Functional and mutational analysis of the identification of py-binding residues ON PDE6

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Functional and mutational analysis of the identification of py-binding residues ON PDE6'

Granovski Olexiy

PhD, Associate Professor

Department of Anatomy, Physiology of Human and Animals Luhansk Taras Shevchenko National University, Poltava, Ukraine

Boiarchuk Olena

Candidate of Biological Sciences, Associate Professor Department of Anatomy, Physiology of Human and Animals Luhansk Taras Shevchenko National University, Poltava, Ukraine

Summary

catalytic phosphodiesterase inhibition

Photoreceptor cGMP phosphodiesterase (PDE 6) is an effector enzyme in the G-protein mediated visual transduction cascade. In the dark, PDE6 activity is turned off by an inhibitory y- subunit (Py). To study the mechanism of PDE6 catalytic activity inhibition by Py chimeric proteins PDE6a/PDE5 were created. The catalytic properties of chimeric PDE remained the same as those of PDE5. Mutational analysis of the Pg-binding region, PDE6a' - (750-760), using ala-scan revealed PDE6a' residues required for interaction. The M758A mutation markedly impaired and the Q752A mutation moderately impaired Py inhibition of the chimeric PDE. Analysis of the catalytic properties of the mutant PDE and the PDE6 catalytic domain model suggest that the Met758 and Gln752 residues bind Py directly. The PDE6 catalytic site model shows that PDE6a'-(750-760) forms a loop at the entrance to the cGMP-binding pocket. Binding of Py to Met758 effectively blocks cGMP access to the catalytic cavity, providing a structural basis for the PDE6 inhibition mechanism.

Key words: PDE6, PDE5, Y-subunit, catalytic site, inhibition.

Photoreceptor cGMP phosphodiesterases (PDE6 family) function as effector proteins in vertebrate visual transduction, which is mediated by the rhodopsin- coupled G-protein, transducin. The retinal rod PDE6 is composed of two PDE6 catalytic ap subunits, each of which is tightly coupled to a smaller inhibitory у subunit (Py) [1]. The cone PDE consists of two identical PDEa' subunits combined in a complex with two copies of the cone-specific Py subunit. The catalytic subunits of rods and cones of PDE, as well as the corresponding subunits of Py, have a high degree of homology [2]. The key role of Py is to inhibit the hydrolysis of cGMP by catalytic subunits in the dark. Upon light stimulation of photoreceptors, PDE6 is activated by GTP-bound transducin a , which displaces Py from the catalytic core of the enzyme.

Two Py regions are mainly involved in interactions with PDE6 catalytic subunits, the central polycationic region (residues 24-45 of the Py stem ) and the C- terminal Py residue. The C-terminus of Py is a key inhibitory domain, while the polycationic region enhances the overall affinity of Py for PDE6 catalytic subunits. Crosslinking studies located the C-terminal Py binding site on PDE6a with residues 751-763 (PDE6P or PDE6a' residues 749-761) within the broader catalytic domain of PDE6. Our further analysis of the interaction between fluorescently labeled Py and PDE6 ap suggests that the C-terminus of Py inhibits PDE6 activity by physically blocking the PDE catalytic site [4].

Progress in studying the structure/function of PDE6 and the mechanism of PDE6 inhibition by Py has slowed down due to the lack of an effective PDE6 expression system [5]. Our approach to designing a system for the expression and mutagenesis of PDE6 involved the creation of chimeras between PDE6a' and the binding cGMP, cGMP specific to PDE (PDE5 family) [6]. PDE5 and PDE6 have a common domain organization, i.e. two non-catalytic cGMP binding sites are located at the N-terminus of the conserved PDE catalytic domain. In addition, PDE5 and PDE6 show a high degree of homology (45-48% identity) between catalytic domains. Unlike PDE6, PDE5 is readily expressed using the baculovirus /insect cell system [7]. In our previous studies, we created PDE6a'/PDE5 chimeric enzymes that contained a C- terminal Py binding site and were highly inhibited by Py. Ala scanning mutation analysis of the Py binding site using a chimeric PDE as a template identified key interacting residues and provided a structural rationale for the mechanism of PDE6 inhibition [6].

The PDE a'-(737-784) includes the PDEa'-(749-761) segment, which was previously identified as the PyC -terminal binding site. The sequence corresponding to PDEa'-(749-761) is unique to the PDE of photoreceptors, which show strong conservatism at this site [3]. In contrast to PDE5 and Chi4 [6], the catalytic activity of Chi16 was effectively inhibited by Py. K value , equal to 3.6 nM , indicates that Py binds to Chi16 only with an affinity 20 times lower than the affinity of its interaction with native PDE6a' (Table 1).

To test the potential role of the non-catalytic cGMP-binding domain of PDE6, the PDE6a'-(737-784) region was also replaced by the PDE5 cDNA. The resulting chimera, Chi17, had catalytic properties similar to those of PDE5 and Chi 16 (Km 1.9 |uM and kcat 9.8 s-1) (Table 1). The IC50 value for inhibition of Chi17 with zaprinast (0.77 |uM ) was similar to the IC50 value for PDE5 but slightly higher than the IC50 value for Chi16 (Table 1). Py inhibited the hydrolysis of cGMP by Chi17 to a lesser extent than the catalytic activity of Chi16. The maximum inhibition was up to 70% of Chi17 activity and the K value was 142 nM. These results suggest that the non-catalytic cGMP- binding domain of PDE6a' promotes a high-affinity interaction with Py.

Ala-scanning mutagenesis of the C-terminal Py binding site in Chi 16 was performed to identify the Py-binding residues of PDE6a'. Eleven consecutive residues starting at position 750 have been replaced by alanine. All Chi 16 mutants were analyzed for their ability to hydrolyze cGMP. Two mutants, L751A and D760A, were catalytically inactive. Two other mutants, P755A and I756A, showed a markedly reduced rate of catalysis (Table 1). In addition to reducing the kcat value for cGMP hydrolysis, the P755A substitution also resulted in an increase in the Km value from 2.8 to 42 |uM (Table 1). The catalytic properties of P755A indicate that this mutation likely affected the overall folding of the catalytic site in Chi16. The Km values for cGMP hydrolysis for the remaining Chi16 mutants were in the range of 4-15 pM (Table 1). Inhibition of Chi16 mutants with zaprinast did not reveal large variations in their IC50 values, which were comparable to the IC50 value for Chi16 (Table 1).

Table 1

Functional properties of Chi16 mutants

* Data from Granovsky et al.[6] ** N/A, not applicable.

All catalytically active Chi16 mutants were then tested for Py inhibition. Most of the mutants retained their functional interaction with Py at Ki 0.8 to 5 nM (Table 1). Two mutants, Q752A and M758A, were defective in Py binding. The Q752A mutation had a moderate effect on interaction with Py. Py was able to completely inhibit the catalytic activity of Q752A, but the Ki value was increased to 29 nM. For the M758A mutant, a significant disruption of the Py interaction was observed. Inhibition of M758A by Py was incomplete (~75%) with a Ki value of 97 nM. Since the catalytic properties of Q752A and M758A were similar to those of Chi16, the Py binding defects were unlikely to be caused by changes in the overall catalytic domain folding in these mutants.

The vertebrate visual transduction cascade is one of the most studied and best understood G-protein signaling systems. However, PDE6, a key vision enzyme, remains perhaps one of the most understudied effectors of the G protein. The construction of chimeric enzymes between PDE6a' and related PDE5 has been shown to be a useful tool for studying PDE 6. We have previously demonstrated that a fully functional PDE6a'/PDE5 chimeric enzyme containing non-catalytic cGMP binding sites PDE6a' and a PDE5 catalytic domain can be efficiently expressed in the baculovirus /insect cell system [6]. This chimeric enzyme exhibited similar catalytic and non-catalytic cGMP binding properties to those of PDE5 and PDE6a', respectively. The PDE6a '/PDE5 chimeric proteins containing the PDE6a'-active site were catalytically inactive, which indicates that the catalytic domain contains specific sequences that prevent its functional folding in insect cells. Based on these results, we generated and analyzed a number of PDE6a'/PDE5 chimeric proteins with substitutions of various segments of the PDE5 catalytic domain with the corresponding PDE6a' sequences. The sequence PDE6a'-(737-784) containing the Py C-terminal binding site Pa'-(749-761) [3] was introduced into one of these chimeras, Chi16. Not only was Chi 16 catalytically active with Km and kcat values are similar to PDE5, but have also acquired Py sensitivity. Ki value Chi 16 for Py (3.6 nM) was only 10-20 times higher than Ki values native PDE6a', which have been previously reported [2, 6]. Contacts between Py and the PDE6a' catalytic domain beyond PDE6a'-(737-784) may explain the lower value Ki native enzyme. Non- catalytic cGMP binding sites are allosterically associated with Py binding sites and can regulate the affinity of Py for PDE catalytic subunits [8]. To test the role of the cGMP-binding domain, PDE6a'-(737-784) was also replaced with a wild type PDE5 sequence (Chi17). Py inhibited Chi17 (Ki 142 nM) is less potent than Chi 16, indicating that the non-catalytic cGMP-binding domain of PDE6a' is allosterically or owing to additional contacts, it enhances the interaction of Py with the catalytic domain.

We have previously demonstrated that binding of the PyC-terminus to the catalytic domain of PDE6 blocks cGMP access to the catalytic site. Therefore, it was concluded that the residues involved in the binding/hydrolysis of cGMP and the binding of competitive inhibitors are in close proximity to the C-terminal Py binding residues in the three-dimensional structure of PDE6 [4]. In this study, the introduction of a Py binding site into the catalytic domain of PDE5 did not result in a noticeable change in the catalytic properties. Therefore, Py binding residues are not directly involved in cGMP binding/hydrolysis by PDE6 and likely form a domain other than the catalytic pocket.

Both conclusions, namely the proximity of the Py site to the catalytic pocket and its structural independence from the catalytic pocket, are supported by the PDE6 catalytic site model. The model was created based on the recently determined structure of the PDE 4 catalytic domain, the first crystal structure of the PDE enzyme [9]. According to this model, the Py binding site, PDE6a' -(749-761), forms a loop near the entrance to the cGMP binding catalytic pocket. However, PDE6a'-(749-761) residues are not involved in the formation of the catalytic pocket itself. The latter mainly consists of residues stored in the PDE superfamily.

Ala-scanning mutation analysis of PDE6a'-(750-760) in Chi 16 identified two mutants, Q752A and M758A, with impaired inhibition Py. The M758A substitution resulted in a particularly deep defect in Py binding. Both mutants retained catalytic properties (Km and kcat) for cGMP hydrolysis and IC50 values for zaprinast inhibition similar to those of Chi 16, suggesting their intact overall folding. The PDE6a' catalytic domain model shows that the Gln752 and Met758 side chains are similarly oriented on the surface of the molecule. Therefore, in all likelihood, these residues interact directly with Py. If the PyC end is aligned along the plane formed by the Gln752 and Met758 side chains, it can also contact Pro755. Our data do not exclude the possibility of this contact. The P755A mutant had a significantly reduced rate of cGMP hydrolysis and therefore its inhibition by Py cannot be directly compared to that of Chi16. Of the residues, Met758 is located at the very tip of the Py -binding loop facing the opening of the catalytic pocket. This arrangement of the Py binding residue will allow Py to effectively block the entry of cGMP into the catalytic pocket.

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