PDBsum entry 2ev1

Lyase

PDB id: 2ev1

Contents

Protein chains

185 a.a. *

Ligands

OLA ×2

1PE ×2

Waters ×353

* Residue conservation analysis

PDB id:

2ev1

Name:

Lyase

Title:

Structure of rv1264n, the regulatory domain of the mycobacterial adenylyl cylcase rv1264, at ph 6.0

Structure:

Hypothetical protein rv1264/mt1302. Chain: a, b. Fragment: n-terminal domain. Synonym: adenylyl cyclase. Engineered: yes

Source:

Mycobacterium tuberculosis. Organism_taxid: 1773. Gene: rv1264. Expressed in: escherichia coli. Expression_system_taxid: 469008.

Biol. unit:

Tetramer (from PQS)

Resolution:

1.60Å R-factor: 0.189 R-free: 0.216

Authors:

F.Findeisen,I.Tews,I.Sinning

Key ref:

F.Findeisen et al. (2007). The structure of the regulatory domain of the adenylyl cyclase Rv1264 from Mycobacterium tuberculosis with bound oleic acid. J Mol Biol, 369, 1282-1295. PubMed id: 17482646 DOI: 10.1016/j.jmb.2007.04.013

Date:

30-Oct-05 Release date: 07-Nov-06

Protein chains

P9WMU9  (Y1264_MYCTU) -  pH-sensitive adenylate cyclase Rv1264 from Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)

Seq: 397 a.a.

Struc: 185 a.a.

Enzyme reactions

• Enzyme class: E.C.4.6.1.1 - adenylate cyclase.
• Reaction: ATP = 3',5'-cyclic AMP + diphosphate
• Cofactor: Pyridoxal 5'-phosphate

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

reference

DOI no: 10.1016/j.jmb.2007.04.013

J Mol Biol 369:1282-1295 (2007)

PubMed id: 17482646

The structure of the regulatory domain of the adenylyl cyclase Rv1264 from Mycobacterium tuberculosis with bound oleic acid.

F.Findeisen, J.U.Linder, A.Schultz, J.E.Schultz, B.Brügger, F.Wieland, I.Sinning, I.Tews.

ABSTRACT

The universal secondary messenger cAMP is produced by adenylyl cyclases (ACs). Most bacterial and all eukaryotic ACs belong to class III of six divergent classes. A class III characteristic is formation of the catalytic pocket at a dimer interface and the presence of additional regulatory domains. Mycobacterium tuberculosis possesses 15 class III ACs, including Rv1264, which is activated at acidic pH due to pH-dependent structural transitions of the Rv1264 dimer. It has been shown by X-ray crystallography that the N-terminal regulatory and C-terminal catalytic domains of Rv1264 interact in completely different ways in the active and inhibited states. Here, we report an in-depth structural and functional analysis of the regulatory domain of Rv1264. The 1.6 A resolution crystal structure shows the protein in a tight, disk-shaped dimer, formed around a helical bundle, and involving a protein chain crossover. To understand pH regulation, we determined structures at acidic and basic pH values and employed structure-based mutagenesis in the holoenzyme to elucidate regulation using an AC activity assay. It has been shown that regulatory and catalytic domains must be linked in a single protein chain. The new studies demonstrate that the length of the linker segment is decisive for regulation. Several amino acids on the surface of the regulatory domain, when exchanged, altered the pH-dependence of AC activity. However, these residues are not conserved amongst a number of related ACs. The closely related mycobacterial Rv2212, but not Rv1264, is strongly activated by the addition of fatty acids. The structure resolved the presence of a deeply embedded fatty acid, characterised as oleic acid by mass spectrometry, which may serve as a hinge. From these data, we conclude that the regulatory domain is a structural scaffold used for distinct regulatory purposes.

Selected figure(s)

Figure 3.
Figure 3. Enzyme regulation requires integrity of the linker segment that forms the αN10-switch in the Rv1264 holoenzyme. (a) Comparison of the region forming the αN10-switch in the active state (PDB entry 1Y11, shown in green and ochre) and the inhibited state (PDB entry 1Y10, shown in blue and red). The overlay is based on superposition of the regulatory domains; the catalytic domains are offset by 55° rotation and 6 Å movement between active and inhibited conformations.^8 The ribbon diagrams show with helix α10 starting at residue 160, the linker with the αN10-switch, and the first β-strand (β1) of the catalytic domains. The position of residue 204, where additional amino acids were inserted in the mutagenesis study, is indicated for active and inhibited states. Positions of residues Met193 and Met194 that have been exchanged in the earlier study are indicated.^8 (b) pH activity profiles of insertion of one, two, three or nine amino acids at position 204 can be seen. Standard deviations are shown as vertical bars if they exceed the symbol size, which corresponds to SD = 10%. While one or two inserted amino acids ((●) +S204 and (○) +SA204) result in an overall inhibited phenotype, insertion of three residues ((▪) +SAA204) displays a phenotype similar to the wild-type enzyme (broken line). Figure 3. Enzyme regulation requires integrity of the linker segment that forms the αN10-switch in the Rv1264 holoenzyme. (a) Comparison of the region forming the αN10-switch in the active state (PDB entry 1Y11, shown in green and ochre) and the inhibited state (PDB entry 1Y10, shown in blue and red). The overlay is based on superposition of the regulatory domains; the catalytic domains are offset by 55° rotation and 6 Å movement between active and inhibited conformations.[3]^8 The ribbon diagrams show with helix α10 starting at residue 160, the linker with the αN10-switch, and the first β-strand (β1) of the catalytic domains. The position of residue 204, where additional amino acids were inserted in the mutagenesis study, is indicated for active and inhibited states. Positions of residues Met193 and Met194 that have been exchanged in the earlier study are indicated.[4]^8 (b) pH activity profiles of insertion of one, two, three or nine amino acids at position 204 can be seen. Standard deviations are shown as vertical bars if they exceed the symbol size, which corresponds to SD = 10%. While one or two inserted amino acids ((●) +S204 and (○) +SA204) result in an overall inhibited phenotype, insertion of three residues ((▪) +SAA204) displays a phenotype similar to the wild-type enzyme (broken line). Introduction of the nonapeptide SAAGPSGAA ((□) +9aa-204) leads to a decoupling of regulatory and catalytic domains, as this enzyme is, like the M193P/M194P mutant, no longer pH-responsive.[5]^8

Figure 7.
Figure 7. Model for the mechanism of Rv1264 regulation, illustrated on the active (green) and inhibited states (blue) of the holoenzyme, shown in surface representation. The two structural features that accompany enzyme regulation are the extension of α10 by the αN10-switch region and the different placement of the fatty acid molecule, accompanied by a movement of helix α4. Figure 7. Model for the mechanism of Rv1264 regulation, illustrated on the active (green) and inhibited states (blue) of the holoenzyme, shown in surface representation. The two structural features that accompany enzyme regulation are the extension of α10 by the αN10-switch region and the different placement of the fatty acid molecule, accompanied by a movement of helix α4.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2007, 369, 1282-1295) copyright 2007.

Figures were selected by an automated process.