title: Crystallization and preliminary X-ray diffraction analysis of kanamycin-binding β-lactamase in complex with its ligand.
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.
authors.affiliation: Department of Molecular and Cellular Interactions, Vrije Universiteit Brussel, Pleinlaan 2, Brussels, Belgium.
authors.forename: Karen
authors.initials: K
authors.lastname: Van de Water
authors.forename: Sameh H
authors.initials: SH
authors.lastname: Soror
authors.forename: Alexandre
authors.initials: A
authors.lastname: Wohlkonig
authors.forename: Nico A J
authors.initials: NA
authors.lastname: van Nuland
authors.forename: Alexander N
authors.initials: AN
authors.lastname: Volkov
mesh_terms: D003460:Crystallization; D018360:Crystallography, X-Ray; D007612:Kanamycin / Q000378:metabolism; D008024:Ligands; D011485:Protein Binding; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D008024:Ligands; D007612:Kanamycin; D001618:beta-Lactamases