1OI8

5'-Nucleotidase (E. coli) with an Engineered Disulfide Bridge (P90C, L424C)

Summary for 1OI8

• Entry DOI: 10.2210/pdb1oi8/pdb
• Related: 1HO5 1HP1 1HPU 1OID 1OIE 1USH 2USH
• Descriptor: PROTEIN USHA, MANGANESE (II) ION, SULFATE ION, ... (5 entities in total)
• Functional Keywords: metalloprotein, hydrolase, domain movement, disulfide engineering, udp-sugar hydrolase
• Biological source: ESCHERICHIA COLI
• Total number of polymer chains: 2
• Total formula weight: 118995.15

Authors

Schultz-Heienbrok, R.,Maier, T.,Straeter, N. (deposition date: 2003-06-10, release date: 2004-06-10, Last modification date: 2023-12-13)

Primary citation

Schultz-Heienbrok, R.,Maier, T.,Straeter, N.
Trapping a 96 Degree Domain Rotation in Two Distinct Conformations by Engineered Disulfide Bridges
Protein Sci., 13:1811-, 2004

PubMed Abstract: Engineering disulfide bridges is a common technique to lock a protein movement in a defined conformational state. We have designed two double mutants of Escherichia coli 5'-nucleotidase to 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 crystal forms with three independent conformers, which differ from each other by a rotation of up to 12 degrees of the C-terminal domain with respect to the N-terminal domain. This finding, as well as an analysis of the domain motion in the crystal, indicates that the enzyme still exhibits considerable residual domain flexibility. In the double mutant that was designed to trap the enzyme in the closed conformation, the structure analysis reveals an unexpected intermediate conformation along the 96 degrees rotation trajectory between the open and closed enzyme forms. A comparison of the five independent conformers analyzed in this study shows that the domain movement of the variant enzymes is characterized by a sliding movement of the residues of the domain interface along the interface, which is in contrast to a classical closure motion where the residues of the domain interface move perpendicular to the interface.

PubMed: 15215524
DOI: 10.1110/PS.04629604

Experimental method

X-RAY DIFFRACTION (2.1 Å)