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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

Farber, P.J., Mittermaier, A.* (2015) Relaxation Dispersion NMR spectroscopy for the study of protein allostery. Biophysical Reviews 7:191-200Ìý

Freiburger, L., Auclair, K., and Mittermaier, A.* (2015) Global ITC fitting methods in studies of protein allostery MethodsÌý 76:149-161Ìý

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

Freiburger, L., Baettig, O.M., Berghuis, A.M., Sprules, T., Auclair, K., and Mittermaier, A.* (2011) Competing allosteric mechanisms modulate substrate binding in a dimeric enzyme. Nature Structural and Molecular Biology 18:288–294Ìý

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

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 289:27911-27923Ìý

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

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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

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 inÌýhuman cells. Journal of Medicinal Chemistry 55:6306-6315Ìý

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.

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 ()

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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 (À¦°óSMÉçÇø 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 )Ìý