PDBsum entry 1j3h

Transferase

PDB id: 1j3h

Contents

Protein chains

332 a.a. *

Ligands

MPD ×2

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Dynamic features of camp-Dependent protein kinase revealed by apoenzyme crystal structure.

Authors

P.Akamine, Madhusudan, J.Wu, N.H.Xuong, L.F.Ten eyck, S.S.Taylor.

Ref.

J Mol Biol, 2003, 327, 159-171. [DOI no: 10.1016/S0022-2836(02)01446-8]

PubMed id

12614615

Abstract

To better understand the mechanism of ligand binding and ligand-induced conformational change, the crystal structure of apoenzyme catalytic (C) subunit of adenosine-3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) was solved. The apoenzyme structure (Apo) provides a snapshot of the enzyme in the first step of the catalytic cycle, and in this unliganded form the PKA C subunit adopts an open conformation. A hydrophobic junction is formed by residues from the small and large lobes that come into close contact. This "greasy" patch may lubricate the shearing motion associated with domain rotation, and the opening and closing of the active-site cleft. Although Apo appears to be quite dynamic, many important residues for MgATP binding and phosphoryl transfer in the active site are preformed. Residues around the adenine ring of ATP and residues involved in phosphoryl transfer from the large lobe are mostly preformed, whereas residues involved in ribose binding and in the Gly-rich loop are not. Prior to ligand binding, Lys72 and the C-terminal tail, two important ATP-binding elements are also disordered. The surface created in the active site is contoured to bind ATP, but not GTP, and appears to be held in place by a stable hydrophobic core, which includes helices C, E, and F, and beta strand 6. This core seems to provide a network for communicating from the active site, where nucleotide binds, to the peripheral peptide-binding F-to-G helix loop, exemplified by Phe239. Two potential lines of communication are the D helix and the F helix. The conserved Trp222-Phe238 network, which lies adjacent to the F-to-G helix loop, suggests that this network would exist in other protein kinases and may be a conserved means of communicating ATP binding from the active site to the distal peptide-binding ledge.

Figure 1.
Figure 1. (A) ApoA and ApoB superimposed. The two molecules in the asymmetric unit are superimposed to show that they differ in overall domain rotation. ApoA is black and ApoB is gold. Broken lines are distances of representative areas of the small lobe. The distance between the Ser53 C^a atoms, in the Gly-rich loop, is 1.9 Å. The distance between the C^a atoms of Ser339 is 3.4 Å. An MPD molecule and a covalently attached b-ME group were seen in both structures. The ApoA MPD and b-ME modified Cys199 are pink. Residues 128-300 were superimposed. (B) The F[o] -F[c] omit map of Cys199 and the covalently attached b-ME, contoured at 3s. Oxygen, red; nitrogen, blue; carbon, gray; sulfur, green.

Figure 2.
Figure 2. (A) Closed conformation and ApoA. Superposition of ApoA (black) and C:AlF:SP20 (green),[9] in the open and closed conformations, respectively. Broken lines show the distances of two representative parts of the small lobe; the Gly-rich loop (Ser53 C^a) and the C-terminal tail (Ser339 C^a). Residues 128-300 were superimposed. (B) Intralobe hydrophobic contacts. The hydrophobic patch between the small and large lobes, which may provide the "grease" for the shearing motion associated with domain rotation, is shown. Glu91, a conserved residue in the C helix (C), which is important for orienting the phosphate groups of ATP during phosphoryl transfer, is preformed and is within hydrogen-bonding distance from the amide hydrogen atom of Phe185 in the large lobe. From the small lobe (gold ribbon) are residues Glu91, Ile94, Val98, Phe100, Phe102, Leu103, and Val104 (side-chains, blue). From the large lobe, the residues shown are Thr153, Tyr156, Leu162, Tyr179, Gln181, and Phe185 (side-chains, pink). Residues that come into close contact are Ile94-Leu162, Phe185; Val98, Phe100 and Leu103-Tyr156, Leu103-Phe185, and Val104-Gln181. Other hydrogen bonding pairs are: Asn99 amide group to Tyr156 hydroxyl group, and Val104 amide hydrogen atom to Val182 carbonyl oxygen atom. The gray ribbon represents the E helix (E) and the black ribbon includes the Mg-positioning loop (Mg), both from the large lobe. Helix A (A) is shown, since Phe18, Ala22, and Phe26 contribute, peripherally.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2003, 327, 159-171) copyright 2003.

Secondary reference #1

Title

Crystal structure of a transition state mimic of the catalytic subunit of camp-Dependent protein kinase.

Authors

Madhusudan, P.Akamine, N.H.Xuong, S.S.Taylor.

Ref.

Nat Struct Biol, 2002, 9, 273-277. [DOI no: 10.1038/nsb780]

PubMed id

11896404

Figure 1.
Figure 1. Overall view of the Mg[2]ADP -SP20 -AlF[3] complex of the catalytic subunit (cAPK) with the difference density at the position of AlF[3]. a, The disordered region of the catalytic subunit consisting of residues 5 -13 is indicated by dashes. SP20 and Mg2+ ions are displayed in yellow and red, respectively, and ADP and AlF[3] are shown in green. The Gly-rich loop is colored in magenta, and black spheres indicate the three phosphorylation sites observed in the structure. The MPD molecule is displayed in cyan. This figure was generated using MOLSCRIPT33. b, Stereo view of the annealed F[o] - F[c] omit map contoured at 6.0 . This figure was generated from BOBSCRIPT33, 34. Dashed lines indicate the aluminum (Al) coordination with the -phosphate of ADP and the hydroxyl group of Ser from the SP20.

Figure 3.
Figure 3. Schematic representation depicting the detailed interactions of aluminum fluoride with Mg[2]ADP, active site residues of the catalytic subunit, water molecules and the phosphorylation site Ser from SP20. Mg2+ ions and water molecules are indicated in large and small spheres, respectively. Residues displayed in ball-and-stick representation exhibit the exact conformation and relative orientation as observed in the crystal structure; however, they have been displaced with respect to one another for clarity.

The above figures are reproduced from the cited reference with permission from Macmillan Publishers Ltd

Secondary reference #2

Title

Structure of the mammalian catalytic subunit of camp-Dependent protein kinase and an inhibitor peptide displays an open conformation.

Authors

R.Karlsson, J.Zheng, N.Xuong, S.S.Taylor, J.M.Sowadski.

Ref.

Acta Crystallogr D Biol Crystallogr, 1993, 49, 381-388. [DOI no: 10.1107/S0907444993002306]

PubMed id

15299513

Figure 2.
ig. 2. Stereoview of the environ- ment of His87. The small lobe is indicated in red and the large lobe in blue. The inhibitor is indicated in black. (a) In the recombinant mouse C subunit, His87 of the small lobe interacts with the stble phosphoryation site of Thr197 of the large lobe and the carbonyl group of the main chain of Glu86 interacts with the side chain of Asng0. (b) In the pocine heart C subunit, His87 moves away from the phosphate of Thr197 and the side chain of Asng0 of the small lobe interacts with the carbonyl of Ala188 of the large lobe. istances are given in A.

The above figure is reproduced from the cited reference with permission from the IUCr

Secondary reference #3

Title

Crystal structures of the myristylated catalytic subunit of camp-Dependent protein kinase reveal open and closed conformations.

Authors

J.Zheng, D.R.Knighton, N.H.Xuong, S.S.Taylor, J.M.Sowadski, L.F.Ten eyck.

Ref.

Protein Sci, 1993, 2, 1559-1573. [DOI no: 10.1002/pro.5560021003]

PubMed id

8251932

Figure 3.
Fig. 3. Active-siteregionin the open conformational state. A: Theactive regionof the binary complex from the mamma- lian enzyme(red)issuperimposedinstereowith the correspondingregionfrom the ternary complexof therecombinantenzyme (blue). The ATP in the ternary complex is shon in black. B: Some f the distances that hangemostgoing to open confor- mation of the mammalian binary complex are indicated. Distancesin A betweenseveralkey residuesin the two structures are as follows (numbers in parentheses correspond to the closed conformation): Asp 184 to Gly 52 or, 10.5 (6.5); sp 184 to Lys 72 NZ, 6. (3.7); 54 CZ to His 87 ND1, 7.4 His 87 NE2 to P-Thr 197 OE2,6.0 (2.7); lu 91 OEl to Lys 72 HZ, 4.1

Figure 6.
Fig. 6. The Ca-backbone of heclosedand open conformations, highlightingcriticalchangesathe cleft interface.The binary complex of therecombinantC-subunit,representingthe closed conformation, isshown on heright.The open conformation associatedwiththemamalianbinarycomplexisshown on the left. TheN-terminalregions(1-127inthemammalianC-subunit and 9-127 in therecombinantenzymeareshowninred.TheC-terminalregions(resiues128-350)areshowninblue.Thepep- tidesare shown inblack. The fattyacidinthemammaliancomplexandtheMEGA-8detergentintherecombinantcomlex areshowningreen. Key residues at he cleft interface(His87, Asn 90, Thr 197),whoseenironmentchangesasaconsequence of cleft opening,are also indicated in reen.

The above figures are reproduced from the cited reference with permission from the Protein Society

Secondary reference #4

Title

Crystal structure of the catalytic subunit of camp-Dependent protein kinase complexed with mgatp and peptide inhibitor.

Authors

J.Zheng, D.R.Knighton, L.F.Ten eyck, R.Karlsson, N.Xuong, S.S.Taylor, J.M.Sowadski.

Ref.

Biochemistry, 1993, 32, 2154-2161. [DOI no: 10.1021/bi00060a005]

PubMed id

8443157

Secondary reference #5

Title

A binary complex of the catalytic subunit of camp-Dependent protein kinase and adenosine further defines conformational flexibility.

Authors

N.Narayana, S.Cox, X.Nguyen-Huu, L.F.Ten eyck, S.S.Taylor.

Ref.

Structure, 1997, 5, 921-935. [DOI no: 10.1016/S0969-2126(97)00246-3]

PubMed id

9261084

Figure 7.
Figure 7. The C-terminal tail. (a) Schematic picture of the three distinct segments of the C-terminal tail (residues 301-350): the large lobe anchor, the C-terminal gate, and the small lobe anchor. Tyr330 (shown in yellow) makes contact with both the nucleotide and the P -3 arginine in PKI(5-24). (b) The three sections of the C-terminal tail are shown in pink, purple and red in the open mC·I structure; the closed ternary rC·I·ATP is shown in white.

The above figure is reproduced from the cited reference with permission from Cell Press