Abstract
Purpose::
The objective of this work is to identify the structural similarities and functional differences between PDE5 and PDE6 based on comparative genomic analysis and homology modeling. These class I phosphodiesterases share overall sequence similarities, homologous cGMP-binding GAF domains, a catalytic domain specific for cGMP as substrate, and a similar selectivity profile for PDE inhibitors. The structural basis for the high catalytic efficiency and mechanism of activation of PDE6 that distinguish it from PDE5 is not well understood.
Methods::
Using saturated evolutionary trace (SET) analysis, we identified invariant and class-specific sites in the vertebrate PDE5 and 6 genes. Sequences from genomic predictions and mRNA confirmations were compiled and aligned using Clustal W. The sequences were grouped phylogenetically and tested (using PAUP) for saturation to determine if sequence diversity was sufficient. Unanimous sites in each group were then compared to generate the trace sequence. Homology modeling was used to map the trace sites and identify 3-D regions representing functional differences between PDE5 and rod/cone PDE6.
Results::
The SET analysis used 51 protein sequences from 14 vertebrate taxa. This diverse set of sequences represents a >80% likelihood that the sites designated as invariant are indeed identical throughout vertebrate evolution. The trace of these PDE genes identified ~90 invariant sites and ~80 class-specific sites. About 20 of the class-specific sites were clustered within the GAFb domain. A set of residues unique to PDE6 was also found, as well as numerous other class-specific sites throughout the sequence that provide evidence for functional differences in the regulatory and catalytic domains of the two PDE families. Some of the class-specific sites identified here confirm previous site-directed mutagenesis studies that reported on individual sites responsible for some PDE5/PDE6 differences.
Conclusions::
This study represents the first use of SET to understand PDE structure/function. Sequences collected to date are sufficient to positively identify the great majority of invariant and class-specific sites in PDE5 and PDE6. This work reveals novel regions of PDE6 that distinguish it from PDE5, and which may confer the unique catalytic and regulatory properties of PDE6 during visual excitation.
Keywords: photoreceptors • protein structure/function • enzymes/enzyme inhibitors