Trapping a 96{degrees} domain rotation in two distinct conformations by engineered disulfide bridges

doi:10.1110/ps.04629604

Trapping a 96° domain rotation in two distinct conformations by engineered disulfide bridges

Robert Schultz-Heienbrok¹, Timm Maier¹ and Norbert Sträter²

¹ Institut für Chemie/Kristallographie, Freie Universität Berlin, 14195 Berlin, Germany
² Biotechnologisch-Biomedizinisches Zentrum der Universität Leipzig, 04103 Leipzig, Germany

(RECEIVED January 14, 2004; FINAL REVISION March 23, 2004; ACCEPTED March 23, 2004)

Abstract

Engineering disulfide bridges is a common technique to locka protein movement in a defined conformational state. We havedesigned two double mutants of Escherichia coli 5'-nucleotidaseto trap the enzyme in both an open (S228C, P513C) and a closed(P90C, L424C) conformation by the formation of disulfide bridges.The mutant proteins have been expressed, purified, and crystallized,to structurally characterize the designed variants. The S228C,P513C is a double mutant crystallized in two different crystalforms with three independent conformers, which differ from eachother by a rotation of up to 12° of the C-terminal domainwith respect to the N-terminal domain. This finding, as wellas an analysis of the domain motion in the crystal, indicatesthat the enzyme still exhibits considerable residual domainflexibility. In the double mutant that was designed to trapthe enzyme in the closed conformation, the structure analysisreveals an unexpected intermediate conformation along the 96°rotation trajectory between the open and closed enzyme forms.A comparison of the five independent conformers analyzed inthis study shows that the domain movement of the variant enzymesis characterized by a sliding movement of the residues of thedomain interface along the interface, which is in contrast toa classical closure motion where the residues of the domaininterface move perpendicular to the interface.

Keywords: X-ray crystallography; 5'-nucleotidase; protein engineering; disulfide trapping; disulfide geometry; domain rotation; protein movement; protein flexibility; TLS refinement; protein conformation

Abbreviations: 5'-NT, 5'-nucleotidase from E. coli • 5'NT–C, 5'-nucleo-tidase designed to be trapped in a closed conformation • 5'NT–O, designed to be trapped in an open conformation • BPNP, bis-para-nitrophenyl phosphate • DTT, dithiothreitol • EDTA, ethylene-di-amine-tetra-acetic acid • IPTG, isopropylthiogalactosid • LB, Luria-Bertani broth • PEG, polyethylene glycol • PNPP, para-nitrophenyl phosphate • RMSD, root-mean-square deviation • TLS, translation libration screw.

Reprint requests to: Norbert Sträter, Biotechnologisch-Biomedizinisches Zentrum der Universität Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany; e-mail: strater{at}bbz.uni-leipzig.de; fax: 49 (0)341-97-31319.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04629604.

Add to CiteULike CiteULike Add to Connotea Connotea Add to Del.icio.us Del.icio.us Add to Digg Digg Add to Reddit Reddit Add to Technorati Technorati What's this?

This article has been cited by other articles:

I. Alves-Pereira, J. Canales, A. Cabezas, P. Martin Cordero, M. J. Costas, and J. C. Cameselle
CDP-Alcohol Hydrolase, a Very Efficient Activity of the 5'-Nucleotidase/UDP-Sugar Hydrolase Encoded by the ushA Gene of Yersinia intermedia and Escherichia coli
J. Bacteriol., September 15, 2008; 190(18): 6153 - 6161.
[Abstract] [Full Text] [PDF]

S. Mouilleron and B. Golinelli-Pimpaneau
Domain motions of glucosamine-6P synthase: Comparison of the anisotropic displacements in the crystals and the catalytic hinge-bending rotation
Protein Sci., March 1, 2007; 16(3): 485 - 493.
[Abstract] [Full Text] [PDF]

				S. Mouilleron and B. Golinelli-Pimpaneau Domain motions of glucosamine-6P synthase: Comparison of the anisotropic displacements in the crystals and the catalytic hinge-bending rotation Protein Sci., March 1, 2007; 16(3): 485 - 493. [Abstract] [Full Text] [PDF]