News and Publication Highlights

New paper in PNAS: Photocycle-dependent conformational changes in the proteorhodopsin cross-protomer Asp–His–Trp triad revealed by DNP-enhanced MAS-NMR

Proteorhodopsin (PR) is a highly abundant, pentameric, light-driven proton pump. Proton transfer is linked to a canonical photocycle typical for microbial ion pumps. Although the PR monomer is able to undergo a full photocycle, the question arises whether the pentameric complex formed in the membrane via specific cross-protomer interactions plays a role in its functional mechanism. Here, we use dynamic nuclear polarization (DNP)-enhanced solid-state magic-angle spinning (MAS) NMR in combination with light-induced cryotrapping of photointermediates to address this topic. The highly conserved residue H75 is located at the protomer interface. We show that it switches from the (τ)- to the (π)-tautomer and changes its ring orientation in the M state. It couples to W34 across the oligomerization interface based on specific His/Trp ring orientations while stabilizing the pKa of the primary proton acceptor D97 within the same protomer. We further show that specific W34 mutations have a drastic effect on D97 and proton transfer mediated through H75. The residue H75 defines a cross-protomer Asp–His–Trp triad, which potentially serves as a pH-dependent regulator for proton transfer. Our data represent light-dependent, functionally relevant cross talk between protomers of a microbial rhodopsin homo-oligomer.

Maciejko, J.; Kaur, J.; Backer-Baldus, J.; Glaubitz, C., Photocycle-dependent conformational changes in the proteorhodopsin cross-protomer Asp–His–Trp triad revealed by DNP-enhanced MAS-NMR, PNAS 2019, 201817665,

New paper in Scientific Reports: Global response of diacylglycerol kinase towards substrate binding observed by 2D and 3D MAS NMR

Escherichia coli diacylglycerol kinase (DGK) is an integral membrane protein, which catalyses the ATP-dependent phosphorylation of diacylglycerol (DAG) to phosphatic acid (PA). It is a unique trimeric enzyme, which does not share sequence homology with typical kinases. It exhibits a notable complexity in structure and function despite of its small size. Here, chemical shift assignment of wild-type DGK within lipid bilayers was carried out based on 3D MAS NMR, utilizing manual and automatic analysis protocols. Upon nucleotide binding, extensive chemical shift perturbations could be observed. These data provide evidence for a symmetric DGK trimer with all of its three active sites concurrently occupied. Additionally, we could detect that the nucleotide substrate induces a substantial conformational change, most likely directing DGK into its catalytic active form. Furthermore, functionally relevant interprotomer interactions are identified by DNP-enhanced MAS NMR in combination with site-directed mutagenesis and functional assays.

Möbius, K.;  Kazemi, S.;  Güntert, P.;  Jakob, A.;  Heckel, A.;  Becker-Baldus, J.; Glaubitz, C., Global response of diacylglycerol kinase towards substrate binding observed by 2D and 3D MAS NMR. Scientific Reports 2019, 9 (1), 3995.

New paper in JACS: Unexplored nucleotide binding modes for the ABC exporter MsbA

The ATP-binding cassette (ABC) transporter MsbA is an ATP-driven lipid-A flippase. It belongs to the ABC protein superfamily whose members are characterized by conserved motifs in their nucleotide binding domains (NBDs), which are responsible for ATP hydrolysis. The structural foundations underlying the nucleotide binding to MsbA were explored using a concerted approach based on conventional- and DNP-enhanced solid-state NMR, pulsed-Mn2+-EPR (in collaboration with Thomas Prisner, Frankfurt) and MD simulations (with Claudio Soares, Lisbon). MsbA reconstituted into lipid bilayers was trapped in various catalytic states. Our data provide evidence for an additional nucleotide-binding site in close proximity to the Q-loop and the His-Switch, which is required for the recently discovered reverse adenylate kinase (rAK)-like reaction catalyzed by MsbA in addition to ATP hydrolysis.

Kaur H, Abreu B, Akhmetzyanov D, Lakatos-Karoly A, Soares CM, Prisner T, Glaubitz C (2018) Unexplored nucleotide binding modes for the ABC exporter MsbA. J Am Chem Soc in press, doi: 10.1021/jacs.8b067

18th Internatinal Conference on Retinal Proteins: Ontario, Canada, Sep 24-29, 2018

Presentations from the lab: Orawan Jakdetchai Clemens Glaubitz

40th FGMR Annual Discussion Meeting: Leipzig, Sep 10—13, 2018

Presentations from the lab: Jiafei Mao

XXVIII ICMRBS: Dublin, Aug 19—24, 2018

Presentations from the lab: Clara Kriebel, Jiafei Mao

Night of Science: Frankfurt, June 8th, 2018

Presentations from the lab: Clemens Glaubitz

SPP 1601 Workshop: Schmitten, May 7—9, 2018

Presentations from the lab: Clemens Glaubitz

FEBS 2018: ABC Proteins, Innsbruck, Mar 6—12, 2018

Presentations from the lab: Hundeep Kaur, Clemens Glaubitz

28.2.2018: Congratulations to Dr. Hundeep Kaur on the successful completion of her PhD!

15.1.2018: New Paper in Nature Chemical Biology - Subtype Specificity in Human Peptide GPCRs

GPCRs are the most important signal transducers in higher eukaryotes. Despite considerable progress, the molecular basis of subtype-specific ligand selectivity, especially for peptide receptors, remains unknown. Here, by integrating DNP-enhanced solid-state NMR with advanced molecular modeling and docking, the mechanism of the subtype selectivity of human bradykinin receptors for their peptide agonists has been resolved. The detailed molecular picture obtained by this approach opens a new gateway for exploring the complex conformational and chemical space of peptides and peptide analogs for designing GPCR subtype-selective biochemical tools and drugs.

Paper: Lisa Joedicke*, Jiafei Mao*, Georg Kuenze*, Christoph Reinhart, Tejaswi Kalavacherla,Hendrik R A Jonker, Christian Richter, Harald Schwalbe, Jens Meiler, Julia Preu, Hartmut Michel* & Clemens Glaubitz*: The molecular basis of subtype selectivity of human kinin G-protein-coupled receptors. Nature Chemical Biology, published online 15.1.2018, doi: 10.1038/nchembio.2551

Keystone Symposium: GPCR Structure and Function, Santa Fe, Feb 16—20, 2018

Presentations from the lab: Jiafei Mao

56th Meeting of the NMR Society of Japan, Tokyo Nov 14-16

Presentations from the lab: Clemens Glaubitz

17.10.2017: New Paper in BBA - DNP-enhanced MAS NMR on ABC exporter MsbA

MsbA, a homodimeric ABC exporter, translocates its native substrate lipid A as well as a range of smaller, amphiphilic substrates across the membrane. Magic angle sample spinning (MAS) NMR, in combination with dynamic nuclear polarisation (DNP) for signal enhancement, has been used to probe two specific sites in transmembrane helices 4 and 6 of full length MsbA embedded in lipid bilayers. Significant chemical shift changes in both sites were observed in the vanadate-trapped state compared to apo state MsbA. The reduced spectral line width indicates a more confined conformational space upon trapping. In the presence of substrates Hoechst 33342 and daunorubicin, further chemical shift changes and line shape alterations mainly in TM6 in the vanadate trapped state were detected. These data illustrate the conformational response of MsbA towards the presence of drugs during the catalytic cycle.

Paper: Spadaccini et al. BBA 2017, epub ahead of print, DOI: 10.1016/j.bbamem.2017.10.017

13.10.2017: New Paper in JACS - First NMR Data on PR Photointermediates (JACS Spotlight)

Proteorhodopsin (PR) is the most abundant retinal protein on earth and functions as a light-driven proton pump. Despite of extensive efforts, structural data for PR photointermediate states have not been obtained. Based on DNP-enhanced solid-state NMR, we were able to analyze the retinal polyene chain between positions C10 and C15 as well as the Schiff base nitrogen in the ground state in comparison to light induced, cryo-trapped K- and M-states. A high M-state population could be achieved by preventing reprotonation of the Schiff base through a mutation of the primary proton donor (E108Q). Our data reveal unexpected large and alternating 13C chemical shift changes in the K-state propagating away from the Schiff base along the polyene chain. Furthermore, two different M-states have been observed reflecting the Schiff base reorientation after the de-protonation step. Our study provides novel insight into the photocycle of PR and also demonstrates the power of DNP-enhanced solid-state NMR to bridge the gap between functional and structural data and models.

Paper: Mehler et al. JACS 2017, epub ahead of print, DOI: 10.1021/jacs.7b05061

Photoreceptors - from Actitivation to Interaction, Ringberg Oct 8-11

Presentations from the lab: Jiafei Mao, Clemens Glaubitz

New Horizons in Membrane Transport and Communication, Frankfurt Oct 4-6

Presentations from the lab: Clemens Glaubitz, Johanna Becker-Baldus, Kristin Möbius, Julian de Mos, Clara Kriebel, Hundeep Kaur, Orawan Jakdetchai, Jakob Maciejko

Alpine Solid-state NMR, Chamonix Sep 10-14

Presentations from the lab:Johanna Becker-Baldus, Jiafei Mao

Biology meets NMR, German-Indian Meeting, Quebec July 23-28

Speaker from the lab: Clemens Glaubitz

Design and Light Control 2017, Aschaffenburg Aug 30 - Sep 1

Speaker from the lab: Clemens Glaubitz

ISMAR Conference 2017, Quebec July 23-28

Speakers from the lab: Kristin Möbius, Clemens Glaubitz

GRC "Mechanisms on Membrane Transport", New London, June 25-30

Presenter from the lab: Hundeep Kaur

27.2.2017: Congratulations to Dr. Michaela Mehler on the successful completion of her PhD!

23.12.2016: New paper in Nature Communications!

Coupled ATPase-adenylate kinase activity in ABC transporters:ATP-binding cassette (ABC) transporters form a superfamily of integral membrane proteins found in all kingdoms of life, which translocate essential substrates across cellular membranes. Many of these proteins are responsible for multidrug resistance and genetic disorders in humans. These diseases can be a result of either a mutation resulting in dysfunctional protein or overexpression under certain conditions. The generally accepted paradigm links the transport process carried out by specialised transmembrane domains with ATP hydrolysis as the power stroke catalysed by highly conserved, intracellular nucleotide binding domains. In the current study Kaur et al. demonstrate by solid-state NMR combined with biochemical data that ATP hydrolysis in MsbA, a typical ABC exporter involved in lipid A translocation, is intrinsically linked with an adenylate kinase mechanism by which some of the consumed ATPs get recycled. Both mechanisms are associated with the same conserved motifs of the nucleotide-binding domains. The same observations were made for two additional ABC exporters from other organisms suggesting that the coupled mechanism is a general feature at least for members of this protein superfamily subclass. Such a coupled and cyclic catalytic activity adds another layer of complexity to the general molecular mechanism of ABC transporters. However, the presented data also indicate that these proteins might be able to adjust their catalytic capabilities to altered cellular situations: Under conditions of ATP excess, ATP hydrolysis alone dominates the catalytic cycle, while in cases of cellular stress associated with ATP depletion events, a coupling with an adenylate kinase mechanism is advantageous in order to maintain the protein’s transport activity, which is essential for cellular survival.

Hundeep Kaur, Andrea Lakatos-Karoly, Ramona Vogel, Anne Nöll, Robert Tampé and Clemens Glaubitz (2016). Coupled ATPase-adenylate kinase activity in ABC transporters. Nature Communications, published online 22 December 2016, DOI 10.1038/NCOMMS13864. Link

2016 Meetings with Contributions from the Lab

  • International Ringberg Discussion Meeting 'Connecting EPR, ssNMR and DNP for the study of complex biomolecules'

    • Speakers from the lab: Hundeep Kaur, Jiafei Mao
  • Bruker NMR Meeting, November 2016

    • Speaker from the lab: Clemens Glaubitz
  • EMBO Conference Retinal Proteins, Potsdam, October 2016

    • Speaker from the lab: Clemens Glaubitz

  • ICMRBS, Kyoto, August 2016

    • Speaker from the lab: Clemens Glaubitz

  • IUPAB School Receptors and Signalling, Greece, May 2016

    • Lecturer from the lab: Clemens Glaubitz

  • ENC, Pittsburgh, April 2016

    • Speaker from the lab: Clemens Glaubitz

  • Protein Engineering Summit, Boston, April 2016

    • Speaker from the lab: Michaela Mehler

  • ABC Transporters 2016, Innsbruck, April 2016

    • Presenters from the lab: Hundeep Kaur, Andrea Lakatos, Clemens Glaubitz

    GRC Ligand Recognition and Molecular Gating, Il Ciocco, February 2016

    • Speaker form the lab: Jiafei Mao

Recent Lab Highlights

  • October 25th 2015: Glaubitz Lab at Frankfurt Marathon

    • We joint the competition succesfully with two teams (Ramona, Christian, Julian, Jakob M & Jakob L., Hundeep, Jagdeep, Clemens)!

"Old News"

  • Current Developments and Future Challanges in Photoreceptor Research, Fraunchiemsee, 9.-12..10.2015

    • Speaker from the lab: Clemens Glaubitz

  • 9th Alpine Conference on solid-state NMR, Chamonix, 13.-17.9.2015

    • Speaker from the lab: Johanna Becker-Baldus

  • FGMR GDCH 37th Annual Meeting, Darmstadt, 7.-10.9.2015

    • Speaker from the lab: Clemens Glaubitz

  • 5th International DNP Symposium, Egmond aan Zee, 31.8.-4.9.2015

    • Speaker from the lab: Jiafei Mao, Clemens Glaubitz

  • Biomedical Transporters 2015, Lugano, 9.8.-13.8.2015

    • Speaker from the lab: Hundeep Kaur

  • ISMAR 2015, Shanghai, 16.-21.8.2015

    • Speaker from the lab: Jiafei Mao

  • 15th CCPN Meeting, Buxton, 20.-22.7.2015

    • Presenter from the lab: Kristin Möbius, Jagdeep Kaur

  • Gordon Research Conference on Membrane Transport, Lewiston, 28.6.-3.7.2015

    • Speaker and presenter from the lab: Clemens Glaubitz, Hundeep Kaur