© RCSB

I. Signal transduction and cAMP protein phosphorylation

Representative pathway for the activation of cAMP-dependent protein kinase (PKA). In this example glucagon binds to its' cell-surface receptor, thereby activating the receptor. Activation of the receptor is coupled to the activation of a receptor-coupled G-protein (GTP-binding and hydrolyzing protein) composed of 3 subunits. Upon activation the alpha subunit dissociates and binds to and activates adenylate cyclase . Adenylate cylcase then converts ATP to cyclic-AMP (cAMP). The cAMP thus produced then binds to the regulatory subunits of PKA leading to dissociation of the associated catalytic subunits. The catalytic subunits are inactive until dissociated from the regulatory subunits. Once released the catalytic subunits of PKA phosphorylate numerous substrate using ATP as the phosphate donor.

                                   
Figure 2: Ribbon Diagram of Secondary Structures                                       Figure 3: Ball and Stick Representation  of Chains E and I                            

Data obtained from the crystal structure of a ternary complex consisting of the catalytic subunit complexed with the inhibitor and MgATP (magnesium adenosine triphosphate) described the presence of a catalytic core which spans through residues 40-280 as shown in blue in Figure 5. The catalytic core is divided into two lobes as shown by the blue and green atoms in Figure 4. The big lobe (blue) is designated as the peptide binding site while the small lobe (green) correspond to the nucleotide binding site. The structure also revealed a deep cleft between the lobes which was occupied  by MgATP as shown in green in Figure 5.

Figure 4: Space filling model of the calatlytic core (blue)                            Figure 5:  Model of the subunit complexed with MgATP (green)

and  nucleotide binding site (green)                                                                         inside the catalytic  core (blue)

III. Structure Classification (SCOP and CATH) 

     This  cAMP dependent protein kinase was isolated from the recombinant murine catalytic subunit expressed from E.coli. It belongs to a family of Serine/Threonine kinases, the family of enzymes that catalyze the phosphorylation of Serine and Threonine residues. The catalytic core of the enzyme was determined to be concentrated around  40 through 280. This constitute a conserved catalytic core which is shared by more than 100 protein kinases. The structure has both beta and alpha helices and is grouped with the Protein-kinase like family whose structures consist of two alpha and beta domains, with the C terminal domain mostly alpha helical.  SCOP classification is shown as follows:

Lineage:

1. Root: scop
2. Class: Alpha and beta proteins (a+b)
   Mainly antiparallel beta sheets (segregated alpha and beta regions)
3. Fold: Protein kinase-like (PK-like)
   consists of two alpha+beta domains, C-terminal domain is mostly alpha helical
4. Superfamily: Protein kinase-like (PK-like)
   shares functional and structural similarities with the ATP-grasp fold and PIPK
5. Family: Serine/threonin kinases
6. Protein: cAMP-dependent PK, catalytic subunit
7. Species: Mouse (Mus musculus)

        The protein structure is classified into two domains. The first domain consist of mostly alpha helices arranged in an orthogonal bundle. This domain is believed to be responsible for peptide binding and catalysis. The same number of secondary structures and similar clustering patterns are observed in the catalytic domains other enzymes in the Transferase(Phosphotransferase) family. A smaller domain consisting of 127 residues contains alpha and beta residues arranged in a two layer sandwich. This domain is believed to be associated with nucleotide binding  and is shared by other proteins in the kinase family. The CATH classification for the two domains are shown below:

omain	CATH code	Length	Image
2cpkE1	1.10.510.10	209
2cpkE2	3.30.200.20	127

Domain 1

C1		Mai nly Alpha
A10		Orthogonal Bundle
T510		Transferase(Phosphotransferase) domain 1
H10		TRANSFERASE(PHOSPHOTRANSFERASE)

C3		Alpha Beta
A30		2-Layer Sandwich
T200		KINASE
H20		KINASE

IV. Crystallographic Data

The crystallogtaphic work were carried out on the recombinant murine (mouse) catalytic subunit expressed from E.coli. The recombinant enzyme was cocrystallized with a peptide inhibitor PKI 5-24 (TTYADFIASGRTGRRNAIHD) derived from the N-terminal sequence of  the protein  kinase inhibitor protein (PKI). The binary complex crystallized in an orthorhombic space group (P2<1>2<1>2<1>). The crystals diffracts to 2.7 A  resolution to an R factor of 0.212.  More infromation on the crystallographic data can be otained from this link .

As described by Zheng, et al, the recombinant mouse C subunit crsytals (as binary complex with PKI(5-24) or as ternary complex with MgATP and PKI(5-24)) were prepared using the following conditions. The purified murine recombinant subunit was brought to a final concentration of 10 mg/ml  and dialyzed against 50mM Bicine buffer and 150 mM ammonium acetate. Crystals were grown at 4 degrees by hanging drop vapor diffusion after mixing equal volumes of protein solution, reservoir solution and 10mM DTT. The reservoir contained 10mM DTT and 8% polyethylene glycol 400. Before sealing the coverslip, methanol was added to the reservoir to a concentration of 15%. The peptide inhibitors and MgATP were introduced by mixing equal volumes  of the protein, the reservoir solution and 10mM DTT with MgATP and the given inhibitor.

Unit cell dimension
A	B	C	alpha	beta	gamma	Z
73.62	76.52	80.14	90	90	90	4

Space group:

P2<1>2<1>2<1> - Orthorhombic

Crystal Density: 2.62V

Diffraction limit (Angstroms): 2.7

Diffraction life time (Hours): 0

Crystal dimensions in mm: 0 x 0 x 0

Reference's designation of this crystal form: complex with 5-24 peptide inhibitor

V. Structural Alignments

	ID	Z-Score	RMSD(Å)	Seq.(%)	Aligned / Size	Gap	Exp.	Name
	2CPK:E	Query					X-Ray	$C-/AMP$-DEPENDENT PROTEIN KINASE (E.C.2.7.1.37) ($C/APK$) (CATALYTIC SUBUNIT)
	1APM:E Neighbors	7.8	0.3	99.7	350 / 350	0	X-Ray	$C-/AMP$-DEPENDENT PROTEIN KINASE (E.C.2.7.1.37) ($C/APK$) (CATALYTIC SUBUNIT) "
	1JLU:E Neighbors	7.8	0.4	99.4	350 / 350	0	X-Ray	MOL_ID: 1; MOLECULE: AMP-DEPENDENT PROTEIN KINASE, ALPHA-CATALYTIC SUBUNIT; CHAI
	1FMO:E Neighbors	7.8	0.4	99.4	350 / 350	0	X-Ray	MOL_ID: 1; MOLECULE: CAMP-DEPENDENT PROTEIN KINASE; CHAIN: E; FRAGMENT: CATALYTI
	1JBP:E Neighbors	7.8	0.4	99.1	350 / 350	0	X-Ray	MOL_ID: 1; MOLECULE: CAMP-DEPENDENT PROTEIN KINASE, ALPHA-CATALYTIC SUBUNIT; CHA
	1ATP:E Neighbors	7.8	0.4	100.0	336 / 350	0	X-Ray	$C-/AMP$-DEPENDENT PROTEIN KINASE (E.C.2.7.1.37) ($C/APK$) (CATALYTIC SUBUNIT) C
	1CDK:B Neighbors	7.8	0.4	97.0	343 / 352	0	X-Ray	MOL_ID: 1; MOLECULE: CAMP-DEPENDENT PROTEIN KINASE; CHAIN: A, B; DOMAIN: CATALYT