PDBsum entry: 1tl7

Lyase

PDB id: 1tl7

Contents

Protein chains

189 a.a. *

189 a.a. *

330 a.a. *

Ligands

FOK

ONM

GSP

Metals

_MN ×2

_MG

_CL

Waters ×18

* Residue conservation analysis

PDB id:

1tl7

Name:

Lyase

Title:

Complex of gs- with the catalytic domains of mammalian adenylyl cyclase: complex with 2'(3')-o-(n- methylanthraniloyl)-guanosine 5'-triphosphate and mn

Structure:

Adenylate cyclase, type v. Chain: a. Fragment: c1a domain of adenylyl cyclase. Synonym: atp pyrophosphate-lyase, ca2+, -inhibitable adenylyl cyclase. Engineered: yes. Adenylate cyclase, type ii. Chain: b. Fragment: c2a domain of adenylyl cyclase.

Source:

Canis lupus familiaris. Dog. Organism_taxid: 9615. Strain: familiaris. Gene: name=adcy5. Expressed in: escherichia coli bl21. Expression_system_taxid: 511693. Rattus norvegicus. Norway rat.

Biol. unit:

Trimer (from PQS)

Resolution:

2.80Å R-factor: 0.254 R-free: 0.298

Authors:

T.C.Mou,A.Gille,R.J.Seifert,S.R.Sprang

Key ref:

T.C.Mou et al. (2005). Structural basis for the inhibition of mammalian membrane adenylyl cyclase by 2 '(3')-O-(N-Methylanthraniloyl)-guanosine 5 '-triphosphate. J Biol Chem, 280, 7253-7261. PubMed id: 15591060 DOI: 10.1074/jbc.M409076200

Date:

09-Jun-04 Release date: 14-Dec-04

Protein chain

P30803  (ADCY5_CANFA) -  Adenylate cyclase type 5

Seq: 1265 a.a.

Struc: 189 a.a.*

Protein chain

P26769  (ADCY2_RAT) -  Adenylate cyclase type 2

Seq: 1090 a.a.

Struc: 189 a.a.

Protein chain

P04896  (GNAS2_BOVIN) -  Guanine nucleotide-binding protein G(s) subunit alpha isoforms short

Seq: 394 a.a.

Struc: 330 a.a.

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: Chains A, B: E.C.4.6.1.1 - Adenylate cyclase.
• Reaction: ATP = 3',5'-cyclic AMP + diphosphate

ATP (Bound ligand (Het Group name = ONM) matches with 73.00% similarity) = 3',5'-cyclic AMP (Bound ligand (Het Group name = GSP) matches with 68.00% similarity) + diphosphate

• Cofactor: Pyridoxal 5'-phosphate

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

 Gene Ontology (GO) functional annotation 

• Cellular component: membrane (5 terms)
• Biological process: intracellular signal transduction (25 terms)
• Biochemical function: nucleotide binding (10 terms)

reference

DOI no: 10.1074/jbc.M409076200

J Biol Chem 280:7253-7261 (2005)

PubMed id: 15591060

Structural basis for the inhibition of mammalian membrane adenylyl cyclase by 2 '(3')-O-(N-Methylanthraniloyl)-guanosine 5 '-triphosphate.

T.C.Mou, A.Gille, D.A.Fancy, R.Seifert, S.R.Sprang.

ABSTRACT

Membrane-bound mammalian adenylyl cyclase (mAC) catalyzes the synthesis of intracellular cyclic AMP from ATP and is activated by stimulatory G protein alpha subunits (Galpha(s)) and by forskolin (FSK). mACs are inhibited with high potency by 2 '(3')-O-(N-methylanthraniloyl) (MANT)-substituted nucleotides. In this study, the crystal structures of the complex between Galpha(s).GTPgammaS and the catalytic C1 and C2 domains from type V and type II mAC (VC1.IIC2), bound to FSK and either MANT-GTP.Mg(2+) or MANT-GTP.Mn(2+) have been determined. MANT-GTP coordinates two metal ions and occupies the same position in the catalytic site as P-site inhibitors and substrate analogs. However, the orientation of the guanine ring is reversed relative to that of the adenine ring. The MANT fluorophore resides in a hydrophobic pocket at the interface between the VC1 and IIC2 domains and prevents mAC from undergoing the "open" to "closed" domain rearrangement. The K(i) of MANT-GTP for inhibition of VC1.IIC2 is lower in the presence of mAC activators and lower in the presence of Mn(2+) compared with Mg(2+), indicating that the inhibitor binds more tightly to the catalytically most active form of the enzyme. Fluorescence resonance energy transfer-stimulated emission from the MANT fluorophore upon excitation of Trp-1020 in the MANT-binding pocket of IIC2 is also stronger in the presence of FSK. Mutational analysis of two non-conserved amino acids in the MANT-binding pocket suggests that residues outside of the binding site influence isoform selectivity toward MANT-GTP.

Selected figure(s)

Figure 1.
FIG. 1. Global and detailed views of G [s]·VC1·IIC2 complex with MANT-GTP. A, complex of G [s]-activated mAC with MANT-GTP and two Mn2+ ions in the catalytic site. FSK, MANT-GTP, and two metal ions are bound in the ventral cleft between the C1 and C2 domains. The following coloring scheme is used here and in Figs. 2 and 3: the VC1 and IIC2 domains are colored tan and mauve, respectively; the switch II helix of G [s]·GTP S is shown as a red cylinder; FSK and MANT-GTP are drawn as stick models; carbon atoms are gray, nitrogen atoms are blue, oxygen atoms are red, phosphorus atoms are green, and the two Mn2+ ions are shown as metallic orange spheres. B, substrate-binding site of VC1·IIC2 showing MANT-GTP and two Mn2+ ions. Secondary structure elements are labeled as defined previously (16). The positions of the 1- 1- 2 and 3- 4 of VC1 and 7'- 8' of IIC2 in the mAC complex with (closed conformation) or without (open conformation) the competitive inhibitor, -L-2',3'-dd-5'-ATP (Protein Data Bank codes 1CJU [PDB] and 1AZS, respectively) (16, 23), were superimposed and depicted as transparent, rose- and gray-colored elements, respectively. C, interactions are shown among protein residues, MANT-GTP, and two metal ions in the VC1·IIC2 substrate-binding site. MANT-GTP and protein residues are shown as stick models. Stick models of -L-2',3'-dd-5'-ATP and selected side chains in Asn-1025 in 1CJU (see A) are shown in yellow. The gray dashed lines depict the hydrogen bonds between MANT-GTP and protein residues and coordination of the metal ions at sites A and B (see text). Because of the high thermal parameters of the nucleotide (see text), the hydrogen bond distances are approximate. The coordination between metal ions and MANT-GTP is similar to that in the complex with -L-2',3'-dd-5'-ATP; metal A has four ligands, whereas metal B has five ligands. D, detailed view of the purine-binding pocket in the mAC substrate-binding site (see text) The superimposed adenosine group of -L-2',3'-dd-5'-ATP is shown in yellow. The distances of hydrogen bond between guanine ring and surrounding protein residues are as indicated.

Figure 2.
FIG. 2. Difference electron density for 3·-O-MANT-GTP in complex with G [s]·VC1·IIC2. The complexes formed in the presence with Mn2+ or Mg2+ are shown in A and B, respectively. The blue wire cage corresponds to |F[o]| - |F[c]| electron density contoured at 2.5 . Coordinates for MANT-GTP and the two metal ions were omitted from the phasing model.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2005, 280, 7253-7261) copyright 2005.

Figures were selected by the author.