A05

A05 Project movie

A05

In project A05 a novel binding site for calmodulin located in the C-terminal NUDT9H domain of TRPM2 was identified. Replacement of the anchor residues of the CaM binding motif abrogated binding of CaM and largely reduced the temperature-induced increase in current in response to ADPR, suggesting a role of this CaM-binding site in the temperature sensitivity of TRPM2. Further, project A05 focused on the analysis of the ligand binding domains of TRPM2. During the 1st funding period, the first cryo-EM structure of TRPM2 from zebra fish was determined and a second binding site for ADPR in the N-terminal MHR1/MHR2 domain was discovered by Dr. Du’s group. The situation appears to be complex since in a somewhat later published structure of human TRPM2, ADPR was not detected in the novel binding site. However, project A05 recently expressed, purified and crystallized the MHR1/2 domain of TRPM2 from zebra fish and confirmed binding of ADPR and 2dADPR. Further, a novel high-affinity binding site for a Zn2+ ion in a region that has not been resolved in previous cryo-EM structures and is conserved among the TRPM subfamily of TRP channels was discovered. In vitro mutant studies and whole-cell patch clamp experiments suggest a stabilizing role of this Zn2+-binding domain with functional relevance. Based on previous work, project A05 also developed specific antagonists for the activation of TRPM2 by 2dADPR and ADPR. Of note, agonist-specific antagonism was discovered: whereas monodeoxy analogs of the terminal ribose of ADPR decreased ADPR evoked currents, dideoxy analogs located at both ribose moieties diminished TRPM2 currents evoked by 2dADPR. Finally, project A05 also showed that ADPR and 2dADPR are both degraded by the nucleotide pyrophosphatases NUDT9 or NUDT5. Of note, small angle X-ray scattering and NMR spectroscopy revealed that binding of the nucleotide substrates induced a substantial conformational change that may be used to engineer a FRET sensor for ADPR and 2dADPR using an enzymatically inactive variant of NUDT9.

Dr. Ralf Fliegert

Selected publications (*equal contribution)

 

  1. Fliegert R, Riekehr WM, Guse AH (2020) Does Cyclic ADP-Ribose (cADPR) Activate the Non-selective Cation Channel TRPM2?. Front Immunol 11:2018
  2. Baszczyňski O, Watt JM, Rozewitz MD, Guse AH, Fliegert R, Potter BVL (2019) Synthesis of Terminal Ribose Analogues of Adenosine 5'-Diphosphate Ribose as Probes for the Transient Receptor Potential Cation Channel TRPM2. J Org Chem  84:6143-6157
  3. Fliegert R*, Heeren J*, Koch-Nolte F*, Nikolaev VO*, Lohr C*, Meier C*, Guse AH* (2019) Adenine nucleotides as paracrine mediators and intracellular second messengers in immunity and inflammation. Biochem Soc Trans 47(1):329-337.
  4. Gattkowski E, Johnsen A, Bauche A, Möckl F, Kulow F, Garcia Alai M, Rutherford TJ, Fliegert R*, Tidow H* (2019) Novel CaM-binding motif in its NudT9H domain contributes to temperature sensitivity of TRPM2. Biochim Biophys Acta Mol Cell Res 66(7):1162-1170.
  5. Fliegert R, Bauche A, Wolf Pérez AM, Watt JM, Rozewitz MD, Winzer R, Janus M, Gu F, Rosche A, Harneit A, Flato M, Moreau C, Kirchberger T, Wolters V, Potter BVL, Guse AH (2017) 2'-Deoxyadenosine 5'-diphosphoribose is an endogenous TRPM2 superagonist. Nat Chem Biol 13:10361044.
  6. Fliegert R, Watt JM, Schöbel A, Rozewitz MD, Moreau C, Kirchberger T, Thomas MP, Sick W, Araujo AC, Harneit A, Potter BVL, Guse AH (2017) Ligand-induced activation of human TRPM2 requires the terminal ribose of ADPR and involves Arg1433 and Tyr1349. Biochem J 474:2159-2175.
  7. Guse AH, Ernst IM, Fliegert R (2013) NAADP signaling revisited. Curr Top Med Chem 13:29782990.
  8. Schwarz N*, Fliegert R*, Adriouch S*, Seman M, Guse AH, Haag F, Koch-Nolte F (2009) Activation of the P2X7 ion channel by soluble and covalently bound ligands. Purinergic Signal 5:139-149.
  9. Fliegert R, Gasser A, Guse AH (2007) Regulation of calcium signalling by adenine-based second messengers. Biochem Soc Trans 35:109-114.
  10. Fliegert R, Glassmeier G, Schmid F, Cornils K, Genisyuerek S, Harneit A, Schwarz JR, Guse AH (2007) Modulation of Ca2+ entry and plasma membrane potential by human TRPM4b. FEBS J 274:704713.

Prof. Dr. Henning Tidow

Selected publications

 

  1. Gattkowski E, Johnsen A, Bauche A, Möckl F, Kulow F, Garcia Alai M, Rutherford TJ, Fliegert R, Tidow H (2019) Novel CaM-binding motif in its NudT9H domain contributes to temperature sensitivity of TRPM2. Biochim Biophys Acta Mol Cell Res 1866:1162-1170
  2. Veith K, Molledo MM, Hernandez YA, Josts I, Nitsche J, Löw C, Tidow H (2017) Lipid-like peptides can stabilize integral membrane proteins for biophysical and structural studies. Chembiochem 18:17351742.
  3. Josts I, Almeida Hernandez Y, Andreeva A, Tidow H (2016) Crystal Structure of a Group I Energy Coupling Factor Vitamin Transporter S Component in Complex with Its Cognate Substrate. Cell Chem Biol 23:827-836.
  4. Tidow H, Nissen P (2013) Structural diversity of CaM-binding to its target sites. FEBS J 280:55515565.
  5. Tidow H, Poulsen LR, Andreeva A, Knudsen M, Hein KL, Wiuf C, Palmgren MB, Nissen P (2012) A bimodular mechanism of calcium control in eukaryotes. Nature 491:468-472.
  6. Tidow H, Andreeva, A, Rutherford, TJ, Fersht, AR (2009) Solution structure of U11-48K CHHC zinc finger domain that specifically binds the 5’ splice site of U12-type introns. Structure 17:294-302.
  7. Andreeva A, Tidow H (2008) A novel CHHC Zn-finger domain found in spliceosomal proteins and tRNA modifying enzymes. Bioinformatics 24:2277-2280.
  8. Wells M, Tidow H, Rutherford TJ, Markwick P, Jensen MR, Mylonas E, Svergun DI, Blackledge M, Fersht AR (2008) Structure of tumor suppressor p53 and its intrinsically disordered N-terminal transactivation domain. Proc Natl Acad Sci USA 105:5762-5767.
  9. Tidow H, Melero R, Mylonas E, Freund SM, Grossmann, JG, Carazo KM., Svergun DI, Valle M, Fersht AR (2007) Quaternary structures of tumor suppressor p53 and a specific p53 DNA complex. Proc Natl Acad Sci USA 104: 12324-12329.
  10. Tidow H, Andreeva A., Rutherford TJ, Fersht AR (2007) Solution Structure of ASPP2 N-terminal Domain (N-ASPP2) Reveals a Ubiquitin-like Fold. J Mol Biol 371:948-958.

Our Team

Prof. Dr. Henning Tidow

Dept. of Chemistry & The Hamburg Centre for Ultrafast Imaging (CUI)
 
University of Hamburg

Dr. Ralf Fliegert

Department of Biochemistry and Molecular Cell Biology

University Medical Centre Hamburg-Eppendorf (UKE)

M.Sc. Winnie Riekehr

Department of Biochemistry and Molecular Cell Biology

University Medical Centre Hamburg-Eppendorf (UKE)

M.Sc. Simon Sander

Dept. of Chemistry & The Hamburg Centre for Ultrafast Imaging (CUI)
 
University of Hamburg

Jelena Pick

Department of Biochemistry and Molecular Cell Biology

University Medical Center Hamburg-Eppendorf (UKE)

Alumni

Dr. Ellen Gattkowski

Dept. of Chemistry & The Hamburg Centre for Ultrafast Imaging (CUI)
 
University of Hamburg

Contact

University Medical Center Hamburg-Eppendorf 
Department of Biochemistry and Molecular Cell Biology 
Martinistrasse 52
20246 Hamburg

Scientific Coordinator

Dr. Björn-Philipp Diercks
Fon: +49  (0) 40 7410 54338
E-Mail: b.diercks©uke.de

Administration

Laura Mitsching
Fon: +49  (0) 40 7410 50301
E-Mail: l.mitsching©uke.de