A generalized allosteric mechanism for cis-regulated cyclic nucleotide binding domains

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 beta(2,3) 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 beta(2,3) 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.

Figure 1. Current understanding of the major cAMP–CNB domain interactions represented by the PKA regulatory subunit type Iα: A-domain.

(A) Correlated movement of the PBC and the Hinge. cAMP-bound conformation (B-form) is colored yellow; holoenzyme conformation (H-form) is colored cyan. The cAMP and the capping residue positions are shown. (B) N-terminal part of the α-subdomain performs reverse motion (with respect to the Hinge), contacting the PBC in the H-form.

Figure 2. Study of the cAMP-induced conformational changes in PKA∶RIα by LSP-alignment.

High involvement scores correspond to relatively rigid parts of the molecule. Low values of the score characterize elements, which are the most sensitive to the presence of cAMP. Secondary structure is shown by red rectangles (α-helix), magenta rectangles (3₁₀-helix), and yellow arrows (β-strands). Four allosteric “hot spots” for each domain are shown by arrows.

Figure 3. The conserved N3A-motif in the N-terminal part of the α-subdomain.

(A) Stereo picture of N3A-motifs from 9 different CNB domains: A and B domains of PKA types RIα, RIIα, and RIIβ; Epac2; ionic channel HCN; and potassium channel MloK1. (B) Sequence alignment of the 9 N3A-motifs. α-helical regions are shaded magenta. Residues with negative chirality are shaded yellow. Hydrophobic residues or residues with large aliphatic segments are shown in bold. Colored circles correspond to the coloring on the stereo picture. (C) Hydrophobic interactions between residues of the N3A-motif provide integrity of the structural element. N3A-motif of PKA∶RIα A-domain is colored tan. Interacting residues are colored yellow. Connelly surfaces around their aliphatic parts are shown. (D) Residues on the tip of the 3₁₀-loop are involved in protein-protein interactions in the PKA holoenzyme. The PBC is colored cyan. C-subunit is colored grey.

Figure 4. Accumulated involvement scores obtained by LSP alignment different CNB domains.

B- form of RIα∶A was compared to B-forms of RIα∶B, RIIβ∶A, RIIβ∶B, HCN, and MloK1 (red bars); and H-forms of RIα∶B, RIIα∶A, RIIα∶B, Epac, and MloK1 (blue bars). Colored circles indicate the “three-shell” model residues: 1^st shell, red; 2^nd shell, yellow; and 3^rd shell, green. Dark grey circles indicate the residues, which were found to be conserved previously .

Figure 5. Sequence alignment of β_2,3-loops for different CNB domains.

Contacts formed by the residues are shown either on the upper left side (side chain) or lower right side (main chain). Contacts to the R²⁰⁹ are indicated by capital R: green, hydrophobic; red, polar. Also contacts to the PBC, B-helix, β_4,5-loop, and β₇-strand are indicated. Question mark signifies that the β_4,5-loop is not resolved in the HCN structure. Dashed arrows show important hydrogen bonds: above the residue letters, between side chains; under the letters, between their main chains. The last row presents main chain chirality sign for the residues.

Figure 6. Highly conserved β_2–3-loop secures the PBC-arginine side chain position.

(A) Major polar interactions of the β_2–3-loop (RIα∶A case). R²⁰⁹ is colored tan. Other residues involved in the interactions are colored yellow. (B) Nonpolar polar interactions surrounding the PBC-arginine. Hydrophobic residues are colored blue, and their Connelly surfaces are shown. CH-π interaction between the conserved G¹⁶⁹ and R²⁰⁹ is indicated by an arrow.

Figure 7. CNB domains of CAP are trans-regulated.

Interface between two monomers of CAP is shown (PDB ID, 1CGP). β-subdomains are shown as black and white contours. The PBC and the Hinge of each monomer are shown as cartoons and colored cyan (first monomer) and yellow (second monomer). Residues which form the hydrophobic interface between PBCs and hinges are shown as sticks with Connelly surfaces.

Figure 8. General allosteric mechanism for different CNB domains (RIα∶A case).

(A) Major interactions between cAMP, the PBC (red) and the β_2,3-loop (black) in cAMP-bound state. Red circles represent residues forming polar bonds (red arrows); yellow circles show residues making hydrophobic contacts (green arrows). The most important bond between cAMP and R²⁰⁹ is shown by a double red arrow. Residues and structure elements changing their positions upon cAMP binding are shaded grey. (B) cAMP-free configuration. R²⁰⁹ becomes much less restricted. (C) General diagram of major interactions in the CNB domain. The PBC controls cAMP, the 3₁₀-loop controls R²⁰⁹, and their interaction provides correct orientation of the hinge region and the N3A motif, which form a protein-protein interface.