Mix-and-inject XFEL crystallography reveals gated conformational dynamics during enzyme catalysis

How changes in enzyme structure and dynamics facilitate passage along the reaction coordinate is a fundamental unanswered question. Here, we use time-resolved mix-and-inject serial crystallography (MISC) at an X-ray free electron laser (XFEL), ambient-temperature X-ray crystallography, computer simulations, and enzyme kinetics to characterize how covalent catalysis modulates isocyanide hydratase (ICH) conformational dynamics throughout its catalytic cycle. We visualize this previously hypothetical reaction mechanism, directly observing formation of a thioimidate covalent intermediate in ICH microcrystals during catalysis. ICH exhibits a concerted helical displacement upon active-site cysteine modification that is gated by changes in hydrogen bond strength between the cysteine thiolate and the backbone amide of the highly strained Ile152 residue. These catalysis-activated motions permit water entry into the ICH active site for intermediate hydrolysis. Mutations at a Gly residue (Gly150) that modulate helical mobility reduce ICH catalytic turnover and alter its pre-steady-state kinetic behavior, establishing that helical mobility is important for ICH catalytic efficiency. These results demonstrate that MISC can capture otherwise elusive aspects of enzyme mechanism and dynamics in microcrystalline samples, resolving long-standing questions about the connection between nonequilibrium protein motions and enzyme catalysis.

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Proton uptake mechanism in bacteriorhodopsin captured by serial synchrotron crystallography

Science ◽

10.1126/science.aaw8634 ◽

2019 ◽

Vol 365 (6448) ◽

pp. 61-65 ◽

Cited By ~ 32

Author(s):

Tobias Weinert ◽

Petr Skopintsev ◽

Daniel James ◽

Florian Dworkowski ◽

Ezequiel Panepucci ◽

...

Keyword(s):

Structural Changes ◽

Conformational Dynamics ◽

Uptake Mechanism ◽

Time Resolved ◽

X Ray ◽

Membrane Pump ◽

Protein Motions ◽

Hydrophobic Barrier ◽

Proton Uptake ◽

Laser Experiments

Conformational dynamics are essential for proteins to function. We adapted time-resolved serial crystallography developed at x-ray lasers to visualize protein motions using synchrotrons. We recorded the structural changes in the light-driven proton-pump bacteriorhodopsin over 200 milliseconds in time. The snapshot from the first 5 milliseconds after photoactivation shows structural changes associated with proton release at a quality comparable to that of previous x-ray laser experiments. From 10 to 15 milliseconds onwards, we observe large additional structural rearrangements up to 9 angstroms on the cytoplasmic side. Rotation of leucine-93 and phenylalanine-219 opens a hydrophobic barrier, leading to the formation of a water chain connecting the intracellular aspartic acid–96 with the retinal Schiff base. The formation of this proton wire recharges the membrane pump with a proton for the next cycle.

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Proton uptake mechanism in bacteriorhodopsin captured by serial synchrotron crystallography

10.1101/576629 ◽

2019 ◽

Author(s):

Tobias Weinert ◽

Petr Skopintsev ◽

Daniel James ◽

Florian Dworkowski ◽

Ezequiel Panepucci ◽

...

Keyword(s):

Structural Changes ◽

Conformational Dynamics ◽

Proton Pumping ◽

Uptake Mechanism ◽

Time Resolved ◽

X Ray ◽

Membrane Pump ◽

Protein Motions ◽

Hydrophobic Barrier ◽

Laser Experiments

AbstractConformational dynamics are essential for proteins to function. Here we describe how we adapted time-resolved serial crystallography developed at X-ray lasers to visualize protein motions using synchrotrons. We recorded the structural changes upon proton pumping in bacteriorhodopsin over 200 ms in time. The snapshot from the first 5 ms after photoactivation shows structural changes associated with proton release at comparable quality to previous X-ray laser experiments. From 10-15 ms onwards we observe large additional structural rearrangements up to 9 Å on the cytoplasmic side. Rotation of Leu93 and Phe219 opens a hydrophobic barrier leading to the formation of a water chain connecting the intracellular Asp96 with the retinal Schiff base. The formation of this proton wire recharges the membrane pump with a proton for the next cycle.

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Mix-and-inject XFEL crystallography reveals gated conformational dynamics during enzyme catalysis

10.1101/524751 ◽

2019 ◽

Cited By ~ 1

Author(s):

Medhanjali Dasgupta ◽

Dominik Budday ◽

Saulo H.P. de Oliveira ◽

Peter Madzelan ◽

Darya Marchany-Rivera ◽

...

Keyword(s):

Ambient Temperature ◽

Computer Simulations ◽

Active Site ◽

Enzyme Catalysis ◽

Conformational Dynamics ◽

Covalent Modification ◽

Enzyme Structure ◽

Conformational Ensemble ◽

Cysteine Modification ◽

X Ray

Summary ParagraphProtein dynamics play an important role in enzyme catalysis1-4. Many enzymes form covalent catalytic intermediates that can alter enzyme structure and conformational dynamics5,6. How these changes in enzyme structure and dynamics facilitate passage along the reaction coordinate is a fundamental unanswered question in structural enzymology. Here, we use Mix-and-Inject Serial Femtosecond X-ray Crystallography (MISC) at an X-ray Free Electron Laser (XFEL)7-10, ambient temperature X-ray crystallography, computer simulations, and enzyme kinetics to characterize how covalent modification of the active site cysteine residue in isocyanide hydratase (ICH) alters the enzyme’s conformational ensemble throughout the catalytic cycle. With MISC, we directly observe formation of a thioimidate covalent intermediate during ICH catalysis. The intermediate exhibits changes in the active site electrostatic environment, disrupting a hydrogen bond and triggering a cascade of conformational changes in ICH. X-ray-induced formation of a cysteine-sulfenic acid at the catalytic nucleophile (Cys101-SOH) with conventional crystallography at ambient temperature induces similar conformational shifts, demonstrating that these enzyme motions result from cysteine modification. Computer simulations show how cysteine modification-gated structural changes allosterically propagate through the ICH dimer. Mutations at Gly150 that modulate helical mobility reduce ICH catalytic turnover and alter its pre-steady state kinetic behavior, establishing that helical mobility is important for ICH catalytic efficiency. Taken together, our results demonstrate the potential of mix-and-inject XFEL crystallography to capture otherwise elusive mechanistic details of enzyme catalysis and dynamics from microcrystalline samples7,11. This approach can connect conformational dynamics to function for the large class of systems that rely on covalently modified cysteine residues for catalysis or regulation, resolving long-standing questions about enzyme mechanism and functionally relevant non-equilibrium enzyme motions.

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Mapping Enzyme Landscapes by Time-Resolved Crystallography with Synchrotron and X-Ray Free Electron Laser Light

Annual Review of Biophysics ◽

10.1146/annurev-biophys-100421-110959 ◽

2021 ◽

Vol 51 (1) ◽

Author(s):

Mark A. Wilson

Keyword(s):

Free Electron ◽

Free Electron Laser ◽

Enzyme Catalysis ◽

Energy Landscape ◽

Annual Review ◽

Publication Date ◽

Ambient Conditions ◽

Time Resolved ◽

X Ray ◽

X Ray Crystallography

Directly observing enzyme catalysis in real time at the molecular level has been a long-standing goal of structural enzymology. Time-resolved serial crystallography methods at synchrotron and X-ray free electron laser (XFEL) sources have enabled researchers to follow enzyme catalysis and other nonequilibrium events at ambient conditions with unprecedented time resolution. X-ray crystallography provides detailed information about conformational heterogeneity and protein dynamics, which is enhanced when time-resolved approaches are used. This review outlines the ways in which information about the underlying energy landscape of a protein can be extracted from X-ray crystallographic data, with an emphasis on new developments in XFEL and synchrotron time-resolved crystallography. The emerging view of enzyme catalysis afforded by these techniques can be interpreted as enzymes moving on a time-dependent energy landscape. Some consequences of this view are discussed, including the proposal that irreversible enzymes or enzymes that use covalent catalytic mechanisms may commonly exhibit catalysis-activated motions. Expected final online publication date for the Annual Review of Biophysics, Volume 51 is May 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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Crystal Structure of Escherichia coli Agmatinase: Catalytic Mechanism and Residues Relevant for Substrate Specificity

International Journal of Molecular Sciences ◽

10.3390/ijms22094769 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4769

Author(s):

Pablo Maturana ◽

María S. Orellana ◽

Sixto M. Herrera ◽

Ignacio Martínez ◽

Maximiliano Figueroa ◽

...

Keyword(s):

Escherichia Coli ◽

Active Site ◽

Catalytic Mechanism ◽

Conformational Dynamics ◽

High Specificity ◽

Arginine Decarboxylase ◽

Space Groups ◽

X Ray Crystallography ◽

High Dynamics ◽

Dynamics Simulations

Agmatine is the product of the decarboxylation of L-arginine by the enzyme arginine decarboxylase. This amine has been attributed to neurotransmitter functions, anticonvulsant, anti-neurotoxic, and antidepressant in mammals and is a potential therapeutic agent for diseases such as Alzheimer’s, Parkinson’s, and cancer. Agmatinase enzyme hydrolyze agmatine into urea and putrescine, which belong to one of the pathways producing polyamines, essential for cell proliferation. Agmatinase from Escherichia coli (EcAGM) has been widely studied and kinetically characterized, described as highly specific for agmatine. In this study, we analyze the amino acids involved in the high specificity of EcAGM, performing a series of mutations in two loops critical to the active-site entrance. Two structures in different space groups were solved by X-ray crystallography, one at low resolution (3.2 Å), including a guanidine group; and other at high resolution (1.8 Å) which presents urea and agmatine in the active site. These structures made it possible to understand the interface interactions between subunits that allow the hexameric state and postulate a catalytic mechanism according to the Mn2+ and urea/guanidine binding site. Molecular dynamics simulations evaluated the conformational dynamics of EcAGM and residues participating in non-binding interactions. Simulations showed the high dynamics of loops of the active site entrance and evidenced the relevance of Trp68, located in the adjacent subunit, to stabilize the amino group of agmatine by cation-pi interaction. These results allow to have a structural view of the best-kinetic characterized agmatinase in literature up to now.

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