Structural and biophysical studies on bacterial UMP kinase as anti - TB targetPatrick Walter [patrick.walter@pasteur.fr] and Hélène Munier-Lehmann [helene.munier-lehmann@pasteur.fr]

Institut Pasteur Paris, Unit of Chemistry and Biocatalysis, Department of Structural Biology and Chemistry

Bacterial UMP kinases (UMPKs) are of particular interest, as they have no counterpart in eukaryotes, making them potential anti-infectivetargets. UMPKs from G- and G+ bacteria exhibit different regulatory mechanisms: i) only these latter are cooperative with respect to Mg-ATP; ii) even though UTP (negative) and GTP (positive) are common effectors, their binding-sites are distinct in UMPKs from G- bacteria,and probably overlapping in UMPKs from G+ bacteria (no structural data yet). We have focused our attention on the Mycobacteriumtuberculosis enzyme as a representative of G+ UMPKs in order to better characterize the effector binding properties and to identify theUTP-binding site for further inhibitor discovery and drug design.

+UDP Mg-ADP+UMP Mg-ATPUMP kinase

“This project has received funding from the European Union’s Framework Programme forResearch and Innovation Horizon 2020 (2014-2020) under the Marie Skłodowska-Curie GrantAgreement No. 675555, Accelerated Early staGe drug discovery (AEGIS).”

INSIGHT INTO THE BINDING PROPERTIES OF THE NATURAL EFFECTORSKinetics and thermodynamic characterization of enzyme ligand interactions offer valuable clues to affinity parameters (KD values),thermal stability or oligomerization state. The obtained data can be used for potent inhibitor discovery, crystal production andoptimization respectively.

FIRST THERMODYNAMIC CHARACTERIZATIONThermal shift assay (TSA) Differential scanning calorimetry (DSC) and

circular dichroism (CD) for closer informationon the Tm and its structural characteristics.

LIGAND AFFINITY STUDIESSurface Plasmon Resonance (SPR)

Temperature [°C]

Temperature [°C]

Sign

alSi

gnal

UMPK-apo – UMPK-GTP – UMPK-UTP

UMPK-apo – UMPK-Mg-ATP – UMPK-UMP

Increaseofthermalstability.Unexpectedcurveshapes– furtheranalysisinprogress.FurtherSPRandITCmeasurementstospecifyKD values.

UMP

Concentration [M]

Res

pons

e [R

U]

△H

[kca

l/mol

]

Molar Ratio

Mg-ATPIsothermalCalorimetry(ITC)

The resolution of the unknown negative effector binding-site serves as a starting pointfor crystallographic based allosteric inhibitor development (soaking of fragmentlibraries).

INSIGHT INTO THE NEGATIVE EFFECTOR BINDING-SITE

COMPUTATIONAL APPROACH X-RAY APPROACHKinetic studies suggest an overlapping of thetwo effector binding-sites.

G. Labesse et al., Nucleic Acids Res. 39, 2011, 3458

ModellingofthePDBUMPKcrystalstructure(GTPcomplex).

DockingapproacheswithUTPonthewholesurfaceandcavitiesofthemodelledstructure.

Firstcrystalsafterageneralscreeningforcrystallization.

Manualreproductionandcrystaloptimization.

Low resolution datasets require furthercrystal optimization.

New constructs to remove the tag.

Additionalstepsforproteinpurification.

IDENTIFICATION OF THE BACTERIAL UMPK INTERACTOME

Based on the results obtained from electronmicroscopy, we hypothesized that there are as-yet unidentified intracellular PPIs. The searchfor interacting partners using the BacterialAdenylate Cyclase Two-Hybrid system (BACTH)is ongoing.

Proof of BACTH functionality by UMPK(hexameric) self-interaction (blue clones).Negative control (white clones) by co-transformation of a vector-UMPK fusionprotein and the corresponding emptyvector.

Karimove et al., PNAS, 1998, 95, 10

M. tuberculosis genome library screening.

ACKNOWLEDGEMENTSInstitut Pasteur,Paris:-Unit of Biochemistry of Macromolecular Interactions:

D. Ladant, G. Karimova-Unit of Structural Bioinformatics:M. Nilges, A. Blondel, L. Ortega Varga-Platform of Molecular Biophysics:P. England, B. Raynal, B. Baron, S. Brulé-Platform of Crystallography:A. Haouz, P. Weber, C. Pissis

-Computational Chemistry, Chemistry Innovation Centre:H. Boyd, H. Chen, S. Geschwindner, P. Hansson

-Institute of Pharmaceutical Chemistry, AG Klebe:G. Klebe, A. Heine, E. Hassaan, F. Magari

-Center of Structural Biochemistry:G. Labesse, P. Bron

AstraZeneca,Mölndal:

PhilippsUniversity,Marburg:

CNRSMontpellier:

UMPK – apo form

Temperature [°C]

Cp

[kca

l/mol

/°C]

DSCofUMPK-apo form

Dic

hroi

sm [m

deg]

Wavelength [nm]

CDofUMPK-apo form