA
- George Calvey
- School of Applied and Engineering Physics, Cornell University, 254 Clark Hall, Ithaca, NY 14853, USA
- Andrea M. Katz
- School of Applied and Engineering Physics, Cornell University, 254 Clark Hall, Ithaca, NY 14853, USA
- Tek Narsingh Malla
- Physics Department, University of Wisconsin-Milwaukee, 3135 North Maryland Avenue, Milwaukee, WI 53211, USA
- Faisal H. M. Koua
- Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, Notkestrasse 85, 22607 Hamburg, Germany
- Jose M. Martin-Garcia
- School of Molecular Sciences and Biodesign Center for Applied Structural Discovery, Arizona State University, Tempe, AZ 85287-1604, USA
- Ishwor Poudyal
- Physics Department, University of Wisconsin-Milwaukee, 3135 North Maryland Avenue, Milwaukee, WI 53211, USA
- Jay-How Yang
- School of Molecular Sciences and Biodesign Center for Applied Structural Discovery, Arizona State University, Tempe, AZ 85287-1604, USA
- Mohammad Vakili
- European XFEL, Holzkoppel 4, 22869 Schenefeld, Germany
- Oleksandr Yefanov
- Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, Notkestrasse 85, 22607 Hamburg, Germany
- Kara A. Zielinski
- School of Applied and Engineering Physics, Cornell University, 254 Clark Hall, Ithaca, NY 14853, USA
- Sasa Bajt
- Deutsches Elektronen-Synchrotron DESY, Notkestrasse 85, 22607 Hamburg, Germany
- Salah Awel
- Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, Notkestrasse 85, 22607 Hamburg, Germany
- Katarina Doerner
- European XFEL, Holzkoppel 4, 22869 Schenefeld, Germany
- Matthias Frank
- Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, CA 94550, USA
- Luca Gelisio
- Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, Notkestrasse 85, 22607 Hamburg, Germany
- Rebecca Jernigan
- School of Molecular Sciences and Biodesign Center for Applied Structural Discovery, Arizona State University, Tempe, AZ 85287-1604, USA
- Henry Kirkwood
- European XFEL, Holzkoppel 4, 22869 Schenefeld, Germany
- Marco Kloos
- European XFEL, Holzkoppel 4, 22869 Schenefeld, Germany
- Jayanath Koliyadu
- European XFEL, Holzkoppel 4, 22869 Schenefeld, Germany
- Valerio Mariani
- Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, Notkestrasse 85, 22607 Hamburg, Germany
- Mitchell D. Miller
- Department of BioSciences, Rice University, 6100 Main Street, Houston, TX 77005, USA
- Grant Mills
- European XFEL, Holzkoppel 4, 22869 Schenefeld, Germany
- Garrett Nelson
- Department of Physics, Arizona State University, Tempe, AZ 85287, USA
- Jose L. Olmos Jr
- Department of BioSciences, Rice University, 6100 Main Street, Houston, TX 77005, USA
- Alireza Sadri
- Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, Notkestrasse 85, 22607 Hamburg, Germany
- Tokushi Sato
- European XFEL, Holzkoppel 4, 22869 Schenefeld, Germany
- Alexandra Tolstikova
- Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, Notkestrasse 85, 22607 Hamburg, Germany
- Weijun Xu
- Department of BioSciences, Rice University, 6100 Main Street, Houston, TX 77005, USA
- Abbas Ourmazd
- Physics Department, University of Wisconsin-Milwaukee, 3135 North Maryland Avenue, Milwaukee, WI 53211, USA
- John C. H. Spence
- Department of Physics, Arizona State University, Tempe, AZ 85287, USA
- Peter Schwander
- Physics Department, University of Wisconsin-Milwaukee, 3135 North Maryland Avenue, Milwaukee, WI 53211, USA
- Anton Barty
- Deutsches Elektronen-Synchrotron DESY, Notkestrasse 85, 22607 Hamburg, Germany
- Henry N. Chapman
- Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, Notkestrasse 85, 22607 Hamburg, Germany
- Petra Fromme
- School of Molecular Sciences and Biodesign Center for Applied Structural Discovery, Arizona State University, Tempe, AZ 85287-1604, USA
- Adrian P. Mancuso
- European XFEL, Holzkoppel 4, 22869 Schenefeld, Germany
- George N. Phillips Jr
- Department of BioSciences, Rice University, 6100 Main Street, Houston, TX 77005, USA
- Richard Bean
- European XFEL, Holzkoppel 4, 22869 Schenefeld, Germany
- Lois Pollack
- School of Applied and Engineering Physics, Cornell University, 254 Clark Hall, Ithaca, NY 14853, USA
- Marius Schmidt
- Physics Department, University of Wisconsin-Milwaukee, 3135 North Maryland Avenue, Milwaukee, WI 53211, USA
- DOI
- https://doi.org/10.1107/S2052252521008125
- Journal volume & issue
-
Vol. 8,
no. 6
pp. 878 – 895
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
- substrate diffusion in crystals
- antibiotic resistance
- β-lactamases
- enzyme kinetics
- irreversible inhibition
- mix-and-inject serial crystallography
- serial femtosecond crystallography
- european x-ray free-electron laser
- megahertz pulse-repetition rate
- protein structure determination
- drug discovery
- ceftriaxone
- sulbactam
- x-ray crystallography
- enzyme mechanisms
