Structural change of bovine retinal cGMP phosphodiesterase by release of its γ subunit:: direct imaging by improved low angle rotary shadowing

Cyclic GMP phosphodiesterase (PDE), a key enzyme for phototransduction, contains two catalytic subunits, Pa and PP, and two identical regulatory subunits, Pys. Neither the structure of the subunits of PDE nor their changes in structure during PDE regulation have been known. Here, improved low angle rotary shadowing was applied to depict the three-dimensional structure of bovine PDE (P alpha beta gamma gamma) and its changes by P gamma release. P alpha beta gamma gamma and P alpha beta gamma were isolated from photoreceptor membranes after treatment with a hydrolysis-resistant GTP analogue, and P alpha beta was prepared from P alpha beta gamma gamma by tryptic digestion. Images of P alpha beta gamma gamma consisted of two crooked strands. These two strands faced each other to make a ring shape, but this ring structure was bent at the centre Line between the two strands. In P alpha beta gamma, one of these strands changed its shape toward reducing the central space of the ring structure. This ring appeared to be more bent at the centre line. In P alpha beta, both strands changed their shape such that the ring structure appeared to be a twisted quasi ring shape. These observations suggest that in P alpha beta gamma gamma each P gamma is complexed with a catalytic subunit, and that the shapes of P alpha and P beta are drastically changed by the P gamma release. These shape changes are no doubt: crucial for various PDE regulations, such as activation of cGMP hydrolysis by P alpha beta, interaction of P alpha beta with GARP2 and a GARP2-like protein and cGMP binding to non-catalytic sites on P alpha beta.