Abstract
Cyclic nucleotides (cAMP and cGMP) regulate multiple intracellular processes and are thus of a great general interest for molecular and structural biologists. To study the allosteric mechanism of different cyclic nucleotide binding (CNB) domains, we compared cAMP-bound and cAMP-free structures (PKA, Epac, and two ionic channels) using a new bioinformatics method: local spatial pattern alignment. Our analysis highlights four major conserved structural motifs: 1) the phosphate binding cassette (PBC), which binds the cAMP ribose-phosphate, 2) the “hinge,” a flexible helix, which contacts the PBC, 3) the β2,3 loop, which provides precise positioning of an invariant arginine from the PBC, and 4) a conserved structural element consisting of an N-terminal helix, an eight residue loop and the A-helix (N3A-motif). The PBC and the hinge were included in the previously reported allosteric model, whereas the definition of the β2,3 loop and the N3A-motif as conserved elements is novel. The N3A-motif is found in all cis-regulated CNB domains, and we present a model for an allosteric mechanism in these domains. Catabolite gene activator protein (CAP) represents a trans-regulated CNB domain family: it does not contain the N3A-motif, and its long range allosteric interactions are substantially different from the cis-regulated CNB domains.
Highlights
Cyclic adenosine monophosphate is an important second messenger, which regulates a large variety of cellular processes, including metabolism, cell shape transformation, gene transcription, photoreception and chemosensation [1,2,3,4,5]
We studied changes in different cyclic nucleotide binding (CNB) domains induced by the cyclic nucleotides using a new method for comparison of protein structures: local spatial patterns alignment
Our analysis has shown that there are four elements conserved in all known CNB domains, with the exception of the catabolite gene activator protein (CAP): the Phosphate Binding Cassette (PBC), the hinge, the b2,3-loop and the ‘‘N3A-motif’’
Summary
Cyclic adenosine monophosphate (cAMP) is an important second messenger, which regulates a large variety of cellular processes, including metabolism, cell shape transformation, gene transcription, photoreception and chemosensation [1,2,3,4,5]. All cAMP-binding proteins in both pro- and eukaryotes share a small module – the cyclic nucleotide binding domain (CNB domain), which is typically fused to another domain. The CNB domain contains a contiguous bsubdomain and a non-contiguous a-subdomain (Figure 1). The former is a relatively rigid eight-stranded b-sandwich, which accommodates the cyclic nucleotide molecule. Recent structure studies of cAMPdependent protein kinase (PKA) demonstrated, that CNB domains toggle between two stable conformations: bound to cAMP (so called B-form [7]), or to catalytic subunit of PKA (H-form) [8,9]. The intermediate, non bound form (apo-form) is characterized by high backbone flexibility [10,11,12] and is apparently represented by a dynamic ensemble of multiple configurations
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