A Generalized Allosteric Mechanism for cis-Regulated Cyclic Nucleotide Binding Domains

Open Access
Publisher Website
Google Scholar
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. Cyclic nucleotides are small regulatory molecules which transmit signal from receptors positioned on a living cell membrane into the cell interior and regulate multiple biological processes ranging from bacteria to humans. Such regulation occurs through binding of the cyclic nucleotides to the corresponding proteins. All such proteins contain a relatively small domain responsible for the cyclic nucleotide binding. The most important task is to understand how cyclic nucleotide binding (CNB) domains translate the signal into a biological response. In this work, we studied changes in different CNB domains induced by the cyclic nucleotides using a new method for comparison of protein structures: local spatial patterns alignment. This novel method compares protein molecules and detects conserved spatial patterns comprised of similar amino acid residues. Moreover, it ranks the detected residues with respect to their functional or structural importance. Our results show that there are at least two different families of CNB domains. The first family has four structural elements which perform the signal translation. Two of these elements were known previously and two are novel. The second family can translate the signal using only three elements, but they have to work in pairs to provide interaction between the functional elements.