Crystallization and preliminary X-ray diffraction analysis of kanamycin-binding β-lactamase in complex with its ligand.

TitleCrystallization and preliminary X-ray diffraction analysis of kanamycin-binding β-lactamase in complex with its ligand.
Publication TypeJournal Article
Year of Publication2011
AuthorsVan de Water, K., S. H. Soror, A. Wohlkonig, N. A. J. van Nuland, and A. Volkov
JournalActa Crystallogr Sect F Struct Biol Cryst Commun
Volume67
IssuePt 6
Pagination703-6
Date Published2011 Jun 1
ISSN1744-3091
Keywordsbeta-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.

DOI10.1107/S1744309111013832
Alternate JournalActa Crystallogr. Sect. F Struct. Biol. Cryst. Commun.
PubMed ID21636917
PubMed Central IDPMC3107148