Title | Crystallization and preliminary X-ray diffraction analysis of kanamycin-binding β-lactamase in complex with its ligand. |
Publication Type | Journal Article |
Year of Publication | 2011 |
Authors | Van de Water, K., S. H. Soror, A. Wohlkonig, N. A. J. van Nuland, and A. Volkov |
Journal | Acta Crystallogr Sect F Struct Biol Cryst Commun |
Volume | 67 |
Issue | Pt 6 |
Pagination | 703-6 |
Date Published | 2011 Jun 1 |
ISSN | 1744-3091 |
Keywords | beta-Lactamases, Crystallization, Crystallography, X-Ray, Kanamycin, Ligands, Protein Binding |
Abstract | TEM-1 β-lactamase is a highly efficient enzyme that is involved in bacterial resistance against β-lactam antibiotics such as penicillin. It is also a robust scaffold protein which can be engineered by molecular-evolution techniques to bind a variety of targets. One such β-lactamase variant (BlaKr) has been constructed to bind kanamycin (kan) and other aminoglycoside antibiotics, which are neither substrates nor ligands of native β-lactamases. In addition to recognizing kan, BlaKr activity is up-regulated by its binding via an activation mechanism which is not yet understood at the molecular level. In order to fill this gap, determination of the structure of the BlaKr-kan complex was embarked upon. A crystallization condition for BlaKr-kan was identified using high-throughput screening, and crystal growth was further optimized using streak-seeding and hanging-drop methods. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 47.01, b = 72.33, c = 74.62 Å, and diffracted to 1.67 Å resolution using synchrotron radiation. The X-ray structure of BlaKr with its ligand kanamycin should provide the molecular-level details necessary for understanding the activation mechanism of the engineered enzyme. |
DOI | 10.1107/S1744309111013832 |
Alternate Journal | Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. |
PubMed ID | 21636917 |
PubMed Central ID | PMC3107148 |
- Log in to post comments
- Google Scholar
- PubMed
- DOI