Research

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.

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

Publications

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

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

3. 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. Nature Chemical Biology, 10, 937–942.

4. 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. Nature Structural & Molecular Biology, 18, 288–294.

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

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

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. 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). Analytical Chemistry, 97(12), 6368–6381.

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

3. 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. European Journal of Medicinal Chemistry, 229, 114046.

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

5. 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. Journal of Medicinal Chemistry, 62, 7874–7884.

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

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 human cells. Journal of Medicinal Chemistry 55:6306-6315 read more

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. Nucleic Acids Research, 49(6), 3063–3076.

2. Harkness, R. W., Hennecker, C., Grün, J. T., Blümler, A., Heckel, A., Schwalbe, H., & Mittermaier, A. K. (2021). Parallel reaction pathways accelerate folding of a guanine quadruplex. Nucleic Acids Research, 49(3), 1247–1262.

3. Grün, J. T., Hennecker, C., Klötzner, 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. Journal of the American Chemical Society, 142, 264–273.

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. Journal of the American Chemical Society, 139(46), 16913–16922.

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

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

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. Chemistry – A European Journal, 19, 17836–17845.

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. ChemMedChem, 7, 85–94.

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.

In order extract timescale information, the shapes of the individual ITC peaks are fitted to kinetic models (reproduced from JACS 2012, 134:559-565)

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). Analytical Chemistry, 97(12), 6368–6381.

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

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. Analytical Chemistry, 91, 11803–11811.

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. Nature Communications, 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. Analytical Chemistry, 90, 8430–8435.

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

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 (McGill 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.

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 Biology Notes) 

Publications

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

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

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 3CL^pro dimerization and resistance mutations.

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

Publications

1. 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‐like protease from the SARS‐CoV‐2 virus dramatically weakens the dimer interface. Protein Science, 33(1), e4857.