Biomedical subjects

Hakim Muradov

Publications and source records attributed to Hakim Muradov.

4 recordsLinked to original sources

PubMed2006

The inhibitory gamma subunit of the rod cGMP phosphodiesterase binds the catalytic subunits in an extended linear structure.

The unique feature of rod photoreceptor cGMP phosphodiesterase (PDE6) is the presence of inhibitory subunits (Pgamma), which interact with the catalytic heterodimer (Palphabeta) to regulate its activity. This uniqueness results in an extremely high sensitivity and sophisticated modulations of rod visual signaling where the Pgamma/Palphabeta interactions play a critical role. The quaternary organization of the alphabetagammagamma heterotetramer is poorly understood and contradictory patterns of interaction have been previously suggested. Here we provide evidence that supports a specific interaction, by systematically and differentially analyzing the Pgamma-binding regions on Palpha and Pbeta through photolabel transfer from various Pgamma positions throughout the entire molecule. The Pgamma N-terminal Val16-Phe30 region was found to interact with the Palphabeta GAFa domain, whereas its C terminus (Phe73-Ile87) interacted with the Palphabeta catalytic domain. The interactions of Pgamma with these two domains were bridged by its central Ser40-Phe50 region through interactions with GAFb and the linker between GAFb and the catalytic domain, indicating a linear and extended interaction between Pgamma and Palphabeta. Furthermore, a photocross-linked product alphabetagamma(gamma) was specifically generated by the double derivatized Pgamma, in which one photoprobe was located in the polycationic region and the other in the C terminus. Taken together the evidence supports the conclusion that each Pgamma molecule binds Palphabeta in an extended linear interaction and may even interact with both Palpha and Pbeta simultaneously.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed2005

Analysis of PDE6 function using chimeric PDE5/6 catalytic domains.

cGMP-phosphodiesterases of the PDE6 family are expressed in retinal photoreceptor cells, where they mediate the phototransduction cascade. A system for expression of PDE6 in vitro is lacking, thus straining progress in understanding the structure-function relationships of the photoreceptor enzyme. Here, we report generation and characterization of bacterially expressed chimeric PDE5/6 catalytic domains which are highly soluble, catalytically active, and sensitive to inhibition by the PDE6 Pgamma subunit. Two flexible PDE6 loops, H and M, impart chimeric PDE5/6 catalytic domains with PDE6-like properties. The replacement of the PDE6 H-loop into the PDE5 catalytic domain increases the catalytic rate and the K(m) value for cGMP hydrolysis, whereas the substitution of the M-loop produces catalytic PDE domains responsive to Pgamma. Multiple PDE6 segments preventing functional expression of the catalytic domain are identified, supporting the requirement for specialized photoreceptor chaperones to assist PDE6 folding in vivo.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed2005

Asymmetric interaction between rod cyclic GMP phosphodiesterase gamma subunits and alphabeta subunits.

Rod phosphodiesterase (PDE6) is the central effector enzyme in vertebrate visual transduction. Holo-PDE6 consists of two similar catalytic subunits (Palphabeta) and two identical inhibitory subunits (Pgamma). Palphabeta is the only heterodimer in the PDE superfamily, yet its significance for the function of PDE6 is poorly understood. An unequal interaction of Pgamma with Pbeta as compared with Palpha in the PDE6 complex has not been reported. We investigated the interaction interface between full-length Pgamma and Palphabeta, by differentiating Pgamma interaction with each individual Palphabeta subunit through radiolabel transfer from various positions throughout the entire Pgamma molecule. The efficiency of radiolabel transfer indicates that the close vicinity of serine 40 on Pgamma makes a major contribution to the interaction with Palphabeta. In addition, a striking asymmetry of interaction between the Pgamma polycationic region and the Palphabeta subunits was observed when the stoichiometry of Pgamma versus the Palphabeta dimer was below 2. Preferential photolabeling on Pbeta from Pgamma position 40 and on Palpha from position 30 increased while lowering the Pgamma/Palphabeta ratio, but diminished when the Pgamma/Palphabeta ratio was over 2. Our finding leads to the conclusion that two classes of Pgamma binding sites exist on Palphabeta, each composed of GAF domains in both Palpha and Pbeta, differing from the conventional models suggesting that each Pgamma binds only one of the Palphabeta catalytic subunits. This new model leads to insight into how the unique Palphabeta heterodimer contributes to the sophisticated regulation in visual transduction through interaction with Pgamma.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed2004

Structural determinants of the PDE6 GAF A domain for binding the inhibitory gamma-subunit and noncatalytic cGMP.

Photoreceptor cGMP phosphodiesterases (PDE6 family) are modular enzymes with each catalytic subunit containing two N-terminal regulatory GAF domains, GAF A and GAF B. The GAF A domains contribute to dimerization of the PDE6 catalytic subunits and to binding of the inhibitory Pgamma subunits, and represent candidate sites for noncatalytic binding of cGMP. We performed a mutational analysis of selected residues from the GAF A domain of cone PDEalpha' to identify the cGMP-binding pocket and delineate the Pgamma-binding surface. Results of this analysis establish the noncatalytic cGMP-binding site within the PDE6 GAF A domain and suggest that occupation of the pocket by cGMP is required for high-affinity binding of Pgamma to the proximate contact surface.

3',5'-Cyclic-GMP Phosphodiesterases↗