Research Projects: Multidrug Transporter, Proteorhodopsin, GPCRs, solid-state NMR

Understanding Multidrug Transport Proteins - Solving the Riddle of Multidrug Resistance

Multidrug resistance is an important problem in cancer chemotherapy and in the treatment of infectious diseases. The most distinct mechanism for multidrug resistance is based on secondary and primary active transport proteins which extrude a wide range of antibiotics out of the cell.

The mechanism by which they recognize and transport drugs remains to be resolved. We use solid-state NMR in combination with other spectroscopic and biochemical methods to resolve the structure-function relationship of these proteins directly within the lipid bilayer. We study both ABC transporters (LmrA) and secondary drug-proton antiporters (EmrE, TBsmr, Hsmr). We are interested in key events and structural changes during the transport cycle, in characterising the properties of the drug binding pockets, and investigate the role of lipids and oligomerisation for protein activity.

The key problem of multidrug efflux:

The unusual broad substrate specificity of multidrug efflux pumps and biochemical hints for membrane embedded binding site(s) of these transporters raise the question, whether the membrane could act as a drug selectivity filter. Therefore, an extensive study on the interaction between typical multidrug transporter substrates with model membranes has been carried out. The location profile of these molecules across the membrane was determined by ¹H MAS NMR. We have developed data analysis tools which allow extensive, semi-automatic drug-membrane screens. Although structurally rather diverse, all tested substances are found to have their highest concentration between the phosphate of the lipid headgroup and the upper segments of the lipid hydrocarbon chains (Siarheyeva et al., 2006).

Transport activity and transport cycle of EmrE and other small multidrug resistance proteins:

EmrE and other proteins from the SMR family require pmf to drive substrate transport across the membrane. In order to investigate their transport activity in vitro, we have developed a fluorescence based assay in which a pH gradient is generated through co-reconstitution with the light-driven proton pump bacteriorhodopsin. Sample illumination activates bR and excites ethidium bromide fluorescence. The experiment can be carried out in a standard fluorescence spectrometer. 

The transport cycle must involve various conformational states of the protein needed for substrate binding, translocation and release. A fluorescent substrate will therefore experience a significant change of environment while being transported, which influences its fluorescence properties. Thus the substrate itself can report intermediate states that form during the transport cycle. We have shown the existence of such a substrate-transporter complex for both EmrE and TBsmr (Basting et al. 2007).

Solid-State NMR reveals the formation of an asymmetric EmrE dimer:

Using cell free expression in combination with ¹³C double quantum filtering methods, we were able to selectively observe highly conserved and essential residue Glu-14 in EmrE, functionally reconstituted in E.coli lipids. For E14, two distinct sets of chemical shifts were observed which indicates structural asymmetry in the binding pocket of homodimeric EmrE. Upon addition of ethidium bromide, chemical shift changed and altered line shapes were observed, demonstrating substrate coordination by both E14s in the dimer. Our data show directly that during the exchange reaction E14 of each protomer is involved in substrate binding and coordination (Lehner et al. 2007).

ABC Transporter LmrA:

LmrA, a 590-amino acid ABC-multidrug-transporter native to L. lactis, is a bacterial homologue of the human P-glycoprotein (Pgp). This primary efflux pump is active as a homodimer. Each of its monomers comprises of a six helical transmembrane domain (TMD) and one nucleotide binding domain (NBD), following the general architecture of ABC-transporters. we have for the first time applied solid-state NMR to full length LmrA in order to probe ist dynamic during the catalytic cycle (Siarheyeva et al. 2007).

Recent papers:

• Lehner, I., Basting, D.,  Meyer, B., Haase, W., Manolikas, T., Kaiser, C., Karas, M., Glaubitz, C. (2008) The key residue for substrate transport in the EmrE dimer is asymmetric. J. Biol. Chem. 283, 3281-3288. doi:10.1074/jbc.M707899200

• Basting, D., Lorch, M., Lehner, I., Glaubitz, C. (2008) Transport Cycle Intermediate in Small Multidrug Resistance Protein is revealed by Substrate Fluorescence. FASEB J 22, 365-373, doi:10.1096/fj.07-9162com

• Siarheyeva, A., Lopez, J.J., Lehner, I., Hellmich, U.A., van Veen, H., Glaubitz, C. (2007) Probing the Molecular Dynamics of the ABC Multidrug Transporter LmrA by Deuterium Solid-State NMR. Biochemistry 46, 3075-83., doi:10.1021/bi062109a

• Siarheyeva, A., Lopez, J.J., Glaubitz, C. (2006) Localization of multidrug transporter substrates within model membranes. Biochemistry 45, 6203-11., doi:10.1021/bi0524870

• Basting, D., Lehner, I., Lorch, M., and Glaubitz, C. (2006) Investigating transport proteins by solid state NMR. Naunyn-Schmiedberg's Arch Pharmacol 372, 451-464., doi:10.1007/s00210-006-0039-4

• Mason, A.J., Siarheyeva, A., Haase, W., Lorch, M., van Veen, H. & Glaubitz, C. (2004) Amino acid type selective isotope labelling of the multidrug ABC transporter LmrA for solid-state NMR studies. FEBS Lett 568, 117-121., doi.10.1016/j.febslet.2004.05.016

Proteorhodopsin: Direct Photosynthesis in the Oceans

Proteorhodopsin (PR) is one of a host of newly discovered retinal proteins found to high abundance in marine bacteria. It could be of importance for the energy balance within the ocean’s biosphere considering its high abundance and function as a light driven proton pump. However, the vectoriality of the PR's proton pumping appears to be dependent on pH with a pKa close to the pH of sea water. This raises questions about its natural function. Solid-state NMR in combination with different isotope labeling schemes and site-directed mutagenesis is used to elucidate the 3D structure and the role of different highly conserved residues which serve as proton donors, acceptors or which might have regulatory functions. Furthermore, the mechanism of color tuning by which PR adapts to different environments is investigated.

We have been able to prepare 2D crystals of green PR for NMR and microscopic analysis (Shastri et al. 2007). Surprisingly, crystal formation is found under a wide range of conditions which indicates, that PR might be densely packed in its native environment. AFM has revealed that PR assembles into  a 'donut-shaped' haxameric complex (Klyszejnko et al. 2007). This is in contrast to the well known bR trimer. Using ¹³C and ¹⁵N MAS NMR, we have been able to characterise retinal and Schiff base in green PR (Pfleger et al. 2007). In contrast to bR, retinal is found in mainly all-trans configuration in the dark adapted state. The optical absorption maximum together with retinal and Schiff base chemical shifts indicate a strong interaction network between chromophore and opsin.

Figure: Double quantum filtered ¹³C MAS spectra of PR and bR (left) regenerated with 10,11-¹³C₂ all-trans retinal. Dark adapted PR shows two well resolved resonances for C₁₀ and C₁₁. In contrast, dark adapted bR shows four resonances which correspond to all-trans-C₁₁, 13-cis-C₁₁, all-trans-C₁₀ and 13-cis-C₁₀, respectively. PR forms a 'donut-shpaed' hexamer (right) and has the 7TMH topology of a typical retinal protein.

Recent papers:

• Klyszejnko, A.L., Shastri, S., Mari, S.A., Grubmüller, H., Müller, D.J., Glaubitz, C. (2008) Folding and Assembly of Proteorhodopsin. JMB 376, 35-41. doi: 10.1016/j.jmb.2007.11.030

• Pfleger, N., Lorch, M., Wörner, A., Shastri, S., Glaubitz, C. (2008) Characterisation of Schiff base and Chromophore in Green Proteorhodopsin by Solid-State NMR. J. Biomol. NMR 40, 15-21. doi: 101007/s10858-007-9203-5

• Shastri, S., Vonck, J., Haase, W., Pfleger, N, Kuehlbrandt, W., Glaubitz, C. (2007) Proteorhodopsin: Characterisation of 2D Crystals by Electron Microscopy and Solid-State NMR. BBA Biomem. 1768, 3012-3019. doi:10.1096/10.1016/j.bbamem.2007.10.001

G-Protein coupled Receptors: Understanding Structure and Function

G protein-coupled receptors (GPCRs) are responsible for a large number of physiological processes, such as sensory transduction, mediation of hormonal activity and cell to cell communication. GPCRs are membrane proteins with seven transmembrane helices and are the target of some 50% of today’s modern drugs.  So far, two high resolution GPCR structures (rhodopsin, beta adrenergic receptor) are available. Pharmacological research and rational drug design aimed at GPCRs can be based on homology models derived from these structures but additional data are needed. This limitation could be potentially overcome by determining the structures of bound agonists, which activate GPCRs, and using these as structural templates for drug-design. Solid-state NMR is ideally suited to obtain such structural constraints.

Figure: In a recent study, we have determined the backbone structure of the neuropeptide bradykinin bound to the human G-protein coupled bradykinin subtype 2 receptor by solid state NMR (Lopez et al. 2007). ¹³C chemical shift based torsion angle constraints were used for structure calculation which revealed an elongated conformation with an alpha-helical turn at the N-terminus and a beta-turn at the C-terminus with an  average backbone RMSD value of 0.5Å.

Furthermore, we are interested in the mechanism by which GPCRs transfer information across the membrane.

Recent paper:

• Lopez, J.J., Shukla, A.K., Reinhart, C., Schwalbe, H., Michel, H., Glaubitz, C. (2007) The structure of bradykinin bound to the human GPCR bradykinin-2 as determined by solid-state NMR. Angew. Chem. Int. Ed.47, 1668-1671. doi. 10.1002/anie.200704282

Solid-State NMR: Improving Methodology

Solid-state NMR spectroscopy is at the heart of the above mentioned projects. Technical and methodological improvements are needed to use this powerful technique to its full potential. We mainly use MAS NMR but also investigate the possibility of utilising uniformly aligned samples (static oriented and MAOSS NMR). The intrinsic signal-to-noise problem is addressed by dynamic nuclear polarisation and FAST-NMR methods which allow to make efficient use of data and instrument time. 

Figure: Bruker 600 MHz WB Avance spectrometer (left), subsection of a ¹³C-¹³C MAS correlation spectrum (middle), example for orientation distribution functions calculated for ordered protein samples (right).  

Recent papers:

• Lopez, J.J., Kaiser, C., Shastri, S., Glaubitz, C. (2008) Double quantum filtering homonuclear MAS NMR correlation spectra: A tool for membrane protein studies. J. Biomol. NMR. in press.
• Kaiser, C., Lopez, J.J., Bermel, W., Glaubitz, C. (2007) Dual Transformation of Homonuclear Solid-State NMR Spectra - an Option to Decrease Measuring Time. BBA Biomem., published electronically ahead of print. doi:10.1016/j.bbamem.2007.09.004

• Lopez, J.J., Mason, A.J., Kaiser, C., Glaubitz, C. (2007) Separated Local Field NMR Experiments on Oriented Samples Rotating at the Magic Angle. J. Biomol. NMR, 37, 97-111. doi:10.1007/s10858-006-9109-7

• Lorch, M., Fahem, S., Kaiser, C., Weber, Mason, A.J., I., Bowie, J., Glaubitz, C. (2005) How to prepare membrane propteins for solid-state NMR - A case study on the alpha-helical membrane protein DGK from E.coli. ChemBioChem, 6, 1693-1700. doi:10.1002/cbic.200500054

geändert am 29. April 2008  E-Mail: WebmasterKobylka@chemie.uni-frankfurt.de