Direct Allosteric Regulation between the GAF Domain and Catalytic Domain of Photoreceptor Phosphodiesterase PDE6

Abstract

Photoreceptor cGMP phosphodiesterase (PDE6) is the central enzyme in the visual transduction cascade. The PDE6 catalytic subunit contains a catalytic domain and regulatory GAF domains. Unlike most GAF domain-containing cyclic nucleotide phosphodiesterases, little is known about direct allosteric communication of PDE6. In this study, we demonstrate for the first time direct, inter-domain allosteric communication between the GAF and catalytic domains in PDE6. The binding affinity of PDE6 for pharmacological inhibitors or for the C-terminal region of the inhibitory γ subunit (Pγ), known PDE6 binding the GAF of the of the GAF domains this allosteric communication between GAF and catalytic domains is in binding the catalytic domain cGMP the GAF cGMP binding of the of PDE6 binding the GAF domain cGMP and In of PDE6 or and a in the catalytic domain the binding of the GAF domains. the in visual the direct, inter-domain allosteric in this a in Photoreceptor cGMP phosphodiesterase (PDE6) is the central enzyme in the visual transduction cascade. The PDE6 catalytic subunit contains a catalytic domain and regulatory GAF domains. Unlike most GAF domain-containing cyclic nucleotide phosphodiesterases, little is known about direct allosteric communication of PDE6. In this study, we demonstrate for the first time direct, inter-domain allosteric communication between the GAF and catalytic domains in PDE6. The binding affinity of PDE6 for pharmacological inhibitors or for the C-terminal region of the inhibitory γ subunit (Pγ), known PDE6 binding the GAF of the of the GAF domains this allosteric communication between GAF and catalytic domains is in binding the catalytic domain cGMP the GAF cGMP binding of the of PDE6 binding the GAF domain cGMP and In of PDE6 or and a in the catalytic domain the binding of the GAF domains. the in visual the direct, inter-domain allosteric in this a in The cyclic nucleotide phosphodiesterase (PDE6) cyclic nucleotide catalytic of PDE6 and inhibitory γ subunit of cyclic nucleotide catalytic of PDE6 and inhibitory γ subunit of is the central enzyme in the visual in and is the of the of of the of in of cGMP of and of the of cGMP PDE6 the and of the of in of The PDE6 of a catalytic of and and inhibitory γ of PDE6 of the inhibitory of binding PDE6. The affinity of for the catalytic is in a cGMP binding PDE6 the catalytic and in in and PDE6 is of of the phosphodiesterase regulatory GAF domains and The GAF domains for in and the of The GAF domains of the a cyclic nucleotide binding cGMP is the for and is the for the the domain allosteric of catalytic binding of cyclic the GAF domains for and cGMP binding the GAF domains a of in the of the enzyme the binding affinity of inhibitors the catalytic domains of is cGMP this allosteric between the GAF and catalytic domains is in inhibitors cGMP binding the GAF domains In this direct allosteric communication between GAF and catalytic inhibitors a in the catalytic domain binding affinity in a the between and PDE6 direct, inter-domain allosteric communication between the GAF and catalytic domains is for PDE6. cGMP binding the catalytic of PDE6 a direct the allosteric cGMP binding the GAF domains the or of the enzyme the allosteric GAF and catalytic domains. The region of is known the GAF domains of the catalytic a affinity the affinity of the C-terminal region of for the catalytic domain of The C-terminal region of is for the catalytic binding the catalytic domains of The of GAF and catalytic domains of the regulatory and catalytic domains of PDE6 in binding the catalytic the binding affinity of cGMP the GAF domain cGMP of the GAF domain affinity this study, we first direct allosteric communication between the GAF domains and catalytic domains of PDE6 binding the GAF domains the affinity of inhibitors and of the binding the catalytic inter-domain allosteric is in binding catalytic domains the binding affinity of cGMP the GAF domains. The of this direct allosteric in PDE6 is in and a in PDE6 of and and of inhibitors in and in and and PDE6 catalytic and PDE6 catalytic in a or a The PDE6 the of cGMP of PDE6 and a of the of the enzyme of cGMP this binding of PDE6 a binding The of inhibitors or in the or of cGMP or of and the the and nucleotide and and in The The and of The inhibitory of catalytic the a cGMP the GAF of in binding and for cGMP of and region The of cGMP PDE6 cGMP The of PDE6 cyclic nucleotide in in the of for catalytic the binding of of cGMP and and the time of in the or of a a is the is the and is the in the of for the the in the of the GAF the of the the of PDE6 we first binding the GAF domains the of the of cGMP and the region of known the GAF domains of the PDE6 catalytic and we first the catalytic of PDE6 binding of the GAF domains. The first of the and the of the GAF domains the the binding affinity of the catalytic domain of or cGMP catalytic the of of the GAF a for this the GAF domain the inhibitory of binding the GAF a of the of binding the region of for this inter-domain in binding the PDE6 GAF domains binding affinity the catalytic for cGMP and or catalytic and the inhibitory of the cGMP binding of cGMP the The the of The the a of and The the the of cGMP the cGMP binding the allosteric binding the we the allosteric of cGMP binding in the of the the catalytic of of cGMP cGMP binding the is binding the GAF domain or cGMP of the binding the in the GAF domains is the catalytic of the of the GAF the of the C-terminal region of PDE6 the the catalytic binding the we binding of the region of the GAF domains the binding affinity of the C-terminal region of the catalytic domains of the for of cGMP about or the GAF domains. The and affinity the of the PDE6 GAF domains this inter-domain allosteric is the of the PDE6 catalytic of or the GAF domains the binding affinity of the catalytic or for of The cGMP The the of and the and The the the in the the the GAF allosteric of and binding PDE6 in cGMP binding the GAF domains the for of the is cGMP and the catalytic of of and this we the for cGMP in the of and and of the GAF in and the catalytic domain the for cGMP binding of the for cGMP The in cGMP and binding a the catalytic domain binding of is the inter-domain communication between the GAF and catalytic domains is the region of the of PDE6. or for of of The the of and a the and and The the for the and of the cGMP the GAF in the the direct allosteric the GAF domains the catalytic domain of the of allosteric and of this allosteric communication of the in the GAF domains. In the of cGMP binding the GAF domains of the cGMP binding PDE6 this catalytic we cGMP the of of the catalytic the of the GAF domains a of cGMP the of a of the the the cGMP in the of the The of cGMP in and the binding of inhibitors the catalytic domain of the PDE6 catalytic allosteric in the cGMP binding the domain cGMP binding PDE6. and or for for or cGMP in the or of cGMP The the and the the of the a of the a the a The for and for the The the the the a in catalytic domain binding the we for a allosteric PDE6. and or for the of for a The of this in inhibitory the affinity of for the for PDE6 in the inhibitors the inhibitors a in the catalytic the inhibitors for or catalytic The the the and and and and The the for of the this in the catalytic domain is the GAF we cGMP PDE6 cGMP the of the of time PDE6 this in the catalytic domain is the the domain of the first PDE6 the allosteric catalytic GAF domain-containing the catalytic of PDE6 direct allosteric the GAF domains. is a allosteric between the GAF and catalytic domains. The binding of the of the GAF domains the catalytic domain in affinity of the the affinity of the C-terminal of in the of the binding of the a in the GAF domains binding affinity for direct allosteric in of PDE6 and a for catalytic between and PDE6 allosteric of catalytic is for the of cGMP the GAF domain of cGMP or binding affinity cGMP binding affinity the GAF domains of is inhibitors binding the catalytic domain The of inter-domain allosteric for is we in this for between the allosteric and PDE6. the of the catalytic and domains in and this in PDE6 is inhibitors regulatory subunit and and in the of direct allosteric the GAF domain a in the catalytic domain is inhibitors The of the for cGMP the C-terminal domains of the binding the for this between and PDE6 is is of of in a in the in a in binding affinity of cGMP is the catalytic cGMP of for PDE6. PDE6 a for catalytic PDE6 a regulatory binding is of the PDE6 domains cGMP in in and for the in the of the catalytic domain of PDE6 in this a allosteric communication between the GAF domains and the catalytic of the for cGMP the and a in or affinity is for or for the PDE6 catalytic we the known catalytic domain the for we the PDE6 catalytic domain the catalytic domain or the of the catalytic domain of the a domain in the of the and in in the of the the and for most of the the cGMP in the is of in the for inhibitors cGMP affinity the catalytic domains is or is and of the PDE6 GAF domains the in the catalytic In a binding the of PDE6 in the or the GAF domains the of the cGMP binding in the domain is the for the inter-domain we for PDE6 in this inhibitors and or a in binding affinity is the GAF domains of PDE6. in affinity for The of in the of the the In binding the catalytic domain of of a of the the of the in the of the PDE6 is of binding affinity for of of is of PDE6 is the regulatory for the of visual of the the PDE6 subunit the C-terminal region of is the region of PDE6 catalytic of PDE6 in in and PDE6 for a time for the allosteric communication between the catalytic and GAF domains the of the of the GAF domains of of PDE6 of and of cGMP the PDE6 catalytic the in a of PDE6 In this allosteric communication between domains of the PDE6 catalytic the of PDE6 and a of of PDE6 catalytic known or in in and this the catalytic domain PDE6 and visual or is the in in the of the affinity of for the catalytic of PDE6 the GAF domains of PDE6 inter-domain allosteric communication between the GAF and catalytic domains of in the visual is the first of direct allosteric communication the PDE6 catalytic and is allosteric regulatory GAF domains. inter-domain allosteric communication between the GAF domains and the catalytic domains is in binding domain the of the Unlike direct allosteric of PDE6 is the of PDE6 binding of the subunit the PDE6 catalytic of PDE6 visual this inter-domain communication between the GAF and catalytic domains for the and of PDE6 of The cyclic nucleotide phosphodiesterase (PDE6) cyclic nucleotide catalytic of PDE6 and inhibitory γ subunit of cyclic nucleotide catalytic of PDE6 and inhibitory γ subunit of is the central enzyme in the visual in and is the of the of of the of in of cGMP of and of the of cGMP PDE6 the and of the of in of The PDE6 of a catalytic of and and inhibitory γ of PDE6 of the inhibitory of binding PDE6. The affinity of for the catalytic is in a cGMP binding PDE6 the catalytic and in in and Photoreceptor PDE6 is of of the phosphodiesterase regulatory GAF domains and The GAF domains for in and the of The GAF domains of the a cyclic nucleotide binding cGMP is the for and is the for the the domain allosteric of catalytic binding of cyclic the GAF domains for and cGMP binding the GAF domains a of in the of the enzyme the binding affinity of inhibitors the catalytic domains of is cGMP this allosteric between the GAF and catalytic domains is in inhibitors cGMP binding the GAF domains In this direct allosteric communication between GAF and catalytic inhibitors a in the catalytic domain binding affinity in a the between and PDE6 direct, inter-domain allosteric communication between the GAF and catalytic domains is for PDE6. cGMP binding the catalytic of PDE6 a direct the allosteric cGMP binding the GAF domains the or of the enzyme the allosteric GAF and catalytic domains. The region of is known the GAF domains of the catalytic a affinity the affinity of the C-terminal region of for the catalytic domain of The C-terminal region of is for the catalytic binding the catalytic domains of The of GAF and catalytic domains of the regulatory and catalytic domains of PDE6 in binding the catalytic the binding affinity of cGMP the GAF domain cGMP of the GAF domain affinity In this study, we first direct allosteric communication between the GAF domains and catalytic domains of PDE6 binding the GAF domains the affinity of inhibitors and of the binding the catalytic inter-domain allosteric is in binding catalytic domains the binding affinity of cGMP the GAF domains. The of this direct allosteric in PDE6 is in and a in PDE6 of and and of inhibitors in and in and and PDE6 catalytic and PDE6 catalytic in a or a The PDE6 the of cGMP of PDE6 and a of the of the enzyme of cGMP this binding of PDE6 a binding The of inhibitors or in the or of cGMP or of and the the and nucleotide and and in The The and of The inhibitory of catalytic the a cGMP the GAF of in binding and for cGMP of and region The of cGMP PDE6 cGMP The of PDE6 cyclic nucleotide in in the of for catalytic the binding of of cGMP and and the time of in the or of a a is the is the and is the in the of for the the in the and and of inhibitors in and in PDE6 and and PDE6 catalytic and PDE6 catalytic in a or a The PDE6 the of cGMP of PDE6 and a of the of the enzyme of cGMP this binding of PDE6 a binding The of inhibitors or in the or of cGMP or of and the the and nucleotide and and in The The and of The inhibitory of catalytic the a cGMP the GAF of in binding and for cGMP of and region The of cGMP PDE6 cGMP The of PDE6 cyclic nucleotide in in the of for catalytic the binding of of cGMP and and the time of in the or of a a is the is the and is the in the of for the the in the of the GAF the of the the of PDE6 we first binding the GAF domains the of the of cGMP and the region of known the GAF domains of the PDE6 catalytic and we first the catalytic of PDE6 binding of the GAF domains. The first of the and the of the GAF domains the the binding affinity of the catalytic domain of or cGMP catalytic the of of the GAF a for this the GAF domain the inhibitory of binding the GAF a of the of binding the region of for this inter-domain in of cGMP the cGMP binding the allosteric binding the we the allosteric of cGMP binding in the of the the catalytic of of cGMP cGMP binding the is binding the GAF domain or cGMP of the binding the in the GAF domains is the catalytic of the of the GAF the of the C-terminal region of PDE6 the the catalytic binding the we binding of the region of the GAF domains the binding affinity of the C-terminal region of the catalytic domains of the for of cGMP about or the GAF domains. The and affinity the of the PDE6 GAF domains this inter-domain allosteric is the of the PDE6 catalytic of or the GAF domains the binding affinity of the catalytic or for of The cGMP The the of and the and The the the in the the the GAF allosteric of and binding PDE6 in cGMP binding the GAF domains the for of the is cGMP and the catalytic of of and this we the for cGMP in the of and and of the GAF in and the catalytic domain the for cGMP binding of the for cGMP The in cGMP and binding a the catalytic domain binding of is the inter-domain communication between the GAF and catalytic domains is the region of the of PDE6. or for of of The the of and a the and and The the for the and of the cGMP the GAF in the the direct allosteric the GAF domains the catalytic domain of the of allosteric and of this allosteric communication of the in the GAF domains. In the of cGMP binding the GAF domains of the cGMP binding PDE6 this catalytic we cGMP the of of the catalytic the of the GAF domains a of cGMP the of a of the the the cGMP in the of the The of cGMP in and the binding of inhibitors the catalytic domain of the PDE6 catalytic allosteric in the cGMP binding the domain cGMP binding PDE6. and or for for or cGMP in the or of cGMP The the and the the of the a of the a the a The for and for the The the the the a in catalytic domain binding the we for a allosteric PDE6. and or for the of for a The of this in inhibitory the affinity of for the for PDE6 in the inhibitors the inhibitors a in the catalytic the inhibitors for or catalytic The the the and and and and The the for of the this in the catalytic domain is the GAF we cGMP PDE6 cGMP the of the of time PDE6 this in the catalytic domain is the the domain of PDE6. of the GAF the of the the of PDE6 we first binding the GAF domains the of the of PDE6. cGMP and the region of known the GAF domains of the PDE6 catalytic and we first the catalytic of PDE6 binding of the GAF domains. The first of the and the of the GAF domains the the binding affinity of the catalytic domain of or cGMP catalytic the of of the GAF a for this the GAF domain the inhibitory of binding the GAF a of the of binding the region of for this inter-domain in of cGMP the cGMP binding the allosteric binding the we the allosteric of cGMP binding in the of the the catalytic of of cGMP cGMP binding the is binding the GAF domain or cGMP of the binding the in the GAF domains is the catalytic of the of the GAF the of the C-terminal region of PDE6 the the catalytic binding the we binding of the region of the GAF domains the binding affinity of the C-terminal region of the catalytic domains of the for of cGMP about or the GAF domains. The and affinity the of the PDE6 GAF domains this inter-domain allosteric is the of the PDE6 catalytic in the the the GAF allosteric of and binding PDE6 in cGMP binding the GAF domains the for of the is cGMP and the catalytic of of and this we the for cGMP in the of and and of the GAF in and the catalytic domain the for cGMP binding of the for cGMP The in cGMP and binding a the catalytic domain binding of is the inter-domain communication between the GAF and catalytic domains is the region of of the cGMP the GAF in the the direct allosteric the GAF domains the catalytic domain of the of allosteric and of this allosteric communication of the in the GAF domains. In the of cGMP binding the GAF domains of the cGMP binding PDE6 this catalytic we cGMP the of of the catalytic the of the GAF domains a of cGMP the of a of the the the cGMP in the of the The of cGMP in and the binding of inhibitors the catalytic domain of the PDE6 catalytic allosteric in the cGMP binding the domain cGMP binding the a in catalytic domain binding the we for a allosteric PDE6. and or for the of for a The of this in inhibitory the affinity of for the for PDE6 in the inhibitors the this in the catalytic domain is the GAF we cGMP PDE6 cGMP the of the of time PDE6 this in the catalytic domain is the the domain of PDE6. the first PDE6 the allosteric catalytic GAF domain-containing the catalytic of PDE6 direct allosteric the GAF domains. is a allosteric between the GAF and catalytic domains. The binding of the of the GAF domains the catalytic domain in affinity of the the affinity of the C-terminal of in the of the binding of the a in the GAF domains binding affinity for direct allosteric in of PDE6 and a for catalytic between and PDE6 allosteric of catalytic is for the of cGMP the GAF domain of cGMP or binding affinity cGMP binding affinity the GAF domains of is inhibitors binding the catalytic domain The of inter-domain allosteric for is we in this for between the allosteric and PDE6. the of the catalytic and domains in and this in PDE6 is inhibitors regulatory subunit and and in the of direct allosteric the GAF domain a in the catalytic domain is inhibitors The of the for cGMP the C-terminal domains of the binding the for this between and PDE6 is is of of in a in the in a in binding affinity of cGMP is the catalytic cGMP of for PDE6. PDE6 a for catalytic PDE6 a regulatory binding is of the PDE6 domains cGMP in in and for the in the of the catalytic domain of PDE6 in this a allosteric communication between the GAF domains and the catalytic of the for cGMP the and a in or affinity is for or for the PDE6 catalytic we the known catalytic domain the for we the PDE6 catalytic domain the catalytic domain or the of the catalytic domain of the a domain in the of the and in in the of the the and for most of the the cGMP in the is of in the for inhibitors cGMP affinity the catalytic domains is or is and of the PDE6 GAF domains the in the catalytic In a binding the of PDE6 in the or the GAF domains the of the cGMP binding in the domain is the for the inter-domain we for PDE6 in this inhibitors and or a in binding affinity is the GAF domains of PDE6. in affinity for The of in the of the the In binding the catalytic domain of of a of the the of the in the of the PDE6 is of binding affinity for of of is of PDE6 is the regulatory for the of visual of the the PDE6 subunit the C-terminal region of is the region of PDE6 catalytic of PDE6 in in and PDE6 for a time for the allosteric communication between the catalytic and GAF domains the of the of the GAF domains of of PDE6 of and of cGMP the PDE6 catalytic the in a of PDE6 In this allosteric communication between domains of the PDE6 catalytic the of PDE6 and a of of PDE6 catalytic known or in in and this the catalytic domain PDE6 and visual or is the in in the of the affinity of for the catalytic of PDE6 the GAF domains of PDE6 inter-domain allosteric communication between the GAF and catalytic domains of in the visual is the first of direct allosteric communication the PDE6 catalytic and is allosteric regulatory GAF domains. inter-domain allosteric communication between the GAF domains and the catalytic domains is in binding domain the of the Unlike direct allosteric of PDE6 is the of PDE6 binding of the subunit the PDE6 catalytic of PDE6 visual this inter-domain communication between the GAF and catalytic domains for the and of PDE6 of the first PDE6 the allosteric catalytic GAF domain-containing the catalytic of PDE6 direct allosteric the GAF domains. is a allosteric between the GAF and catalytic domains. The binding of the of the GAF domains the catalytic domain in affinity of the the affinity of the C-terminal of in the of the binding of the a in the GAF domains binding affinity for direct allosteric in of PDE6 and a for catalytic between and PDE6 allosteric of catalytic is for the of cGMP the GAF domain of cGMP or binding affinity cGMP binding affinity the GAF domains of is inhibitors binding the catalytic domain The of inter-domain allosteric for is we in this for PDE6. between the allosteric and PDE6. the of the catalytic and domains in and this in PDE6 is inhibitors regulatory subunit and and in the of direct allosteric the GAF domain a in the catalytic domain is inhibitors The of the for cGMP the C-terminal domains of the binding the for this between and PDE6 is is of of in a in the in a in binding affinity of cGMP is the catalytic cGMP of for PDE6. PDE6 a for catalytic PDE6 a regulatory binding is of the PDE6 domains cGMP in in and for the in the of the catalytic domain of PDE6 in this a allosteric communication between the GAF domains and the catalytic of the for cGMP the and a in or affinity is for or for the PDE6 catalytic we the known catalytic domain the for we the PDE6 catalytic domain the catalytic domain or the of the catalytic domain of the a domain in the of the and in in the of the the and for most of the the cGMP in the is of in the for inhibitors cGMP affinity the catalytic domains is or is and of the PDE6 GAF domains the in the catalytic In a binding the of PDE6 in the or the GAF domains the of the cGMP binding in the domain is the for the inter-domain we for PDE6 in this inhibitors and or a in binding affinity is the GAF domains of PDE6. in affinity for The of in the of the the In binding the catalytic domain of of a of the the of the in the of the PDE6 is of binding affinity for of of is of PDE6 is the regulatory for the of visual of the the PDE6 subunit the C-terminal region of is the region of PDE6 catalytic of PDE6 in in and PDE6 for a time for the allosteric communication between the catalytic and GAF domains the of the of the GAF domains of of PDE6 of and of cGMP the PDE6 catalytic the in a of PDE6 In this allosteric communication between domains of the PDE6 catalytic the of PDE6 and a of of PDE6 catalytic known or in in and this the catalytic domain PDE6 and visual or is the in in the of the affinity of for the catalytic of PDE6 the GAF domains of PDE6 inter-domain allosteric communication between the GAF and catalytic domains of in the visual is the first of direct allosteric communication the PDE6 catalytic and is allosteric regulatory GAF domains. inter-domain allosteric communication between the GAF domains and the catalytic domains is in binding domain the of the Unlike direct allosteric of PDE6 is the of PDE6 binding of the subunit the PDE6 catalytic of PDE6 visual this inter-domain communication between the GAF and catalytic domains for the and of PDE6 of of for the of the