The cAMP binding domain: An ancient signaling module

Abstract

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 ...

Structural Insights on the Plant Salt-Overly-Sensitive 1 (SOS1) Na+/H+ Antiporter

“…5c), suggesting that the latter contributes mainly in the dimerization of the transporter. Thus, we suggest that this region, the distal one, may correspond to the cyclic nucleotide binding domain that has been reported to be involved in the oligomerization of other transporters 28,29 and the area at the vicinity of the TM domain may constitute the first domain (Fig. 5c).…”

Surface comparison of active and inactive protein kinases identifies a conserved activation mechanism

“…Earlier, this method was used to study cAMP-binding proteins and proved to be effective in detecting conserved surface motifs that cannot be found readily by other methods (15). This method is rapid and does not require sequence or structure alignments, nor any preliminary knowledge of the protein function or ligandbinding localization.…”

cAMP activation of PKA defines an ancient signaling mechanism

“…T he cAMP binding domain (CBD) and cAMP are conserved from bacteria to humans as a ubiquitous signaling mechanism to translate extracellular stress signals into appropriate biological responses (1). The major receptor for cAMP in higher eukaryotes, cAMP-dependent PKA (2), is ubiquitous in mammalian cells where it exists in two forms: the inactive tetrameric holoenzyme and the active dissociated catalytic subunit (Csubunit).…”

“…Previous analyses (8)(9)(10)(11)(12)(13)(14)(15)(16)) have led to the proposal of an initial allosteric model in which the ␣-and ␤-subdomains are directly coupled to each other through a salt bridge between E200 and R241 and also possibly through a hydrophobic hinge defined by the L203, I204, and Y229 side-chain cluster (9,11,12). However, mutations (17), sequence conservation analyses (1), structurebased comparisons (1), and genetic screening (18,19) indicate that several other sites, which are not accounted for by the existing model, are also likely to play an active role in the cAMP-mediated activation of PKA. To comprehensively understand this allosteric mechanism, it is therefore necessary to elucidate at high resolution how cAMP remodels the free energy landscape of CBD-A, which serves as the central controlling unit of PKA.…”