Prediction of peptides binding to the PKA RIIalpha subunit using a hierarchical strategy

Abstract

Motivation: Favorable interaction between the regulatory subunit of the cAMP-dependent protein kinase (PKA) and a peptide in A-kinase anchoring proteins (AKAPs) is critical for translocating PKA to the subcellular sites where the enzyme phosphorylates its substrates. It is very hard to identify AKAPs peptides binding to PKA due to the high sequence diversity of AKAPs.

Results: We propose a hierarchical and efficient approach, which combines molecular dynamics (MD) simulations, free energy calculations, virtual mutagenesis (VM) and bioinformatics analyses, to predict peptides binding to the PKA RIIα regulatory subunit in the human proteome systematically. Our approach successfully retrieved 15 out of 18 documented RIIα-binding peptides. Literature curation supported that many newly predicted peptides might be true AKAPs. Here, we present the first systematic search for AKAP peptides in the human proteome, which is useful to further experimental identification of AKAPs and functional analysis of their biological roles.

Fig. 1.

RMSF of backbone atoms in the RIIα/AKAP10 complex. X-axis is the residue number (residues in monomer 1 are indicated by plain numbers and those in monomer 2 by primes).

Fig. 2.

The interaction energy spectrum for the core residues of the AKAP10 peptide obtained by the MM/GBSA decomposition analysis. (a) Total energy, (b) van der Waals energy and (c) polar energy. The residues with interaction energy less than −4.0 kcal/mol are labeled (the amino acid one-letter codes are labeled in Fig. 2a).

Fig. 3.

Alignment of the RII-binding domains of the available AKAPs. Conserved amino acid residues at each position are colored in blue. All but AKAP14 (Kultgen et al., 2002) and AKAPis peptides were taken from Hundsrucker et al. (2006). The two binding peptides of AKAP9 are labeled as AKAP9_1 and AKAP9_2. AKAPis is a high-affinity peptide obtained from bioinformatics and peptide-array screening (Alto et al., 2003). DAKAP550 is a AKAP found in Drosophila, not in human (Han et al., 1997).

Fig. 4.

The hydrophobic binding interface of the RIIα D/D domain. The solvent accessible surface of the RIIα D/D domain is colored by the hydrophobicity of the residues. High hydrophobicity is colored in red and low hydrophobicity in blue. The peptide is shown as a stick model. The picture was generated by Discovery Studio molecular simulation package (2009).

Fig. 5.

The binding free energy difference (ΔΔG) between the peptide in the template and the mutated peptide mutated at positions (a) P5, (b) P8, (c) P9, (d) P12, (e) P13 and (f) P16. The preference of amino acids at each position can be determined based on ΔΔG.

Fig. 6.

The hierarchical approach to identify the putative binding peptides of the RIIα D/D domain.

Fig. 7.

The motifs used in database screening. Little x represents nonproline residues and capital X represents any residue.

Fig. 8.

The alignment of the PKA D/D domains across seven species.

Fig. 9.

The sequence alignments across seven species for the binding peptides of (a) AKAP1 and (b) AKAP14.