PDBsum entry 2j4y

Signaling protein

PDB id: 2j4y

Contents

Protein chains

328 a.a. *

Ligands

RET ×2

NAG ×2

* Residue conservation analysis

PDB id:

2j4y

Name:

Signaling protein

Title:

Crystal structure of a rhodopsin stabilizing mutant expressed in mammalian cells

Structure:

Rhodopsin. Chain: a, b. Engineered: yes. Mutation: yes

Source:

Bos taurus. Cattle. Organism_taxid: 9913. Organ: eye. Tissue: retina. Cell: rod photoreceptor. Expressed in: chlorocebus aethiops. Expression_system_taxid: 9534. Expression_system_cell_line: cos-1 monkey kidney cells.

Resolution:

3.40Å R-factor: 0.290 R-free: 0.330

Authors:

J.Standfuss,G.Xie,P.C.Edwards,M.Burghammer,D.D.Oprian,G.F.X.Schertler

Key ref:

J.Standfuss et al. (2007). Crystal structure of a thermally stable rhodopsin mutant. J Mol Biol, 372, 1179-1188. PubMed id: 17825322 DOI: 10.1016/j.jmb.2007.03.007

Date:

07-Sep-06 Release date: 25-Sep-07

Protein chains

P02699  (OPSD_BOVIN) -  Rhodopsin from Bos taurus

Seq: 348 a.a.

Struc: 327 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

DOI no: 10.1016/j.jmb.2007.03.007

J Mol Biol 372:1179-1188 (2007)

PubMed id: 17825322

Crystal structure of a thermally stable rhodopsin mutant.

J.Standfuss, G.Xie, P.C.Edwards, M.Burghammer, D.D.Oprian, G.F.Schertler.

ABSTRACT

We determined the structure of the rhodopsin mutant N2C/D282C expressed in mammalian cells; the first structure of a recombinantly produced G protein-coupled receptor (GPCR). The mutant was designed to form a disulfide bond between the N terminus and loop E3, which allows handling of opsin in detergent solution and increases thermal stability of rhodopsin by 10 deg.C. It allowed us to crystallize a fully deglycosylated rhodopsin (N2C/N15D/D282C). N15 mutations are normally misfolding and cause retinitis pigmentosa in humans. Microcrystallographic techniques and a 5 mum X-ray beam were used to collect data along a single needle measuring 5 mumx5 mumx90 mum. The disulfide introduces only minor changes but fixes the N-terminal cap over the beta-sheet lid covering the ligand-binding site, a likely explanation for the increased stability. This work allows structural investigation of rhodopsin mutants and shows the problems encountered during structure determination of GPCRs and other mammalian membrane proteins.

Selected figure(s)

Figure 4.
Figure 4. Electron density maps of N2C/D282C rhodopsin. (a) Retinal omit electron density map (0.75 σ) obtained after molecular replacement and density modification. The retinal (red) was not included but appears clearly in the electron density. (b) Disulfide bond and N15 glycosylation. The σA-weighted 2F[o] – F[c] with electron density contoured at 0.9σ. The disulfide bond between the mutated residues C2 and C282 is well resolved, as is the first residue of the N-glycosylation linked to N15. No density is visible adjacent to the mutated residue 2, which would have been glycosylated in wild-type rhodopsin.

Figure 6.
Figure 6. The N-terminal cap domain. Contacts between the N-terminal cap (blue) and the rest of the receptor (cyan). Atoms of the receptor with a minimum distance of 5 Å to residues 1–33 of the N terminus are colored beige. The covalently bound retinal (red) is separated from the N-terminal cap by the lid formed from the E2 loop. The designed disulfide between C2 and C282 connecting the N-terminal cap with loop E3 is shown as yellow sticks. The molecule is viewed with the extracellular site facing up.

The above figures are reprinted from an Open Access publication published by Elsevier: J Mol Biol (2007, 372, 1179-1188) copyright 2007.

Figures were selected by an automated process.