Predicted structures of cAMP binding domains of type I and II regulatory subunits of cAMP-dependent protein kinase

Weber, Irene T.; Steitz, Thomas A.; Bubis, José; Taylor, Susan S.

doi:10.1021/bi00376a003

Cited by 151 publications

(91 citation statements)

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“…Results Sequence Structure Analysis. As shown previously (1,4,12), sequences of CNB-containing domains found in many organisms are well aligned. Structural analyses confirmed that secondary structure assignments are also very similar to what is seen in Fig.…”

Section: Methodssupporting

confidence: 74%

The cAMP binding domain: An ancient signaling module

Berman

Eyck

Goodsell

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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cAMP-binding domains from several different proteins were analyzed to determine the properties and interactions of this recognition motif. Systematic computational analyses, including structure-based sequence comparison, surface matching, affinity grid analysis, and analyses of the ligand protein interactions were carried out. These analyses show distinctive roles of the sugar phosphate and the adenine in the cAMP-binding module. We propose that the cAMPbinding regulatory proteins function by providing an allosteric system in which the presence or absence of cAMP produces a substantial structural change through the loss of hydrophobic interactions with the adenine ring and consequent repositioning of the C helix. The modified positioning of the helix in turn is recognized by a proteinbinding event, completing the allostery.allostery ͉ conserved cyclic nucleotide-binding motif C yclic AMP (cAMP) (Fig. 1a) is an ancient signaling molecule that serves as an indicator of stress from the most primitive bacteria to humans. The module that binds cAMP and translates the signal into a biological response, the cyclic nucleotidebinding (CNB) domain, is also ancient. Although the best-known bacterial CNB domain is associated with the catabolite activator protein (CAP), is linked to a DNA binding domain, and mediates gene transcription in the absence of glucose, the elucidation of many bacterial genomes indicates that this module is much more diverse. There are literally hundreds of proteins that are linked to CNB domains, and these proteins mediate many types of stress responses (1).The CNB domain is a small module, typically Ϸ120 aa, comprised of both ␤ strands and helical elements. The general organization of secondary structure of the essential cAMPbinding site is an eight-stranded ␤ barrel that functions as a basket. The cyclic nucleotide phosphate lies at the base of the ␤ barrel where it is well shielded from solvent and protected from phosphodiesterases (2). The most conserved feature of the ␤ barrel is the phosphate binding cassette (PBC) comprised of ␤ strand 6, a short turn of helix, and ␤ strand 7. A conserved feature of this PBC is a buried arginine that binds to the exocyclic phosphate of cAMP and a glutamate that binds the ribose 2Ј-OH. In addition to the ␤ barrel, the CNB module has a helical subdomain. The A helix precedes the ␤ barrel and is in an antiparallel manner with the B helix that immediately follows ␤ strand 8. The most variable feature of the domain is the C helix ( Fig. 1 b and c).The structure of the CNB domain was first revealed when the structure of CAP was determined (3). Based on sequence similarities, it was predicted that the fold would be conserved in the CNB domains associated with mammalian protein kinases (4), protein kinase A (PKA) and cGMP-dependent protein kinase (5). This prediction was confirmed when the structure of a deletion mutant of the RI␣ subunit of PKA was determined (6). RI␣ has two tandem CNB domains, each with a fold that resembles the CNB domain of CAP. The subsequent ...

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Section: Methodssupporting

confidence: 74%

The cAMP binding domain: An ancient signaling module

Berman

Eyck

Goodsell

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

195

213

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“…However, the nature of the uncoupling in molecular terms is poorly understood. According to the CAP model [8] the side chain of Ala 335 lies in close proximity to the adenine moiety of cAMP. The environment of the adenine binding pocket is mostly hydrophobic as has been shown by cAMPanalog mapping [5,29] and molecular modeling studies [13,30].…”

Section: Discussionmentioning

confidence: 99%

“…The two cAMP binding sites are known to bind cAMP and cAMP analogs with different affinities [5,6]. Sequence alignment of the cAMP binding domains revealed the homology to the catabolite gene activator protein (CAP), and on the basis of the 2.5 A crystal structure of CAP a three-dimensional model for each cAMP binding site has been constructed [7,8].…”

Section: Introductionmentioning

confidence: 99%

Ala³³⁵ is essential for high‐affinity cAMP‐binding of both sites A and B of cAMP‐dependent protein kinase type I

et al. 1995

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A single amino acid substitution (Ala33SAsp) in cAMP binding site B of the regulatory subunit of cAMP-dependent protein kinase type I was sufficient to abolish high affinity cAMP binding for both cAMP binding sites A and B. Furthermore, the Ala33SAsp mutation increased the activation constant for cAMP of the mutant holoenzyme 30-fold and also enhanced the rate of holoenzyme formation. Thus, the substitution was responsible for the dominant negative phenotype of the enzyme. Activation of mutant holoenzyme with site-selective cAMP analogs indicated that the enzyme dissociated through binding to site A only. Our results provide evidence that Ala 335 is an essential residue for high affinity cAMP binding of both sites as well as for the functional integrity of the enzyme.B has been shown to interact with the adenine moiety since alteration of this residue changed the affinity and specificity for cGMP [13]. Recently, a sub-clone of the rat myelocytic leukemia cell line (IPC-81) has been found to harbor a heterozygously expressed point mutation [14]. This mutated clone was selected by treatment with 8-CPT-cAMP and identified as Alato-Asp at the invariant position 335 [14]. In order to study the effects of this mutation on ligand binding and holoenzyme activation we used recombinant protein expression techniques to obtain large quantities of pure recombinant Ala335Asp mutant protein. Materials and methods

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“…X-ray crystallography and homology modeling results show that, despite obvious divergence of sequence among the receptor domains and significantly different biological functions of these proteins, their CNB domains appear to share a common architecture, all consisting of an ␣-helix (helix A), an eight-stranded ␤-roll, and two more ␣-helices (helices B and C). The body of the CNB pocket is mainly located in the ␤-roll, with the C-helix forming the back of the binding pocket (2,8). The superimposition of the structures of CNB domains from CRP and the regulatory subunits of PKA, as shown below in Fig.…”

mentioning

confidence: 99%

Functional Roles of Loops 3 and 4 in the Cyclic Nucleotide Binding Domain of Cyclic AMP Receptor Protein from Escherichia coli

Chen

Lee

2003

Journal of Biological Chemistry

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Cyclic AMP is a ubiquitous secondary message that regulates a large variety of functions. The protein structural motif that binds cAMP is highly conserved with the exception of loops 3 and 4, whose structure and length are variable. The cAMP receptor protein of Escherichia coli, CRP, was employed as a model system to elucidate the functional roles of these loops. Based on the sequence differences between CRP and cyclic nucleotide gated channel, three mutants of CRP were constructed: deletion (residues 54 -56 in loop 3 were deleted), insertion (loop 4 was lengthened by 5 residues between Glu-78 and Gly-79) and double mutants. The effects of these mutations on the structure and function of CRP were monitored. Results show that the deletion and insertion mutations do not significantly change the secondary structure of CRP, although the tertiary and qua- Cyclic AMP serves as an intracellular message in both prokaryotes and eukaryotes by transmitting information through proteins such as protein kinase A (PKA) 1 , cyclic nucleotidegated ion channels (CNGC), and cAMP receptor protein in Escherichia coli (CRP). These proteins are involved in a very diverse set of cellular functions such as signal transduction, excitability, and gene expression (1-6). These proteins of diverse functions all consist of a cAMP binding motif. The structural motif, which serves as cAMP receptor, is found to display a high degree of similarity. X-ray crystallography and homology modeling results show that, despite obvious divergence of sequence among the receptor domains and significantly different biological functions of these proteins, their CNB domains appear to share a common architecture, all consisting of an ␣-helix (helix A), an eight-stranded ␤-roll, and two more ␣-helices (helices B and C). The body of the CNB pocket is mainly located in the ␤-roll, with the C-helix forming the back of the binding pocket (2,8). The superimposition of the structures of CNB domains from CRP and the regulatory subunits of PKA, as shown below in Fig. 1, indicates that the ␤-roll basically assumes the same structure with the exception of loops 3 and 4 between strands 4 and 5, and strands 6 and 7, respectively. In some cases, such as in CNGC and PKA, loop 3 is shortened whereas loop 4 is lengthened, as shown in Fig. 1. Only six residues (Gly-33, Gly-45, Gly-71, Glu-72, Arg-82, and Ala-84 using the CRP sequence as reference) are invariant among all members of the families. It has been suggested that the invariant residues play important and conserved roles in the folding and function of the CNB sites of these diverse proteins. Gly-33, Gly-45, and Gly-71 are involved in turns between strands of the ␤-roll; Arg-82 and Glu-72 contact the cyclic nucleotide, and the function of Ala-84 is uncertain (3). Despite large variation of primary sequences, the sizes of secondary structural elements of the CNB domain are much conserved among the family. For example, the alignment of CRP and CNGCs by keeping the six conserve residues at the same positions shows that the si...

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Predicted structures of cAMP binding domains of type I and II regulatory subunits of cAMP-dependent protein kinase

Cited by 151 publications

References 47 publications

The cAMP binding domain: An ancient signaling module

The cAMP binding domain: An ancient signaling module

Ala³³⁵ is essential for high‐affinity cAMP‐binding of both sites A and B of cAMP‐dependent protein kinase type I

Functional Roles of Loops 3 and 4 in the Cyclic Nucleotide Binding Domain of Cyclic AMP Receptor Protein from Escherichia coli

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Predicted structures of cAMP binding domains of type I and II regulatory subunits of cAMP-dependent protein kinase

Cited by 151 publications

References 47 publications

The cAMP binding domain: An ancient signaling module

The cAMP binding domain: An ancient signaling module

Ala335 is essential for high‐affinity cAMP‐binding of both sites A and B of cAMP‐dependent protein kinase type I

Functional Roles of Loops 3 and 4 in the Cyclic Nucleotide Binding Domain of Cyclic AMP Receptor Protein from Escherichia coli

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Ala³³⁵ is essential for high‐affinity cAMP‐binding of both sites A and B of cAMP‐dependent protein kinase type I