涩里番

Allostery

Allostery is an essential property of many biomacromolecules whereby ligand binding or covalent modification of one site alters the activity at distant sites. Although many theoretical models have been developed to explain this phenomenon, it remains a challenge to determine which, if any, apply to a given system. In collaboration with the Auclair group, we develop new calorimetric and NMR methods to unravel the mechanisms underlying allosteric communication, and have shown that allostery can be governed by competition between opposing mechanisms, or even switch mechanisms in a ligand-dependant manner.

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Aminoglycoside-6'-acetyltransferase, a dimeric enzyme studied in our lab whose two active sites communicate allosterically.

Publications

1.听Farber, P. J., & Mittermaier, A. (2015). Relaxation dispersion NMR spectroscopy for the study of protein allostery.听Biophysical Reviews,听7, 191鈥�200.

2.听Freiburger, L., Auclair, K., & Mittermaier, A. (2015). Global ITC fitting methods in studies of protein allostery.听Methods,听76, 149鈥�161.

3.听Freiburger, L., Miletti, T., Zhu, S., Baettig, O., Berghuis, A., Auclair, K., & Mittermaier, A. (2014). Substrate-dependent switching of the allosteric binding mechanism of a dimeric enzyme.听Nature Chemical Biology,听10, 937鈥�942.

4.听Freiburger, L., Baettig, O. M., Berghuis, A. M., Sprules, T., Auclair, K., & Mittermaier, A. (2011). Competing allosteric mechanisms modulate substrate binding in a dimeric enzyme.听Nature Structural & Molecular Biology,听18, 288鈥�294.

Molecular Recognition

A fundamental property of biomacromolecules is the ability to recognize and bind to specific targets in the cell. In many cases, biological activity requires that this occur rapidly as well as selectively, raising the question, how do binding partners manage to find the correct relative positions and orientations to form a bound complex? We use a combination of NMR dynamics experiments and calorimetry to characterize protein-ligand binding pathways and have shown that transient complexes form differently than tightly-bound long-lived complexes.

Fundamental questions include: Is there an energy barrier to binding? Are there intermediate states besides free and bound, and are these on-pathway or off-pathway?

Publications

1.听Meneses, E., & Mittermaier, A. (2014). Electrostatic interactions in the binding pathway of a transient protein complex studied by NMR and isothermal titration calorimetry.听Journal of Biological Chemistry,听289, 27911鈥�27923.

2.听Demers, J. P., & Mittermaier, A. (2009). Binding mechanism of an SH3 domain studied by NMR and ITC.听Journal of the American Chemical Society,听131, 4355鈥�4367.

Covalent Drug Design

Covalent drugs form covalent bonds with their targets and include some common and successful pharmaceuticals such as aspirin, penicillin, Neratinib, and Zafgen. The benefits of covalent drugs are starting to be recognized, including their high potencies, long residence times, and high specificities. Nevertheless, their modes of action are less well understood than those of non-covalent drugs. In collaboration with the Moitessier group, we use a combination of computation, calorimetry, and NMR to characterize interactions of covalent and non-covalent inhibitors with the cancer targets prolyloligopeptidase (POP) and fibroblast activation protein (FAP), in order to derive general structure-activity relationship principles for this important class of compounds.

A covalent inhibitor containing an aldehyde group forms a covalent bond with a serine residue in the active site of an enzyme. The enzyme (E) and inhibitor (I) must first form a non-covalent intermediate (E...I) before reaching the covalent complex(E-I)

Publications

1.听Hennecker, C., Venegas, F., Wang, G., Stille, J., Milaczewska, A., Moitessier, N., & Mittermaier, A. (2025). Mechanistic Characterization of Covalent Enzyme Inhibition by Isothermal Titration Calorimetry Kinetic Competition (ITC-KC).听Analytical Chemistry,听97(12), 6368鈥�6381.

2.听Wang, G., Moitessier, N., & Mittermaier, A. K. (2023). Computational and biophysical methods for the discovery and optimization of covalent drugs.听Chemical Communications,听59(73), 10866鈥�10882.

3.听Stille, J. K., Tjutrins, J., Wang, G., Venegas, F. A., Hennecker, C., Rueda, A. M., ... & Moitessier, N. (2022). Design, synthesis and in vitro evaluation of novel SARS-CoV-2 3CLpro covalent inhibitors.听European Journal of Medicinal Chemistry,听229, 114046.

4.听Plescia, J., Dufresne, C., Janmamode, N., Wahba, A. S., Mittermaier, A. K., & Moitessier, N. (2020). Discovery of covalent prolyl oligopeptidase boronic ester inhibitors.听European Journal of Medicinal Chemistry,听185, 111783.

5.听Plescia, J., De Cesco, S., Patrascu, M. B., Kurian, J., Di Trani, J., Dufresne, C., Wahba, A. S., Janmamode, N., Mittermaier, A. K., & Moitessier, N. (2019). Integrated synthetic, biophysical, and computational investigations of covalent inhibitors of prolyl oligopeptidase and fibroblast activation protein alpha.听Journal of Medicinal Chemistry,听62, 7874鈥�7884.

6.听De Cesco, S., Kurian, J., Dufresne, C., Mittermaier, A. K., & Moitessier, N. (2017). Covalent inhibitors design and discovery.听European Journal of Medicinal Chemistry,听138, 96鈥�114.

7. De Cesco, S., Deslandes, S., Therrien, E., Levan, D., Cueto, M., Schmidt, R., Cantin, L.D., Mittermaier, A., Juillerat-Jeanneret, L., Moitessier, N.* (2012) Virtual screening and computational optimization for the discovery of covalent prolyl oligopeptidase inhibitors with activity in听human cells. Journal of Medicinal Chemistry 55:6306-6315听

Guanine Quadruplexes

G-quadruplexes are 4-stranded DNA structures built around tetrads of guanine bases that are Hoogsteen hydrogen-bonded in a planar arrangement. G-quaruplex forming DNA sequences are found in telomeres and in the promoter regions of many oncogenes and thus represent promising targets for cancer therapeutics. Understanding their function is complicated by their high degree of structural polymorphism and internal dynamics. We use a combination of calorimetry and NMR spectroscopy to characterize the stabilities and internal dynamics of G-quadruplexes in order to better explain how they function.

G-quadruplexes are composed of stacked G-tetrads (left) four guanine residues held together by Hoogsteen hydrogen bonds. They can adopt different topologies characterized by parallel and anti-parallel chains and edge and diagonal loops.

Publications

1. Carrino, S., Hennecker, C. D., Murrieta, A. C., & Mittermaier, A. K. (2021). Frustrated folding of guanine quadruplexes in telomeric DNA.听Nucleic Acids Research,听49(6), 3063鈥�3076.

2. Harkness, R. W., Hennecker, C., Gr眉n, J. T., Bl眉mler, A., Heckel, A., Schwalbe, H., & Mittermaier, A. K. (2021). Parallel reaction pathways accelerate folding of a guanine quadruplex.听Nucleic Acids Research,听49(3), 1247鈥�1262.

3. Gr眉n, J. T., Hennecker, C., Kl枚tzner, D. P., Harkness, R. W., Bessi, I., Heckel, A., Mittermaier, A. K., & Schwalbe, H. (2020). Conformational dynamics of strand register shifts in DNA G-quadruplexes.听Journal of the American Chemical Society,听142, 264鈥�273.

4. Garci, A., Castor, K. J., Fakhoury, J., Do, J. L., Di Trani, J., Chidchob, P., Stein, R. S., Mittermaier, A. K., Friscic, T., & Sleiman, H. (2018). Efficient and rapid mechanochemical assembly of platinum(II) squares for guanine quadruplex targeting.听Journal of the American Chemical Society,听139(46), 16913鈥�16922.

5. Harkness, R. W., & Mittermaier, A. K. (2017). G-quadruplex dynamics.听Biochimica et Biophysica Acta (BBA) - General Subjects,听1865, 1544鈥�1554.

6. Harkness, R. W., & Mittermaier, A. (2016). G-register exchange dynamics in guanine quadruplexes.听Nucleic Acids Research,听44(8), 3481鈥�3494.

7. Castor, K., Liu, Z., Fakhoury, J., Hancock, M., Mittermaier, A., Autexier, C., Moitessier, N., & Sleiman, H. (2013). A platinum(II) phenylphenanthroimidazole with an extended side-chain exhibits slow dissociation from a c-kit G-quadruplex motif.听Chemistry 鈥� A European Journal,听19, 17836鈥�17845.

8. Castor, K., Kieltyka, R., Englebienne, P., Weill, N., Fakhoury, J., Mancini, J., Avakyan, N., Mittermaier, A., Autexier, C., Moitessier, N., & Sleiman, H. F. (2012). Platinum(II) phenanthroimidazoles for targeting telomeric G-quadruplexes.听ChemMedChem,听7, 85鈥�94.

Kinetics by Isothermal Titration Calorimetry

In addition to providing thermodynamic information, isothermal titration calorimetry (ITC) can yield detailed information on reaction and binding kinetics on the seconds timescale. This aspect of the ITC instrument is only just starting to be explored, and promises to shed new light on biomacromolecular interactions and catalysis.

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In order extract timescale information, the shapes of the individual ITC peaks are fitted to kinetic models ()

Publications

1. Hennecker, C., Venegas, F., Wang, G., Stille, J., Milaczewska, A., Moitessier, N., & Mittermaier, A. (2025). Mechanistic Characterization of Covalent Enzyme Inhibition by Isothermal Titration Calorimetry Kinetic Competition (ITC-KC).听Analytical Chemistry,听97(12), 6368鈥�6381.

2. Wang, Y., & Mittermaier, A. K. (2021). Characterizing bi-substrate enzyme kinetics at high resolution by 2D-ITC.听Analytical Chemistry,听93(37), 12723鈥�12732.

3. Wang, Y., Guan, J. M., Di Trani, J. M., Auclair, K., & Mittermaier, A. K. (2019). Inhibition and activation of kinases by reaction products: A reporter-free assay.听Analytical Chemistry,听91, 11803鈥�11811.

4. Di Trani, J., De Cesco, S., O'Leary, R., Plescia, J., Nascimento, C., Moitessier, N., & Mittermaier, A. (2018). Rapid measurement of inhibitor binding kinetics by isothermal titration calorimetry.听Nature Communications,听9, 893.

5. Di Trani, J. M., Moitessier, N., & Mittermaier, A. K. (2018). Complete kinetic characterization of enzyme inhibition in a single isothermal titration calorimetric experiment.听Analytical Chemistry,听90, 8430鈥�8435.

6. Di Trani, J. M., Moitessier, N., & Mittermaier, A. K. (2017). Measuring rapid time-scale reaction kinetics using isothermal titration calorimetry.听Analytical Chemistry,听89(13), 7022鈥�7030.

Gamma Tubulin

Microtubules are actively controlled polymers of alpha- and beta-tubulin that are crucial to many cellular processes including vesicle trafficking, nuclear positioning, and chromosome segregation during cell division. The protein gamma tubulin has recently been shown to function as a control centre for microtubule dynamics, yet how this is acheived is unknown. In collaboration with the Vogel lab (涩里番 Biology) we study the instrinsically disordered C-terminal region of gamma tubulin by NMR spectroscopy and MD simulation in order to better understand its key role in controlling microtubules.

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X-ray crystal structure of human gamma tubulin highlighting the C-terminal region (left) and its location (blue) the end of a microtubule (figure adapted from )听

Publications

1.听Payliss, B., Vogel, J., & Mittermaier, A. (2019). Side chain electrostatic interactions and pH-dependent expansion of the intrinsically disordered, highly acidic carboxyl-terminus of 纬-tubulin.听Protein Science,听28, 1095鈥�1105.

2. Harris, J., Shadrina, M., Oliver, C., Vogel, J., & Mittermaier, A. (2018). Concerted millisecond timescale dynamics in the intrinsically disordered carboxyl terminus of 纬-tubulin induced by mutation of a conserved tyrosine residue.听Protein Science,听27(2), 531鈥�545.

Coronavirus Proteases

In response to the COVID-19 pandemic, our lab utilized our expertise to study the structural dynamics and resistance mechanisms of coronavirus proteases, PL^pro and 3CL^pro. These proteases are essential for viral replication and have been regarded as valuable drug targets. Despite their importance in drug development for coronaviruses, their structural dynamics in terms of protein-protein interactions, and their potential drug resistance mechanisms remain poorly understood and require further investigation. We use a combination of MD simulations, fluorescence assays, mass spectrometry, and mathematical modelling to reveal the nature of听3CL^pro听dimerization and resistance mutations.

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X-Ray crystal structures of SARS-CoV-2 proteases: PL^pro and 3CL^pro

Publications

1.听Wang, G., Venegas, F. A., Rueda, A. M., Weerasinghe, N. W., Uggowitzer, K. A., Thibodeaux, C. J., Moitessier, N., & Mittermaier, A. K. (2024). A naturally occurring G11S mutation in the 3C鈥恖ike protease from the SARS鈥怌oV鈥�2 virus dramatically weakens the dimer interface.听Protein Science,听33(1), e4857.