Thesis defences

PhD Oral Exam - Vinod Parmar, Chemistry and Biochemistry

Computational Investigation of the Structure, Dynamics and Functions of Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Relevant to Its Reactions with Nitric Oxide Donors

DATE & TIME
Tuesday, August 14, 2018
10 a.m. – 1 p.m.
COST

This event is free

ORGANIZATION

School of Graduate Studies

CONTACT

Virginia Bruce
514-848-2424, ext. 3812

WHERE

Richard J. Renaud Science Complex
7141 Sherbrooke W.
Room SP 185.11

WHEEL CHAIR ACCESSIBLE

Yes

When studying for a doctoral degree (PhD), candidates submit a thesis that provides a critical review of the current state of knowledge of the thesis subject as well as the student’s own contributions to the subject. The distinguishing criterion of doctoral graduate research is a significant and original contribution to knowledge.

Once accepted, the candidate presents the thesis orally. This oral exam is open to the public.

Abstract

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a well-known glycolytic enzyme, exhibits moonlighting functions, including catalysis of glyceryl trinitrate (GTN) to release vasoactive nitrite or nitric oxide. GTN catalysis generates a thionitrate (E-Cys-NO2) at the active-site thiol of GAPDH. Thus, we investigated reaction mechanisms of decomposition of CH3SNO2, a model compound for E-Cys-NO2, using state-of-the-art quantum mechanics techniques. We showed that the well-studied homolysis pathway, which releases nitric oxide (NO), is energetically unfavorable. Instead, hydrolysis and thiolysis pathways that release nitrite (NO2–) are energetically more favorable. Release of NO2– upon the attack of anionic nucleophiles, e.g. OH– or CH3S–, along the S–N bond is barrierless, which could explain the instability of thionitrates in aqueous solutions. We also looked at the effect of protonation on the stability of S–N bond of CH3SNO2 to mimic the effect of proton donation at protein active site. We observed that O- and S-protonation stabilizes and destabilizes S–N bond, respectively. We evaluated electronic structures of the protonated isoforms of CH3SNO2 to explain the observed stability/instability of S–N bond. Further investigation of nitrite release by hydrolysis of thionitrate in the active site of GAPDH using quantum mechanics / molecular mechanics (QM/MM) technique revealed similar activation barrier as shown by QM methods.

Each subunit of the GAPDH homotetramer binds a cofactor NAD+. The tetramer bind NAD+ with negative cooperativity. We carried out normal mode analysis and molecular dynamics (MD) simulations of GAPDH-NAD+ to understand the mechanism of negative cooperativity and define the subunit interactions that contribute to this phenomenon. We compared dynamics of GAPDH-NAD+, GAPDH-NADH and apo-GAPDH. We observed the concerted motions between subunits of GAPDH-NAD+ and GAPDH-NADH but these motions are lost in apo-GAPDH, indicating the cofactor induces these concerted motions, which are dominant and functionally relevant motions of GAPDH. We also observed the changes in the NAD binding site residues and active site residues that are observed in crystal structure of apo- and holo-GAPDH.

Many moonlighting functions of GAPDH depend on its oligomeric state, tetramer, dimer or monomer. MD simulations are thus carried out for the three oligomeric states. Our study shows that functionally important motions are centered on NBDs in tetramer and dimer, whereas they are centered on S-loop in a monomer. The dimer and monomer are less stable than tetramer. Additionally, the study highlights the importance of dynamics of the S-loop, which acts as a disordered region in dimer and monomer. The disordered S-loop could escalate the binding of GAPDH dimer and monomer with multiple protein partners to exhibit different functions.

We modeled GAPDH and the seven in absentia homolog 1 (Siah1) protein-protein interactions and investigated the possibility of formation of a complex with different oligomeric states of each protein. We show that GAPDH-monomer binds more tightly to Siah1 than GAPDH-tetramer. Thus, GAPDH monomer could stabilize Siah1 and the complex could be translocated to the nucleus to initiate apoptosis.

Overall, our study provides molecular and structural level insight into the various functions of GAPDH. We have highlighted importance of dynamics of various structural regions which contributes to different functions of GAPDH.


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