Dynamics connect substrate recognition to catalysis in protein kinase A

Abstract

Atomic resolution studies of protein kinases have traditionally been carried out in the inhibitory state, limiting our current knowledge on the mechanisms of substrate recognition and catalysis. Using NMR, X-ray crystallography and thermodynamic measurements, we analyzed the substrate recognition process of cAMP-dependent protein kinase (PKA), finding that entropy and protein dynamics play a prominent role. The nucleotide acts as a dynamic and allosteric activator by coupling the two lobes of apo PKA, enhancing the enzyme dynamics synchronously and priming it for catalysis. The formation of the ternary complex is entropically driven, and NMR spin relaxation data reveal that both substrate and PKA are dynamic in the closed state. Our results show that the enzyme toggles between open and closed states, which indicates that a conformational selection rather than an induced-fit mechanism governs substrate recognition.

Fig. 1

X-ray crystal structure of the PKA-C ternary complex containing AMP-PNP and PLN_1-19. (a) The asymmetric unit revealed two molecules of PKA-C, an apo (open, grey) form and a ternary (closed, tan) complex. (b) The ternary complex is missing the first 15 residues of the N-terminus, part of the glycine-rich loop and C-terminus, which is likely due to conformational disorder.

Fig. 2

Details of the interaction of isotopically labeled PLN_1-20 with PKA-C. (a) NMR nuclear spin dynamics of PLN_1-20 in the free (black) and bound to PKA-C in the presence of AMP-PNP (blue). (b) Mapping of these dynamics in the bound form shows that ordering around the recognition sequence was observed, while elevated R₂/R₁ values extend the region of interaction to residues 10-17. Data represent fitted values and associated error from non-linear fitting.

Fig. 3

Mapping of the backbone amide dynamics of PKA-C from the apo to ternary complex. Structural elements and loops are indicated. Fast (a) and slow (b) dynamics are shown for PKA-C in the apo (left), binary form (middle), and the ternary complex containing AMP-PNP and PLN_1-20 (right).

Fig. 4

Opening and closing of the enzyme active site cleft. Correlation plots of R_ex for (a) AMP-PNP bound form or (b) PLN_1-20/AMP-PNP bound form with the chemical shift differences of open and closed states. (c) The nucleotide induces opening and closing at the entrance of the enzyme which is reported by contiguous and non-contiguos pathways from the active site (residues denoted in orange). Errors in R_ex were taken from error propagation of equation 1 using the r.m.s noise of the NMR spectra. (d) In the ternary complex, residues which are not linear with opening and closing are distal and are known to interact with the regulatory subunit based on crystal structure 2QCS.

Fig. 5

Model for the mechanism of the formation of a catalytically competent ternary complex. The apo form contains the C-spine residues (red) which are disengaged from the two lobes. Nucleotide binding completes the C-spine architecture and induces the conformational changes throughout the enzyme. The conformational fluctuations (opening and closing) present in the ternary complex limit the rate of catalysis.