R-subunit isoform specificity in protein kinase A: distinct features of protein interfaces in PKA types I and II by amide H/2H exchange mass spectrometry

Abstract

The two isoforms (RI and RII) of the regulatory (R) subunit of cAMP-dependent protein kinase or protein kinase A (PKA) are similar in sequence yet have different biochemical properties and physiological functions. To further understand the molecular basis for R-isoform-specificity, the interactions of the RIIbeta isoform with the PKA catalytic (C) subunit were analyzed by amide H/(2)H exchange mass spectrometry to compare solvent accessibility of RIIbeta and the C subunit in their free and complexed states. Direct mapping of the RIIbeta-C interface revealed important differences between the intersubunit interfaces in the type I and type II holoenzyme complexes. These differences are seen in both the R-subunits as well as the C-subunit. Unlike the type I isoform, the type II isoform complexes require both cAMP-binding domains, and ATP is not obligatory for high affinity interactions with the C-subunit. Surprisingly, the C-subunit mediates distinct, overlapping surfaces of interaction with the two R-isoforms despite a strong homology in sequence and similarity in domain organization. Identification of a remote allosteric site on the C-subunit that is essential for interactions with RII, but not RI subunits, further highlights the considerable diversity in interfaces found in higher order protein complexes mediated by the C-subunit of PKA.

Figure 1

Type II PKA. (A) Domain organization of PKA RIIβ with an N-terminal dimerization/docking domain (D/D domain), substrate/autoinhibitor site, and two cAMP binding domains, Domain A (108–268) and Domain B (269–416). (B) Deletion mutants of RIIβ for probing interactions with the C-subunit, RIIβ(108–268) and RIIβ(108–402). * Increased proteolytic cleavage of C-terminal residues 403-416 during purification of the RIIβ(108–402):C holoenzyme, necessitated use of a more stable, double truncation mutant RIIβ(108–402) for studying interactions with the C-subunit. (C) Structures of the catalytic subunit of PKA (left) (PDB access code 1ATP) [38] and of RIIβ(right) (PDB access code 1CX4) [35]. The inhibitor peptide is green and ATP is red for C-subunit (left). In RIIβ(right), residues 108-252 are in blue, while residues 269-416 are green and the two cAMP molecules are yellow. The α:C helix (residues 253-268), a hotspot for interactions with the C-subunit is in red [17].

Figure 2

MALDI-TOF spectra of one of the peptides spanning the α:C helix (residues 253-268) in RIIβ that showed decreased exchange in complexes of C-subunit with the deletion fragment, RIIβ(108–402). The spectra are expanded so as to show the isotopic distribution for the peptide of interest (m/z = 2281.31). i) Undeuterated sample. The higher mass peaks in the envelope are caused by naturally occurring isotopes. The isotopic envelope for the same peptide after two minutes of deuteration from:- ii) free RIIβ(residues 108-402) (iii) RIIβ(residues 108-402):C (minus Mg²⁺ATP/Mn²⁺AMP-PNP) and (iv) RIIβ(residues 108-402):C (plus Mn²⁺AMP-PNP)

Figure 3

MALDI-TOF spectra of one of the peptides spanning the α:G helix (residues 247-261) in the C-subunit that showed decreased exchange in complexes of C-subunit with the deletion fragment, RIIβ(108–402). The spectra are expanded so as to show the isotopic distribution for the peptide of interest (m/z = 1793.97). i) Undeuterated sample. The higher mass peaks in the envelope are caused by naturally occurring isotopes. The isotopic envelope for the same peptide after two minutes of deuteration from:-ii) C-subunit (minus Mg²⁺ATP/Mn²⁺AMP-PNP) (iii) C-subunit (+ Mg²⁺ATP) (iv) RIIβ(108–268):C complex + Mn²⁺AMP-PNP, (v) RIIβ(108–402):C complex + Mn²⁺AMP-PNP, vi) RIIβ(108–402):C complex (minus Mg²⁺ATP/Mn²⁺AMP-PNP).

Figure 4

Deuterium exchange in four N-terminal fragment peptides of the C subunit bound to Mg²⁺ATP (x), bound to RIIβ(residues 108-268) and Mn²⁺AMP-PNP (open squares)) and bound to RIIβ(residues 108-402) with Mn²⁺AMP-PNP (filled circles)). The Y-axis scale reflects the total number of exchangeable amide hydrogens in each of the peptides analyzed. (A) C-subunit fragment residues 44-54 (glycine-rich loop), (B) C-subunit fragment residues 163-172 (catalytic loop), (C) C-subunit fragment residues 133-145 (α:D) and D) C-subunit fragment residues 92-100 (α:C). (E) Structure of the C subunit (PDB access code: 1ATP) with the N-lobe colored white and the C lobe in wheat and above four fragment peptides in red.

Figure 5

Deuterium exchange in three C-lobe peptides of the C-subunit bound to Mg²⁺ATP (x), bound to RIIβ(residues 108-268) and Mn²⁺AMP-PNP (open squares)) and bound to RIIβ(residues 108-402) with Mn²⁺AMP-PNP (filled circles)). (A) C-subunit residues 278-289 (α:Hα:I loop), (B) C-subunit residues 247-261, (α:G) (C) C-subunit residues 212-221 (APE-α:F loop). (D) Structure of the C subunit (PDB: 1ATP) with the N lobe colored white and the C lobe in wheat and above 3 fragment peptides in red.

Figure 6

(A)–(F) Deuterium exchange in six fragment peptides of the C subunit bound to RIIβ(108–402) with (filled circles)) and without Mn²⁺AMP-PNP (open circles)). (A) Residues 278-289, (B) Residues 247-261, (C) Residues 212-221, (D) Residues 163-172, (E) Residues 133-145, (F) Residues 44-54.

Figure 7

(A)–(F) Deuterium exchange in six peptide fragments of the C subunit when bound to Mg²⁺ATP (x), RIIβ(108–402) and Mn²⁺AMP-PNP (filled circles)), and RIα; (1–379) with Mg²⁺ATP(open squares)). (A) Residues 278-289, (B) Residues 247-261, (C) Residues 212-221, (D) Residues 163-172, (E) Residues 133-145, (F) Residues 44-54, (G) Allosteric coupling of αH-αI loop C(residues 278-289) (in purple) and the ATP binding site (Glycine-rich loop C(residues 44-54) (in yellow)), (H) Close-up of αH-αI loop C(residues 278-289) highlighting residues important for interactions between Domain B of RIIβ and the C-subunit.