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Peter S Brzovic, Clemens C Heikaus,
Leonid Kisselev,
Robert Vernon,
Eric Herbig,
Derek Pacheco,
Linda Warfield,
Peter Littlefield,
David Baker,
Rachel E Klevit,
Steven Hahn
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ABSTRACT: The structural basis for binding of the acidic transcription activator Gcn4 and one activator-binding domain of the Mediator subunit Gal11/Med15 was examined by NMR. Gal11 activator-binding domain 1 has a four-helix fold with a small shallow hydrophobic cleft at its center. In the bound complex, eight residues of Gcn4 adopt a helical conformation, allowing three Gcn4 aromatic/aliphatic residues to insert into the Gal11 cleft. The protein-protein interface is dynamic and surprisingly simple, involving only hydrophobic interactions. This allows Gcn4 to bind Gal11 in multiple conformations and orientations, an example of a "fuzzy" complex, where the Gcn4-Gal11 interface cannot be described by a single conformation. Gcn4 uses a similar mechanism to bind two other unrelated activator-binding domains. Functional studies in yeast show the importance of residues at the protein interface, define the minimal requirements for a functional activator, and suggest a mechanism by which activators bind to multiple unrelated targets.
Molecular cell 12/2011; 44(6):942-53. · 14.61 Impact Factor
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ABSTRACT: GAF domains regulate the catalytic activity of certain vertebrate cyclic nucleotide phosphodiesterases (PDEs) by allosteric, noncatalytic binding of cyclic nucleotides. GAF domains arranged in tandem are found in PDE2, -5, -6, -10, and -11, all of which regulate the cellular concentrations of the second messengers cAMP and/or cGMP. Nucleotide binding to GAF domains affects the overall conformation and the catalytic activity of full-length PDEs. The cyclic nucleotide-bound GAF domains from PDE2, -5, -6, and -10 all adopt a conserved fold but show subtle differences within the binding pocket architecture that account for a large range of nucleotide affinities and selectivity. NMR data and details from the structure of full-length nucleotide-free PDE2A reveal the dynamic nature and magnitude of the conformational change that accompanies nucleotide binding. The discussed GAF domain structures further reveal differences in dimerization properties and highlight the structural diversity within GAF domain-containing PDEs.
Structure 12/2009; 17(12):1551-7. · 6.35 Impact Factor
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ABSTRACT: Phosphodiesterase 5 (PDE5) controls intracellular levels of cGMP through its regulation of cGMP hydrolysis. Hydrolytic activity
of the C-terminal catalytic domain is increased by cGMP binding to the N-terminal GAF A domain. We present the NMR solution
structure of the cGMP-bound PDE5A GAF A domain. The cGMP orientation in the buried binding pocket was defined through 37 intermolecular
nuclear Overhauser effects. Comparison with GAF domains from PDE2A and adenylyl cyclase cyaB2 reveals a conserved overall
domain fold of a six-stranded β-sheet and four α-helices that form a well defined cGMP binding pocket. However, the nucleotide
coordination is distinct with a series of altered binding contacts. The structure suggests that nucleotide binding specificity
is provided by Asp-196, which is positioned to form two hydrogen bonds to the guanine ring of cGMP. An alanine mutation of
Asp-196 disrupts cGMP binding and increases cAMP affinity in constructs containing only GAF A causing an altered cAMP-bound
structural conformation. NMR studies on the tandem GAF domains reveal a flexible GAF A domain in the absence of cGMP, and
indicate a large conformational change upon ligand binding. Furthermore, we identify a region of ∼20 residues directly N-terminal
of GAF A as critical for tight dimerization of the tandem GAF domains. The features of the PDE5 regulatory domain revealed
here provide an initial structural basis for future investigations of the regulatory mechanism of PDE5 and the design of GAF-specific
regulators of PDE5 function.
Journal of Biological Chemistry 08/2008; 283(33):22749-22759. · 4.77 Impact Factor
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ABSTRACT: The photoreceptor phosphodiesterase (PDE6) regulates the intracellular levels of the second messenger cGMP in the outer segments of cone and rod photoreceptor cells. PDE6 contains two regulatory GAF domains, of which one (GAF A) binds cGMP and regulates the activity of the PDE6 holoenzyme. To increase our understanding of this allosteric regulation mechanism, we present the 2.6A crystal structure of the cGMP-bound GAF A domain of chicken cone PDE6. Nucleotide specificity appears to be provided in part by the orientation of Asn-116, which makes two hydrogen bonds to the guanine ring of cGMP but is not strictly conserved among PDE6 isoforms. The isolated PDE6C GAF A domain is monomeric and does not contain sufficient structural determinants to form a homodimer as found in full-length PDE6C. A highly conserved surface patch on GAF A indicates a potential binding site for the inhibitory subunit Pgamma. NMR studies reveal that the apo-PDE6C GAF A domain is structured but adopts a significantly altered structural state indicating a large conformational change with rearrangement of secondary structure elements upon cGMP binding. The presented crystal structure will help to define the cGMP-dependent regulation mechanism of the PDE6 holoenzyme and its inhibition through Pgamma binding.
Journal of Biological Chemistry 08/2008; 283(38):25913-9. · 4.77 Impact Factor
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ABSTRACT: Phosphodiesterase 5 (PDE5) controls intracellular levels of cGMP through its regulation of cGMP hydrolysis. Hydrolytic activity of the C-terminal catalytic domain is increased by cGMP binding to the N-terminal GAF A domain. We present the NMR solution structure of the cGMP-bound PDE5A GAF A domain. The cGMP orientation in the buried binding pocket was defined through 37 intermolecular nuclear Overhauser effects. Comparison with GAF domains from PDE2A and adenylyl cyclase cyaB2 reveals a conserved overall domain fold of a six-stranded beta-sheet and four alpha-helices that form a well defined cGMP binding pocket. However, the nucleotide coordination is distinct with a series of altered binding contacts. The structure suggests that nucleotide binding specificity is provided by Asp-196, which is positioned to form two hydrogen bonds to the guanine ring of cGMP. An alanine mutation of Asp-196 disrupts cGMP binding and increases cAMP affinity in constructs containing only GAF A causing an altered cAMP-bound structural conformation. NMR studies on the tandem GAF domains reveal a flexible GAF A domain in the absence of cGMP, and indicate a large conformational change upon ligand binding. Furthermore, we identify a region of approximately 20 residues directly N-terminal of GAF A as critical for tight dimerization of the tandem GAF domains. The features of the PDE5 regulatory domain revealed here provide an initial structural basis for future investigations of the regulatory mechanism of PDE5 and the design of GAF-specific regulators of PDE5 function.
Journal of Biological Chemistry 07/2008; 283(33):22749-59. · 4.77 Impact Factor
