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Crystal Structure of a Protein Kinase A Complex Print

 

Protein kinase A (PKA) is an enzyme that regulates processes as diverse as growth, memory, and metabolism. In its unactivated state, PKA exists as a tetrameric complex of two catalytic subunits and a regulatory subunit dimer, but when the intracellular signaling molecule cyclic adenosine monophosphate (cAMP) binds to the regulatory subunit, it facilitates dissociation and activation of the catalytic subunits. While separate structures of these subunits were previously known, a group from the University of California, San Diego, is the first to determine (to a resolution of 2.0 Å) the structure of the PKA catalytic subunits bound to the regulatory subunit. The structure of the complex clarifies the mechanism for PKA inhibition, and its comparison with the structure of cAMP bound to the regulatory subunit hints at how cAMP binding drives its activation.

Regulating the Regulator

Turn up the magnification, and the beehive of activity within a biological cell matches that in any large metropolis. Specialized proteins called enzymes drive most of the activity by modifying other proteins in a way that changes how they function. Having done their work, the enzymes return to their original state, ready to repeat this cycle as long as needed. In mammalian cells, including human, ubiquitous enzymes known as protein kinase A (or PKA) regulate a huge number of processes, including growth, development, memory, metabolism, and gene expression (conversion of the information in DNA into proteins ready to go to work).

Failure to keep PKA under control can have disastrous consequences, including diseases such as cancer. The task of riding herd on PKA falls to cyclic adenosine monophosphate (cAMP), a messenger molecule involved in transmitting signals within the cell. Drugs based on inhibiting PKA activity are under development for treating disease, so understanding how cAMP accomplishes this task is of some interest to life scientists. Kim et al. have made a major contribution by determining the structure of PKA in its inactive state. Their structure and its comparison with a structure of cAMP locked in embrace with a key portion of PKA, the regulatory subunit, provides both a molecular mechanism for inhibition of PKA and suggests how cAMP binding leads to activation.

Structure of a PKA complex consisting of a catalytically active C subunit (two lobes in gray and tan) and a regulatory R subunit (cyan). The focal point of the complex interface is the hydrogen bond formed between two highly conserved tyrosine residues, one at the G helix (in green) of the C subunit and the other at the phosphate binding cassette (PBC, in yellow) of the R subunit. In the complex, the inhibitor/linker region (red) of the R subunit blocks the active site of the C subunit between the two lobes. Click on the image to see a movie of the complex as it rotates 360 degrees.

The PKA family of enzymes is ubiquitous in mammalian cells (e.g., it constitutes approximately 2% of the human genome) and is a prototype for the entire kinase superfamily. Poorly regulated kinase activity can cause many diseases such as cancer. To probe the molecular mechanism of PKA regulation, the group crystallized a complex between the catalytic (C) subunits and the regulatory (R) subunit of PKA. For this purpose, they used a deletion mutant, RIα, that contains key portions of the R subunit. The complex was stabilized against dissociation by crystallizing it in the presence of AMP-PNP, a nonhydrolyzable analog of ATP, and excess Mn2+.

From previous work, it was known that the C subunit consists of a small and large lobe with the active site forming a cleft between the two lobes. The small lobe provides the binding site for adenosine triphosphate (ATP), while the large lobe provides catalytic residues and a docking surface for peptide/protein substrates. In the modular R subunit, two tandem cAMP-binding domains (CBD-A and CBD-B) at the C-terminus are joined to an N-terminal dimerization domain by a flexible linker that includes a substrate-like inhibitor sequence. The inhibitor docks to the active site cleft of the C subunit in the absence of cAMP. In the presence of cAMP, a phosphate binding cassette (PBC) anchors the cAMP and shields it from solvent.

Electrostatic surface potential of the complex (left) and with its interface opened up to view the surfaces of individual subunits (right). The linker segment complements site 3, the PBC complements site 2, and the inhibitor site complements site 1 of the C subunit.

The architecture of the RIα:C complex reveals an extended interface that covers nearly 3000 Å2. Although the C subunit assumes a fully closed conformation with Mn2+AMP-PNP bound at the active site cleft, it does not undergo other major conformational changes as a result of complex formation. The binding surface extends from the inhibitor binding site at the active site cleft (site 1), across the G helix (site 2) and through to the activation loop (site 3).

In contrast to the C subunit, RIα undergoes major conformational changes upon complex formation. Three general features describe the binding: the inhibitor sequence docks to the active site cleft; the linker segment that connects the inhibitor peptide to CBD-A becomes ordered; and the helical subdomain within CBD-A docks onto the large lobe of the C subunit. The inhibitor peptide and linker region are disordered in the crystal structure of cAMP-bound RIα, whereas in the complex this segment binds as an extended chain along the surface of the active site cleft and closely interacts with PBC, another important binding site to the C subunit.

Transition of the R subunit from the cAMP-bound (left) to the C-bound (right) conformation. Relative to the cAMP-bound form with the cAMP (ball and stick) anchored by the phosphate binding cassette (PBC, yellow), binding of the C subunit accompanies three major changes in the R subunit: (1) The disordered inhibitor peptide and linker region (red) docks to the active-site cleft of the C subunit and becomes ordered. (2) Helices rearrange into an extended conformation. (3) The PBC stretches out, and a tyrosine residue at the center binds tightly to another tyrosine of the C subunit. Competition of cAMP and the C subunit for the PBC lies at the heart of cAMP-dependent regulation of PKA. Click on the image to see a movie of the transition.

The structure of complex shows that the essence of PKA regulation is in the dynamic nature of its R subunit, which is very flexible and exhibits two distinct structures (noninhibiting and inhibiting) depending on whether it binds cAMP or the C subunit. Comparison of this structure with structures of RIα in its cAMP-bound conformation provided insight into the structural basis for cAMP-induced activation of PKA. The researchers believe that cAMP causes the dissociation of the complex by binding to the PBC, and in winning the binding competition drives away the C subunit from the PBC.

 


 

Research conducted by C. Kim, N.-H. Xuong, and S.S. Taylor (University of California, San Diego).

Research funding: National Institutes of Health. Operation of the ALS is supported by the U.S. Department of Energy, Office of Basic Energy Sciences.

Publication about this research: C. Kim, N.-H. Xuong, and S.S. Taylor, "Crystal structure of a complex between catalytic and regulatory (Rlα) subunits of PKA,"Science 307, 690 (2005).