Molecular Modeling of regulatory processes in aspartoacylase enzyme

Kots E.D.

Lomonosov Moscow State University, chemistry department

Human brain aspartoacylase (hAsp) belongs to carboxypeptidase family and catalyzes the hydrolysis of N-acetyl-aspartate (NAA) producing aspartic and acetic acids which further proceeds to myelin synthesis1. The exact metabolic pathway of NAA remains illusive but the necessity to maintain its concentration in relatively narrow limits is commonly accepted. Thus, reduced NAA concentrations is observed in several psychiatric disorders such as multiple sclerosis, Alzheimer’s disease and epilepsy. While abnormally high level of NAA is associated with Canavan disease (CD)2 - a fatal neurodegenerative disorder for which there is currently no treatment. CD effect on CNS is characterized by dysmyelination and spongiform degeneration of white matter.

Kinetic experiments conducted by Le Coq et al.3 revealed presumable activation effect at relatively low NAA concentrations and substantial substrate self-inhibition when NAA is present in a large excess. Application of modern modeling tools enabled us to detect a set of putative allosteric sites and to select the most promising for further investigation. Analysis of multiple long-scale molecular dynamics trajectories (exceeding 1.5 μs in total) disclosed a possible mechanism of self-inhibition – NAA presence in allocated binding site induces significant rigidity to gate-forming protein loops on the way to the active site. Subsequent implementation of dynamical correlation analysis provided an opportunity to build signaling paths from allosteric site to the gate residues. Moreover, crucial interactions responsible for the effect observed was detected as a result of community analysis performed on holo- and apo-hAsp forms.

Computed trajectories were also used to estimate Markov state model4 and compute the transition path from closed to opened hAsp conformation. The mean passage times for apo- and holo- systems and relative equilibrium constants were computed. The states distribution proved destabilization effect on the opened state of NAA binding to the active site.

1. Bitto, E.; Bingman, C.A.; Wesenber, G.E.; McCoy, J.G.; Phillips, G.N. Structures of aspartoacylase , the brain enzyme impaired in Canavan disease. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 456-461.

2. Surendran, S.; Matalon, K.; Tyring, S.K.; Matalon, R. Molecular basis of Canavan disease: from human to mouse. J. Child. Neurol. 2003, 18, 604-610.

3. Le Coq, J.; An, H.-J.; Lebrilla, C.; Viola, R.E. Characterization of Human Aspartoacylase: the brain enzyme responsible for Canavan disease. Biochem. 45 (18), 5878-5884.

4. Malmstrom, R. D.; Lee, C. T.; Van Wart, A. T.; Amaro, R. E. Application of Molecular-Dynamics Based Markov State Models to Functional Proteins. J. Chem. Theory Comput. 2014, 21, 626-632.

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