skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: In cellulo crystallization of Trypanosoma brucei IMP dehydrogenase enables the identification of genuine co-factors

Abstract

Sleeping sickness is a fatal disease caused by the protozoan parasite Trypanosoma brucei (Tb). Inosine-5’-monophosphate dehydrogenase (IMPDH) has been proposed as a potential drug target, since it maintains the balance between guanylate deoxynucleotide and ribonucleotide levels that is pivotal for the parasite. Here we report the structure of TbIMPDH at room temperature utilizing free-electron laser radiation on crystals grown in living insect cells. The 2.80 Å resolution structure reveals the presence of ATP and GMP at the canonical sites of the Bateman domains, the latter in a so far unknown coordination mode. Consistent with previously reported IMPDH complexes harboring guanosine nucleotides at the second canonical site, TbIMPDH forms a compact oligomer structure, supporting a nucleotide-controlled conformational switch that allosterically modulates the catalytic activity. The oligomeric TbIMPDH structure we present here reveals the potential of in cellulo crystallization to identify genuine allosteric co-factors from a natural reservoir of specific compounds.

Authors:
ORCiD logo [1];  [2]; ORCiD logo [3];  [4];  [5]; ORCiD logo [6];  [7];  [8];  [9];  [10]; ORCiD logo [11];  [12];  [4];  [13];  [14];  [15]; ORCiD logo [16]; ORCiD logo [17];  [18]; ORCiD logo [19] more »; ORCiD logo [17]; ORCiD logo [20];  [15]; ORCiD logo [17];  [21]; ORCiD logo [18];  [17];  [19]; ORCiD logo [4];  [22];  [12]; ORCiD logo [23];  [17];  [6]; ORCiD logo [24];  [3] « less
  1. Deutsches Elektronen-Synchrotron (DESY), Hamburg (Germany); Paul Scherrer Inst. (PSI), Villigen (Switzerland)
  2. Inst. of Biochemistry and Molecular Biology, Hamburg (Germany). Joint Lab. for Structural Biology of Infection and Inflammation; Univ. of Hamburg (Germany); Univ. of Lübeck, at Deutsches Elektronen-Synchrotron (DESY), Hamburg (Germany); Univ. of Lübeck (Germany); Deutsches Elektronen Synchrotron (DESY), Hamburg (Germany)
  3. Univ. of Hamburg, at Deutsches Elektronen-Synchrotron (DESY) (Germany); The Hamburg Centre for Ultrafast Imaging (CUI), Hamburg (Germany)
  4. Deutsches Elektronen-Synchrotron (DESY), Hamburg (Germany)
  5. Inst. of Biochemistry and Molecular Biology, Hamburg (Germany). Joint Lab. for Structural Biology of Infection and Inflammation; Univ. of Hamburg (Germany); Univ. of Lübeck, at Deutsches Elektronen-Synchrotron (DESY), Hamburg (Germany); BioAgilytix Europe GmbH, Hamburg (Germany)
  6. Univ. of Tübingen (Germany)
  7. Deutsches Elektronen-Synchrotron (DESY), Hamburg (Germany); Univ. di Roma Tor Vergata, Rome (Italy); Istituto Nazionale di Fisica Nucleare (INFN), Rome (Italy)
  8. Russian Academy of Sciences, Moscow Region (Russia)
  9. Univ. of Lübeck (Germany); Deutsches Elektronen Synchrotron (DESY), Hamburg (Germany)
  10. Inst. of Biochemistry and Molecular Biology, Hamburg (Germany). Joint Lab. for Structural Biology of Infection and Inflammation; Univ. of Hamburg (Germany); Univ. of Lübeck, at Deutsches Elektronen-Synchrotron (DESY), Hamburg (Germany); BODE Chemie GmbH, Hamburg (Germany)
  11. Deutsches Elektronen-Synchrotron (DESY), Hamburg (Germany); Univ. of Lübeck (Germany)
  12. Deutsches Elektronen-Synchrotron (DESY), Hamburg (Germany); SLAC National Accelerator Lab., Menlo Park, CA (United States). Linac Coherent Light Source (LCLS)
  13. Arizona State Univ., Tempe, AZ (United States); European Molecular Biology Lab. (EMBL), Grenoble (France)
  14. Arizona State Univ., Tempe, AZ (United States); Max Planck Inst. for Medical Research, Heidelberg (Germany)
  15. Univ. of Lübeck (Germany)
  16. Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
  17. Arizona State Univ., Tempe, AZ (United States)
  18. Max-Planck-Inst. for Medical Research, Heidelberg (Germany)
  19. Univ. of Gothenburg (Sweden)
  20. Arizona State Univ., Tempe, AZ (United States); Beijing Computational Science Research Center (China)
  21. SLAC National Accelerator Lab., Menlo Park, CA (United States). Linac Coherent Light Source (LCLS); Arizona State Univ., Tempe, AZ (United States)
  22. SLAC National Accelerator Lab., Menlo Park, CA (United States). Linac Coherent Light Source (LCLS); Brookhaven National Lab. (BNL), Upton, NY (United States)
  23. Arizona State Univ., Tempe, AZ (United States); La Trobe Univ., VIC (Australia)
  24. Deutsches Elektronen-Synchrotron (DESY), Hamburg (Germany); The Hamburg Centre for Ultrafast Imaging (CUI), Hamburg (Germany); Univ. of Hamburg (Germany)
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL), Upton, NY (United States). National Synchrotron Light Source II (NSLS-II); Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); SLAC National Accelerator Laboratory, Menlo Park, CA (United States). Linac Coherent Light Source (LCLS)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES); German Research Foundation (DFG); Helmholtz Excellence Network; National Science Foundation (NSF); German Federal Ministry for Education and Research (BMBF); Joachim-Herz-Stiftung Hamburg; UCOP Lab Fee Program; USDOE Laboratory Directed Research and Development (LDRD) Program; Swedish Research Council (VR); Knut and Alice Wallenberg Foundation
OSTI Identifier:
1603290
Alternate Identifier(s):
OSTI ID: 1605174
Report Number(s):
BNL-213686-2020-JAAM
Journal ID: ISSN 2041-1723
Grant/Contract Number:  
SC0012704; MCB-1021557; 01KX0806; 01KX0807; 05K16GUA; 05K18FLA; AC52-07NA27344; 118036; AC02-76SF00515
Resource Type:
Accepted Manuscript
Journal Name:
Nature Communications
Additional Journal Information:
Journal Volume: 11; Journal Issue: 1; Journal ID: ISSN 2041-1723
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; Biochemistry; X-ray crystallography

Citation Formats

Nass, Karol, Redecke, Lars, Perbandt, M., Yefanov, O., Klinge, M., Koopmann, R., Stellato, F., Gabdulkhakov, A., Schönherr, R., Rehders, D., Lahey-Rudolph, J. M., Aquila, A., Barty, A., Basu, S., Doak, R. B., Duden, R., Frank, M., Fromme, R., Kassemeyer, S., Katona, G., Kirian, R., Liu, H., Majoul, I., Martin-Garcia, J. M., Messerschmidt, M., Shoeman, R. L., Weierstall, U., Westenhoff, S., White, T. A., Williams, G. J., Yoon, C. H., Zatsepin, N., Fromme, P., Duszenko, M., Chapman, H. N., and Betzel, C. In cellulo crystallization of Trypanosoma brucei IMP dehydrogenase enables the identification of genuine co-factors. United States: N. p., 2020. Web. doi:10.1038/s41467-020-14484-w.
Nass, Karol, Redecke, Lars, Perbandt, M., Yefanov, O., Klinge, M., Koopmann, R., Stellato, F., Gabdulkhakov, A., Schönherr, R., Rehders, D., Lahey-Rudolph, J. M., Aquila, A., Barty, A., Basu, S., Doak, R. B., Duden, R., Frank, M., Fromme, R., Kassemeyer, S., Katona, G., Kirian, R., Liu, H., Majoul, I., Martin-Garcia, J. M., Messerschmidt, M., Shoeman, R. L., Weierstall, U., Westenhoff, S., White, T. A., Williams, G. J., Yoon, C. H., Zatsepin, N., Fromme, P., Duszenko, M., Chapman, H. N., & Betzel, C. In cellulo crystallization of Trypanosoma brucei IMP dehydrogenase enables the identification of genuine co-factors. United States. doi:https://doi.org/10.1038/s41467-020-14484-w
Nass, Karol, Redecke, Lars, Perbandt, M., Yefanov, O., Klinge, M., Koopmann, R., Stellato, F., Gabdulkhakov, A., Schönherr, R., Rehders, D., Lahey-Rudolph, J. M., Aquila, A., Barty, A., Basu, S., Doak, R. B., Duden, R., Frank, M., Fromme, R., Kassemeyer, S., Katona, G., Kirian, R., Liu, H., Majoul, I., Martin-Garcia, J. M., Messerschmidt, M., Shoeman, R. L., Weierstall, U., Westenhoff, S., White, T. A., Williams, G. J., Yoon, C. H., Zatsepin, N., Fromme, P., Duszenko, M., Chapman, H. N., and Betzel, C. Thu . "In cellulo crystallization of Trypanosoma brucei IMP dehydrogenase enables the identification of genuine co-factors". United States. doi:https://doi.org/10.1038/s41467-020-14484-w. https://www.osti.gov/servlets/purl/1603290.
@article{osti_1603290,
title = {In cellulo crystallization of Trypanosoma brucei IMP dehydrogenase enables the identification of genuine co-factors},
author = {Nass, Karol and Redecke, Lars and Perbandt, M. and Yefanov, O. and Klinge, M. and Koopmann, R. and Stellato, F. and Gabdulkhakov, A. and Schönherr, R. and Rehders, D. and Lahey-Rudolph, J. M. and Aquila, A. and Barty, A. and Basu, S. and Doak, R. B. and Duden, R. and Frank, M. and Fromme, R. and Kassemeyer, S. and Katona, G. and Kirian, R. and Liu, H. and Majoul, I. and Martin-Garcia, J. M. and Messerschmidt, M. and Shoeman, R. L. and Weierstall, U. and Westenhoff, S. and White, T. A. and Williams, G. J. and Yoon, C. H. and Zatsepin, N. and Fromme, P. and Duszenko, M. and Chapman, H. N. and Betzel, C.},
abstractNote = {Sleeping sickness is a fatal disease caused by the protozoan parasite Trypanosoma brucei (Tb). Inosine-5’-monophosphate dehydrogenase (IMPDH) has been proposed as a potential drug target, since it maintains the balance between guanylate deoxynucleotide and ribonucleotide levels that is pivotal for the parasite. Here we report the structure of TbIMPDH at room temperature utilizing free-electron laser radiation on crystals grown in living insect cells. The 2.80 Å resolution structure reveals the presence of ATP and GMP at the canonical sites of the Bateman domains, the latter in a so far unknown coordination mode. Consistent with previously reported IMPDH complexes harboring guanosine nucleotides at the second canonical site, TbIMPDH forms a compact oligomer structure, supporting a nucleotide-controlled conformational switch that allosterically modulates the catalytic activity. The oligomeric TbIMPDH structure we present here reveals the potential of in cellulo crystallization to identify genuine allosteric co-factors from a natural reservoir of specific compounds.},
doi = {10.1038/s41467-020-14484-w},
journal = {Nature Communications},
number = 1,
volume = 11,
place = {United States},
year = {2020},
month = {1}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Save / Share:

Works referenced in this record:

Serial femtosecond crystallography: A revolution in structural biology
journal, July 2016

  • Martin-Garcia, Jose M.; Conrad, Chelsie E.; Coe, Jesse
  • Archives of Biochemistry and Biophysics, Vol. 602
  • DOI: 10.1016/j.abb.2016.03.036

Serial femtosecond crystallography: the first five years
journal, February 2015


Serial crystallography at synchrotrons and X-ray lasers
journal, February 2017


Femtosecond X-ray protein nanocrystallography
journal, February 2011

  • Chapman, Henry N.; Fromme, Petra; Barty, Anton
  • Nature, Vol. 470, Issue 7332, p. 73-77
  • DOI: 10.1038/nature09750

Indications of radiation damage in ferredoxin microcrystals using high-intensity X-FEL beams
journal, February 2015

  • Nass, Karol; Foucar, Lutz; Barends, Thomas R. M.
  • Journal of Synchrotron Radiation, Vol. 22, Issue 2
  • DOI: 10.1107/S1600577515002349

Radiation damage in protein crystallography at X-ray free-electron lasers
journal, January 2019


Protein crystallization in living cells
journal, June 2018

  • Schönherr, Robert; Rudolph, Janine Mia; Redecke, Lars
  • Biological Chemistry, Vol. 399, Issue 7
  • DOI: 10.1515/hsz-2018-0158

Protein crystallization in vivo
journal, April 2006


The molecular organization of cypovirus polyhedra
journal, March 2007

  • Coulibaly, Fasséli; Chiu, Elaine; Ikeda, Keiko
  • Nature, Vol. 446, Issue 7131
  • DOI: 10.1038/nature05628

In vivo protein crystallization in combination with highly brilliant radiation sources offers novel opportunities for the structural analysis of post-translationally modified eukaryotic proteins
journal, July 2015

  • Duszenko, Michael; Redecke, Lars; Mudogo, Celestin Nzanzu
  • Acta Crystallographica Section F Structural Biology Communications, Vol. 71, Issue 8
  • DOI: 10.1107/S2053230X15011450

Natively Inhibited Trypanosoma brucei Cathepsin B Structure Determined by Using an X-ray Laser
journal, November 2012


A Diffraction-Quality Protein Crystal Processed as an Autophagic Cargo
journal, April 2015


An in cellulo-derived structure of PAK4 in complex with its inhibitor Inka1
journal, November 2015

  • Baskaran, Yohendran; Ang, Khay C.; Anekal, Praju V.
  • Nature Communications, Vol. 6, Issue 1
  • DOI: 10.1038/ncomms9681

De novo phasing with X-ray laser reveals mosquito larvicide BinAB structure
journal, September 2016

  • Colletier, Jacques-Philippe; Sawaya, Michael R.; Gingery, Mari
  • Nature, Vol. 539, Issue 7627
  • DOI: 10.1038/nature19825

Inosine monophosphate dehydrogenase as a target for antiviral, anticancer, antimicrobial and immunosuppressive therapeutics
journal, January 2010

  • Braun-Sand, Sonja B.; Peetz, Matthew
  • Future Medicinal Chemistry, Vol. 2, Issue 1
  • DOI: 10.4155/fmc.09.147

Inosine 5′-monophosphate dehydrogenase inhibitors as antimicrobial agents: recent progress and future perspectives
journal, August 2015

  • Shah, Chetan P.; Kharkar, Prashant S.
  • Future Medicinal Chemistry, Vol. 7, Issue 11
  • DOI: 10.4155/fmc.15.72

Repurposing existing drugs: identification of irreversible IMPDH inhibitors by high-throughput screening
journal, November 2018

  • Sarwono, Albertus Eka Yudistira; Mitsuhashi, Shinya; Kabir, Mohammad Hazzaz Bin
  • Journal of Enzyme Inhibition and Medicinal Chemistry, Vol. 34, Issue 1
  • DOI: 10.1080/14756366.2018.1540474

Sleeping Sickness in the ‘Omics Era
journal, April 2018

  • Tiberti, Natalia; Sanchez, Jean-Charles
  • PROTEOMICS - Clinical Applications, Vol. 12, Issue 4
  • DOI: 10.1002/prca.201700041

Characterization of the novel Trypanosoma brucei inosine 5′-monophosphate dehydrogenase
journal, February 2013


Human African trypanosomiasis: pharmacological re-engagement with a neglected disease
journal, December 2007

  • Barrett, M. P.; Boykin, D. W.; Brun, R.
  • British Journal of Pharmacology, Vol. 152, Issue 8
  • DOI: 10.1038/sj.bjp.0707354

High-throughput decoding of antitrypanosomal drug efficacy and resistance
journal, January 2012

  • Alsford, Sam; Eckert, Sabine; Baker, Nicola
  • Nature, Vol. 482, Issue 7384
  • DOI: 10.1038/nature10771

One Fold with Many Functions: The Evolutionary Relationships between TIM Barrel Families Based on their Sequences, Structures and Functions
journal, August 2002


The structure of a domain common to archaebacteria and the homocystinuria disease protein
journal, January 1997


A nucleotide-controlled conformational switch modulates the activity of eukaryotic IMP dehydrogenases
journal, June 2017

  • Buey, Rubén M.; Fernández-Justel, David; Marcos-Alcalde, Íñigo
  • Scientific Reports, Vol. 7, Issue 1
  • DOI: 10.1038/s41598-017-02805-x

MgATP Regulates Allostery and Fiber Formation in IMPDHs
journal, June 2013


A Nucleotide-Dependent Conformational Switch Controls the Polymerization of Human IMP Dehydrogenases to Modulate their Catalytic Activity
journal, March 2019

  • Fernández-Justel, David; Núñez, Rafael; Martín-Benito, Jaime
  • Journal of Molecular Biology, Vol. 431, Issue 5
  • DOI: 10.1016/j.jmb.2019.01.020

In vivo protein crystallization opens new routes in structural biology
journal, January 2012

  • Koopmann, Rudolf; Cupelli, Karolina; Redecke, Lars
  • Nature Methods, Vol. 9, Issue 3
  • DOI: 10.1038/nmeth.1859

Real-time investigation of dynamic protein crystallization in living cells
journal, July 2015

  • Schönherr, R.; Klinge, M.; Rudolph, J. M.
  • Structural Dynamics, Vol. 2, Issue 4
  • DOI: 10.1063/1.4921591

Firefly luciferase is targeted to peroxisomes in mammalian cells.
journal, May 1987

  • Keller, G. A.; Gould, S.; Deluca, M.
  • Proceedings of the National Academy of Sciences, Vol. 84, Issue 10
  • DOI: 10.1073/pnas.84.10.3264

Protein transport into peroxisomes: Knowns and unknowns
journal, August 2017

  • Francisco, Tânia; Rodrigues, Tony A.; Dias, Ana F.
  • BioEssays, Vol. 39, Issue 10
  • DOI: 10.1002/bies.201700047

Cheetah : software for high-throughput reduction and analysis of serial femtosecond X-ray diffraction data
journal, May 2014

  • Barty, Anton; Kirian, Richard A.; Maia, Filipe R. N. C.
  • Journal of Applied Crystallography, Vol. 47, Issue 3
  • DOI: 10.1107/S1600576714007626

CrystFEL : a software suite for snapshot serial crystallography
journal, March 2012

  • White, Thomas A.; Kirian, Richard A.; Martin, Andrew V.
  • Journal of Applied Crystallography, Vol. 45, Issue 2
  • DOI: 10.1107/S0021889812002312

XGANDALF – extended gradient descent algorithm for lattice finding
journal, August 2019

  • Gevorkov, Yaroslav; Yefanov, Oleksandr; Barty, Anton
  • Acta Crystallographica Section A Foundations and Advances, Vol. 75, Issue 5
  • DOI: 10.1107/S2053273319010593

Accurate determination of segmented X-ray detector geometry
journal, January 2015

  • Yefanov, Oleksandr; Mariani, Valerio; Gati, Cornelius
  • Optics Express, Vol. 23, Issue 22
  • DOI: 10.1364/OE.23.028459

IMP Dehydrogenase: Structure, Mechanism, and Inhibition
journal, July 2009


Characteristics and Crystal Structure of Bacterial Inosine-5‘-monophosphate Dehydrogenase ,
journal, April 1999

  • Zhang, Rong-guang; Evans, Gwyndaf; Rotella, Frank J.
  • Biochemistry, Vol. 38, Issue 15
  • DOI: 10.1021/bi982858v

De novo GTP Biosynthesis Is Critical for Virulence of the Fungal Pathogen Cryptococcus neoformans
journal, October 2012


Structure of Pseudomonas aeruginosa inosine 5′-monophosphate dehydrogenase
journal, February 2013

  • Rao, Vincenzo A.; Shepherd, Sharon M.; Owen, Richard
  • Acta Crystallographica Section F Structural Biology and Crystallization Communications, Vol. 69, Issue 3
  • DOI: 10.1107/S1744309113002352

Increased riboflavin production by manipulation of inosine 5′-monophosphate dehydrogenase in Ashbya gossypii
journal, July 2015

  • Buey, Rubén M.; Ledesma-Amaro, Rodrigo; Balsera, Mónica
  • Applied Microbiology and Biotechnology, Vol. 99, Issue 22
  • DOI: 10.1007/s00253-015-6710-2

Two Classes of Bacterial IMPDHs according to Their Quaternary Structures and Catalytic Properties
journal, February 2015


Crystallographic studies of two variants of Pseudomonas aeruginosa IMPDH with impaired allosteric regulation
journal, August 2015

  • Labesse, Gilles; Alexandre, Thomas; Gelin, Muriel
  • Acta Crystallographica Section D Biological Crystallography, Vol. 71, Issue 9
  • DOI: 10.1107/S1399004715013115

First-in-class allosteric inhibitors of bacterial IMPDHs
journal, April 2019


A pipeline for structure determination of in vivo -grown crystals using in cellulo diffraction
journal, March 2016

  • Boudes, Marion; Garriga, Damià; Fryga, Andrew
  • Acta Crystallographica Section D Structural Biology, Vol. 72, Issue 4
  • DOI: 10.1107/S2059798316002369

CBS domains: Ligand binding sites and conformational variability
journal, December 2013

  • Ereño-Orbea, June; Oyenarte, Iker; Martínez-Cruz, Luis Alfonso
  • Archives of Biochemistry and Biophysics, Vol. 540, Issue 1-2
  • DOI: 10.1016/j.abb.2013.10.008

Physiological concentrations of purines and pyrimidines
journal, January 1994

  • Traut, Thomas W.
  • Molecular and Cellular Biochemistry, Vol. 140, Issue 1
  • DOI: 10.1007/BF00928361

First lasing and operation of an ångstrom-wavelength free-electron laser
journal, August 2010


Injector for scattering measurements on fully solvated biospecies
journal, March 2012

  • Weierstall, U.; Spence, J. C. H.; Doak, R. B.
  • Review of Scientific Instruments, Vol. 83, Issue 3
  • DOI: 10.1063/1.3693040

An anti-settling sample delivery instrument for serial femtosecond crystallography
journal, July 2012

  • Lomb, Lukas; Steinbrener, Jan; Bari, Sadia
  • Journal of Applied Crystallography, Vol. 45, Issue 4
  • DOI: 10.1107/S0021889812024557

High-Resolution Protein Structure Determination by Serial Femtosecond Crystallography
journal, May 2012


The Coherent X-ray Imaging (CXI) instrument at the Linac Coherent Light Source (LCLS)
journal, March 2010


Phaser.MRage : automated molecular replacement
journal, October 2013

  • Bunkóczi, Gábor; Echols, Nathaniel; McCoy, Airlie J.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 69, Issue 11
  • DOI: 10.1107/S0907444913022750

Coot model-building tools for molecular graphics
journal, November 2004

  • Emsley, Paul; Cowtan, Kevin
  • Acta Crystallographica Section D Biological Crystallography, Vol. 60, Issue 12, p. 2126-2132
  • DOI: 10.1107/S0907444904019158

Towards automated crystallographic structure refinement with phenix.refine
journal, March 2012

  • Afonine, Pavel V.; Grosse-Kunstleve, Ralf W.; Echols, Nathaniel
  • Acta Crystallographica Section D Biological Crystallography, Vol. 68, Issue 4
  • DOI: 10.1107/S0907444912001308

Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions
journal, November 2004

  • Krissinel, E.; Henrick, K.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 60, Issue 12
  • DOI: 10.1107/S0907444904026460

Repurposing existing drugs: identification of irreversible IMPDH inhibitors by high-throughput screening
text, January 2018


    Works referencing / citing this record:

    In cellulo crystallization of Trypanosoma brucei IMP dehydrogenase enables the identification of genuine co-factors
    text, January 2020

    • Nass, Karol; Redecke, Lars; Perbandt, Markus
    • Deutsches Elektronen-Synchrotron, DESY, Hamburg
    • DOI: 10.3204/pubdb-2020-00621