Date of Completion

Spring 5-1-2016

Project Advisor(s)

Eric May

University Scholar Major

Molecular and Cell Biology

Disciplines

Biophysics | Other Biochemistry, Biophysics, and Structural Biology | Structural Biology

Abstract

Phosphodiesterase 6 (PDE6) is a critical enzyme in the eyesight-signaling pathway. When activated, PDE6 hydrolyzes cGMP to GMP, which deactivates cGMP- gated ion channels, causing hyperpolarization of the cell and activating the sensory neurons responsible for vision. Within the PDE family, PDE6 is the only enzyme known to have an inhibitory subunit (PDE6-γ), which allows for the regulation of cGMP levels. When PDE6-γ is bound to PDE6, the enzyme is turned “off” and cannot catalyze cGMP. The α subunit of the G-protein transducin removes PDE6-γ and activates PDE6. PDE6 has proven problematic to isolate, making it difficult to study experimentally and preventing a structure from being solved. A chimera of the homologous proteins PDE5 and PDE6 (PDE5/6) has been constructed to serve as a model for PDE6 that can be expressed and purified. The validity of PDE5/6 as a model for PDE6 has been demonstrated through mild inhibition by PDE6-γ. To study the native sequence of PDE6, a computational approach was applied by creating a homology model of PDE6. Using this model has allowed us to understand the structural basis for PDE6’s inhibition by PDE6-γ.

We have investigated correlations in protein dynamics possibly responsible for allosteric properties by running over 1 microsecond (μs) of molecular dynamics simulations and carrying out various analyses. PDE6 and PDE5/6 are both inhibited by the PDE5 inhibitor sildenafil. With this knowledge, systems of the homology model of PDE6, PDE5, and PDE5/6 in ligand-free (apo), sildenafil bound, and PDE6-γ bound states were constructed and simulated for 100-300 nanoseconds each. Our analyses were aimed at evaluating the quality of our homology model of PDE6 and showing the affect of sildenafil and PDE6-γ within each system and across different systems. These studies have allowed us to gain insight into the structural characteristics and the specific residues that are involved in allosteric communication and regulation of PDE6’s inhibition by PDE6-γ. Using RMSF and cross-correlational analyses we have discovered a novel region that shows correlation with the H- and M-loops and could be an allosteric therapeutic target for diseases in which PDE6 is implicated, such as retinitis pigmentosa. Through this work, we hope to gain a greater understanding of the molecular dynamics and mechanisms of PDE6 and how these features may relate to the molecular underpinnings of eyesight.

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