Zinc Ion-induced Domain Organization in Metallo-β-lactamases

• Published in: The Journal of biological chemistry Vol. 284; no. 24; pp. 16419 - 16431
• Main Authors: Selevsek, Nathalie, Rival, Sandrine, Tholey, Andreas, Heinzle, Elmar, Heinz, Uwe, Hemmingsen, Lars, Adolph, Hans W.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 12.06.2009
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M109.001305

The reversible unfolding of metallo-β-lactamase from Chryseobacterium meningosepticum (BlaB) by guanidinium hydrochloride is best described by a three-state model including folded, intermediate, and unfolded states. The transformation of the folded apoenzyme into the intermediate state requires only very low denaturant concentrations, in contrast to the Zn2-enzyme. Similarly, circular dichroism spectra of both BlaB and metallo-β-lactamase from Bacillus cereus 569/H/9 (BcII) display distinct differences between metal-free and Zn2-enzymes, indicating that the zinc ions affect the folding of the proteins, giving a larger α-helix content. To identify the regions of the protein involved in this zinc ion-induced change, a hydrogen deuterium exchange study with matrix-assisted laser desorption ionization tandem time of flight mass spectrometry on metal-free and Zn1- and Zn2-BcII was carried out. The region spanning the metal binding metallo-β-lactamases (MBL) superfamily consensus sequence His-X-His-X-Asp motif and the loop connecting the N- and C-terminal domains of the protein undergoes a zinc ion-dependent structural change between intrinsically disordered and ordered states. The inherent flexibility even appears to allow for the formation of metal ion-bridged protein-protein complexes which may account for both electrospray ionization-mass spectroscopy results obtained upon variation of the zinc/protein ratio and stoichiometry-dependent variations of 199mHg-perturbed angular correlation of γ-rays spectroscopic data. We suggest that this flexible “zinc arm” motif, present in all the MBL subclasses, is disordered in metal-free MBLs and may be involved in metal ion acquisition from zinc-carrying molecules different from MBL in an “activation on demand” regulation of enzyme activity.