Research

Our research is focussed on the functional mechanism of complex membrane proteins, their dynamics and regulation by lipids. We are also interested in the way drugs interact with their protein targets for their improvement and an in-depth biochemical understanding of the drug-protein complex. Our lab currently focusses on transporters, ion channels and proteases that we study with biochemical, biophysical and structural techniques.

Membrane Transporters (in Multidrug Resistance)

One fascinating example of the dual nature of membrane proteins as “protectors of the realm” while also harboring the potential for harm is that of the “multidrug resistance transporters”. In their protective function, they remove detrimental substances from the cell. But what happens if you are treating a disease with antibiotics and the bacterium also expresses “multidrug transporters”? From the pathogen’s point of view this is fantastic – the antibiotics cannot harm it! From the patient’s point of view – not so great… Actually, multidrug resistance (MDR) is an increasing health problem in hospitals. A number of bacteria are already resistant to a large number of available antibiotics. Methicilin-resistant Staphylococcus aureus (MRSA), also resistant gainst numerous other antibiotics, is one infamous example.

Mechanisms of bacterial multidrug resistance:  MDR pumps represented by Sav1866 [2HYD], MexB [2V5O] and EmrE [2I68]; porins represented by OmpF [3FYX]. Intra- and extracellular drug modification represented by chloramphenicol acetyltransferase [1PD5], aminoglycoside 3’-phosphotransferase [1ND4] and β-lactamase [2B5R]. Modification of drug target represented by 16S rRNA methylation in E. coli 30S ribosomal subunit [3I1M] (relative size adjusted due to space limitations).

In another scenario imagine a cancer patient undergoing treatment. For years, his/her multidrug transporters have done a splendid job and kept the patient’s cells from harm. However, now, his own mutated cancerous cells use these same multidrug transporters to remove the chemotherapeutics thus making efficient cancer therapy much more difficult, if not futile.

Understanding the molecular details of multidrug transporters (especially their dynamics) is one of our main interests. As it turns out, they can be pretty floppy as we saw when we did pulsed EPR spectrsocopy on an ATP binding cassette (ABC) transporter:

EPR3

Global protein dynamics of a multidrug ABC transporter studied with pulsed EPR spectroscopy               (Hellmich, Lyubenova et al (2012) J. Am. Chem. Soc.)

 

 

 

 

Relevant Publications:

Szöllösi, D., Rose-Sperling, D, Hellmich, U. A., Stockner, T. (2017) Comparison of mechanistic transport cycle models of ABC exporters. BBA Biomembr. accepted.doi: 10.1016/j.bbamem.2017.10.028.

Neumann, J., Rose-Sperling, D. and Hellmich, U. A. (2017) Diverse relations between lipids and ABC transporters: an overview. BBA Biomembr. 1859(4):605-618.

Hellmich, U.A., Mönkemeyer L, Velamakanni S, van Veen HW, Glaubitz C. (2015) Effects of nucleotide binding to LmrA: A combined MAS-NMR and solution NMR study. BBA Biomembr. 1848(12):3158-65.

Mörs, K., Hellmich, U.A., Basting, D., Marchand, P., Wurm, J.P., Haase, W. and Glaubitz, C. (2013) A lipid-dependent link between activity and oligomerization state of the M. tuberculosis SMR protein Tbsmr. BBA Biomembr. 1828(2):561-7.

Hellmich*, U.A., Lyubenova*, S., Kaltenborn, E., Doshi, R., van Veen, H. W., Prisner, T.F. and Glaubitz, C. (2012) Probing the ATP hydrolysis cycle of the ABC multidrug transporter LmrA by pulsed EPR Spectroscopy. J. Am. Chem. Soc., 134(13):5857-62.(*contributed equally)

Hellmich, U.A., Duchardt-Ferner, E., Glaubitz, C. and Wöhnert, J. (2012) Backbone NMR resonance assignments of the nucleotide binding domain of the ABC multidrug transporter LmrA from Lactococcus lactis in its ADP-bound state. Biomol. NMR Assign. 6(1):69-73.

Zutz, A., Hoffman, J., Hellmich, U.A., Glaubitz, C., Ludwig, B., Brutschy, B. and Tampé, R. (2011) Asymmetric ATP hydrolysis cycle of the heterodimeric multidrug ABC complex TmrAB from Thermus thermophiles. J. Biol. Chem. 286(9):7104-15

Hellmich, U.A., Haase, W., Velamakanni, S., van Veen, H. W. and Glaubitz, C. (2008) Caught in the Act: ATP hydrolysis of an ABC multidrug transporter by real-time MAS NMR. Febs Lett. 23(23-24), 3557-3562

Siarheyeva, A., Lopez, J. J., Lehner, I., Hellmich, U.A., van Veen, H. W. and Glaubitz, C. (2007) Probing the Molecular Dynamics of the ABC Multidrug Transporter LmrA by Deuterium Solid-State Nuclear Magnetic Resonance. Biochemistry 46(11); 3075-3083

 

Ion channels

Why study ion channels?

Ion channels are responsible that you are able to read this: they make our eyes see, our hearts beat, our neurons communicate, our brains function.

Yes, they "simply" open up and allow ions to pass across the membrane, but the when and how is highly regulated. There would be mayhem in your body if your ion channels decided to malfunction! Numerous diseases (“channelopathies”) are proof of this: epilepsy, cystic fibrosis, seizures… (to name only a few) are all caused by dysfunctional ion channels.

Ion channel structures - they look much more complex than just a "hole in the membrane".  (Examples shown are TRPV1 [3J5P], Kv1.2/2.1 [2R9R], GIRK [3SYA] and the cytosolic domains of a BK channel [3NAF] (figure from Hellmich & Gaudet (2014) Handbook Pharmacol. Handb Exp Pharmacol. 223:963-90)

Ion channels are not only important in human health. Parasites depend on them as well. This is where we come in: we want to understand how select parasitic ion channels work and whether or not they are different from their mammalian counterparts. If they are not, well, we’ll learn something about ion channels anyway (and we already established how important they are). If they are indeed different, great! Not only will this allow insights into protein evolution and how nature uses different scaffolds to carry out similar functions, they may also turn out to be targets for drugs that are selective for the parasite. It’s a win/win situation!

Relevant publications:

Howard, R.J., Carnevale, V., Delemotte, L., Hellmich, U. A., Rothberg, B. (2017) Permeating disciplines: overcoming barriers between molecular simulations and classical structure-function approaches in biological ion transport. Biochim. Biophys. Acta Biomembr. accepted doi: 10.1016/j.bbamem.2017.12.013.

Hellmich, U.A. and Gaudet, R. (2014) High-resolution views of TRPV1 and their implications for the TRP channel superfamily. In Mammalian Transient Receptor Potential (TRP) cation channels. Handb. Exp. Pharmacol. 223:991-1004.

Hellmich, U.A. and Gaudet, R. (2014) Structural Biology of TRP Channels. In Mammalian Transient Receptor Potential (TRP) cation channels. Handb. Exp. Pharmacol. 223:963-90.

Garcia-Elias, A., Mrkonjic, S., Pardo-Pastor, C., Inada, H., Hellmich, U.A., Rubio-Moscardo, F., Plata, C., Gaudet, R., Vicente, R. and Valverde, M.A. (2013) PIP2-dependent rearrangement of TRPV4 cytosolic tails enables channel activation by physiological stimuli. Proc. Nat. Acad. Sci. USA 110(23):9553-8.

Lipid-Protein Interactions

Membrane proteins are our passion: and they can only be understood in the context of their environment - the lipid bilayer! Therefore, we study how membrane proteins interact with lipids and how lipids shape membrane protein structure and function.

Relevant publications:

Neumann, J., Rose-Sperling, D. and Hellmich, U. A. (2017) Diverse relations between lipids and ABC transporters: an overview. BBA Biomembr. 1859(4):605-618.

Hellmich, U.A., Mönkemeyer L, Velamakanni S, van Veen HW, Glaubitz C. (2015) Effects of nucleotide binding to LmrA: A combined MAS-NMR and solution NMR study. BBA Biomembr. 1848(12):3158-65.

Hacker*, C., Christ*, N. A.Duchardt-Ferner,E., Korn, S., Göbl, C., Berninger, L., Düsterhus, S., Hellmich, U. A.Madl, T., Kötter, P., Entian, K., Wöhnert, J. (2015) The solution structure of the lantibiotic immunity protein NisI and its interactions with nisin. J. Biol. Chem. 290(48):28869-86. (*contributed equally)

Garcia-Elias, A., Mrkonjic, S., Pardo-Pastor, C., Inada, H., Hellmich, U.A., Rubio-Moscardo, F., Plata, C., Gaudet, R., Vicente, R. and Valverde, M.A. (2013) PIP2-dependent rearrangement of TRPV4 cytosolic tails enables channel activation by physiological stimuli. Proc. Nat. Acad. Sci. USA 110(23):9553-8.

Mörs, K., Hellmich, U.A., Basting, D., Marchand, P., Wurm, J.P., Haase, W. and Glaubitz, C. (2013) A lipid-dependent link between activity and oligomerization state of the M. tuberculosis SMR protein Tbsmr. BBA Biomembr. 1828(2):561-7.

Christ, N.A., Bochmann, S., Gottstein, D., Duchardt-Ferner, E., Hellmich, U.A., Düsterhus, S., Kötter, P., Güntert, P., Entian, K.D. and Wöhnert, J. (2012) The first structure of a LanI protein, SpaI: The protein conferring autoimmunity against the lantibiotic subtilin in Bacillus subtilis reveals a novel fold.  J. Biol. Chem. 287(42):35286-98.

Ullrich*, S.J., Hellmich*, U.A., Ullrich, S. and Glaubitz, C. (2011) Interfacial enzyme kinetics of a membrane bound kinase analyzed by real-time MAS-NMR. Nature Chem. Biol. 7(5):263-70. (*contributed equally)

Drug-Protein interactions

It is intuitive to see why the functional consequences of a drug (how it inhibits its target protein) should be studied in detail. However, understanding the molecular consequences of drug interactions (structure and dynamics of the drug-protein complex) is just as important to improve the drug and to reduce potential off-target effects. We collaborate with labs specialized on pharmacology, organic synthesis and drug design to reach a comprehensive picture how a drug interacts with its target.

Our favorite organism to test our drugs on is the parasite Trypanosoma brucei. This single cell eukaryote causes African Sleeping Sickness, a devastating disease on the WHO's list of Neglected Tropical Diseases. On the one hand, we are interested in finding new ways to exploit parasite-specific metabolic pathways to kill the parasite without harrming its human host. On the other hand, parasites are fascinating creatures for a biochemist: how do they escape the host's immune system? How do they scavenge nutrients and how do they switch between hosts? There are so many open questions...

Relevant publications:

Preveti S, Ettari R, Cosconati S, Amendola G, Chouchene K, Wagner A, Hellmich UA, Ulrich K, Krauth-Siegel RL, Wich PR, Schmid I, Schirmeister T, Gut J, Rosenthal PJ, Grasso S, Zappalà M. (2017) Development of novel peptide-based Michael acceptors targeting rhodesain and falcipain-2 for the treatment of Neglected Tropical Diseases (NTDs). J. Med. Chem. 60(16):6911-6923.

Wagner, A., Diehl, E., R.L. Krauth-Siegel., Hellmich, U. A. (2017) Backbone NMR assignments of tryparedoxin, the central protein in the hydroperoxide detoxification cascade of African trypanosomes, in the oxidized and reduced form. Biomol. NMR Assign. accepted, doi: 10.1007/s12104-017-9746-7.

Latorre, A., Schirmeister, T., Kesselring, J., Jung, S., Johé, P., Hellmich, U. A., Heilos, A., Engels, B., Krauth-Siegel, R. L., Dirdjaja, N., Bou-Iserte, L., Rodríguez, S., González, F. V. (2016) Dipeptidyl Nitroalkenes as Potent Reversible Inhibitors of Cysteine Proteases Rhodesain and Cruzain. ACS Med. Chem. Lett. 7(12):1073-1076.

Schirmeister, T., Kesselring, J., Jung, S., Schneider, T., Weickert, A., Becker, J., Lee, W., Bamberger, D., Wich, P. R., Distler, U., Tenzer, S., Johé, P., Hellmich, U. A., Engels, B. (2016) Quantum chemical-based Protocol for the rational Design of covalent Inhibitors. J. Am. Chem. Soc., 138(27):8332-5.

 

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NMR Spectroscopy

We like using Nuclear Magnetic Resonance (NMR) Spectroscopy as a powerful and versatile technique for the investigation of complex macromolecules, including proteins and drug-protein interaction studies.

NMR allows the detection of individual atoms (such as hydrogens, carbons and nitrogens) and in conventional protein NMR experiments, each amino acid is therefore represented by at least one reporter. This is different from other powerful structural techniques that make use of single site-specific labels (such as EPR spectroscopy or FRET – that we also love, make no mistake!).

Some examples of NMR applications to proteins include the characterization of protein-protein interactions, mapping of binding sites of ligands and high-resolution structures.

In addition, because NMR studies proteins in solution (not frozen or otherwise immobilized), we can visualize dynamics and conformational changes. By looking either at the protein or a substrate, we can also determine affinities of interactions and even follow enzymatic activity in real time!