The role of phosphorylated residues in peptide-peptide noncovalent complexes formation

Abstract

Electrospray mass spectrometry (ESI-MS) has become the tool of choice for the study of noncovalent complexes. Our previous work has highlighted the role of phosphorylated amino acid residues in the formation of noncovalent complexes through electrostatic interaction with arginine residues' guanidinium groups. In this study, we employ tandem mass spectrometry to investigate the gas-phase stability and dissociation pathways of these noncovalent complexes. The only difference in the three phosphopeptides tested is the nature of the phosphorylated amino acid residue. In addition the absence of acidic residues and an amidated carboxyl terminus insured that the only negative charge came from the phosphate, which allowed for the comparison of the noncovalent bond between arginine residues and each of the different phosphorylated residues. Dissociation curves were generated by plotting noncovalent complex ion intensities as a function of the nominal energy given to the noncovalent complex ion before entering the collision cell. These results showed that noncovalent complexes formed with phosphorylated tyrosine were the most stable, followed by serine and threonine, which had similar stability. (J Am Soc Mass Spectrom 2008Spectrom , 19, 1535Spectrom -1541 Published by Elsevier Inc. on behalf of American Society for Mass Spectrometry P hosphorylation is one of the most prevalent and important post-translational modifications. Phosphorylation/dephosphorylation events function as a molecular switch for signal transduction and enzyme catalysis. In eukaryotes, serine, threonine, and tyrosine are the three amino acid residues that can be phosphorylated. Kinase consensus sites, which promote phosphorylation of amino acid residues, are some of the most abundant domains in eukaryotic organisms, thus illustrating the importance of phosphorylation [1]. Phosphorylation also greatly influences the function of proteins in which protein-phosphate interactions affect their structure [1].The phosphate group in phosphorylated amino acid residues readily interacts noncovalently with the guanidinium group of arginine (Arg) [2]. The guanidinium group of Arg, which has a delocalized positive charge that is distributed over the entire group and a pK a of ϳ12.5 has a strong electrostatic attraction to the phosphate group of phosphorylated amino acid residues due to its strong negative charge and a pK a2 of ϳ6.7. A recent review has addressed the formation of noncovalent complexes (NCX) between the cationic guanidinium group and the anionic phosphate group [3]. The use of mass spectrometry to study noncovalent bonding between biomolecules has increased significantly. Several reviews have addressed the use of electrospray mass spectrometry and matrix-assisted laser/desorption mass spectrometry to study the noncovalent interactions between protein, peptides, nucleic acids, and drugs [4 -7].In our previous work [8 -11], we have demonstrated that motifs composed of at least two adjacent arginine residues on one peptide and a phosphorylated residu...

How adenylate cyclase choreographs the pas de deux of the receptors heteromerization dance

“…In all cases, the major dissociation pathway for the NCX is the disruption of the noncovalent bond formed by the interaction of the phosphate group with the Arg residues guanidinium group, resulting in the generation of ions corresponding to the intact epitopes 25-27 (figure 3a). An alternative pathway was observed for the noncovalent complexes between the A2AR and D2R/D4R epitopes in which the NCX complex is fragmented along the covalent bond between the oxygen from Ser and the phosphorus of the phosphate from the A2AR epitope 8,9 (figure 3b). Similar pathways have been previously observed for NCXs formed by Arg and phosphorylated amino acid residues 8,9,25-27 , In addition, this pathway was favored by 40% more in the nonphosphorylated epitopes compared to the phosphorylated ones 26,27 .…”

“…An alternative pathway was observed for the noncovalent complexes between the A2AR and D2R/D4R epitopes in which the NCX complex is fragmented along the covalent bond between the oxygen from Ser and the phosphorus of the phosphate from the A2AR epitope 8,9 (figure 3b). Similar pathways have been previously observed for NCXs formed by Arg and phosphorylated amino acid residues 8,9,25-27 , In addition, this pathway was favored by 40% more in the nonphosphorylated epitopes compared to the phosphorylated ones 26,27 . The absence of the observation of this pathway in the NCXs between D1R and NMDAR-NR1 epitopes suggests that the electrostatic attraction between them are weaker compared to the other epitopes in this study.…”

Dipole-Guided Electron Capture Causes Abnormal Dissociations of Phosphorylated Pentapeptides

“…Such interactions have been considered to explain the stability of arginine-phosphopeptide ion clusters [46][47][48] but do not seem to play a role in isolated peptide ions in which the phosphate group is neutral. This is presumably due to the donor-acceptor nature of the neutral phosphate group, where the P = O oxygen atom serves as a p-electron donor in solvating acidic protons of the peptide groups, and the acidic P-OH protons are internally solvated by suitable electron donors from the peptide amide or carboxyl groups.…”

Interactions between Intracellular Domains as Key Determinants of the Quaternary Structure and Function of Receptor Heteromers

“…In studies using synthetic peptides, it has been shown that these electrostatic interactions are particularly stable. Thus, the Arg-phosphate interaction is so stable that when using collision-induced dissociation, the noncovalent interactions between the Arg guanidinium groups and the phosphate group remain intact even though the covalent bond between the serine and phosphate breaks (16,(31)(32)(33).…”