Selevsek Nathalie, Rival Sandrine, Tholey Andreas, Heinzle Elmar, Heinz Uwe, Hemmingsen Lars, Adolph Hans W
From the Departments of Biochemical Engineering, 66041 Saarbrücken, Germany.
Biochemistry, Saarland University, 66041 Saarbrücken, Germany.
J Biol Chem. 2009 Jun 12;284(24):16419-16431. doi: 10.1074/jbc.M109.001305. Epub 2009 Apr 24.
The reversible unfolding of metallo-beta-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-beta-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 alpha-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-beta-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 gamma-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.
脑膜败血金黄杆菌金属β-内酰胺酶（BlaB）在盐酸胍作用下的可逆去折叠过程，用包含折叠态、中间态和去折叠态的三态模型来描述最为合适。与锌离子结合的酶相比，折叠态的脱辅基酶转变为中间态仅需要极低浓度的变性剂。同样，脑膜败血金黄杆菌的BlaB和蜡样芽孢杆菌569/H/9的金属β-内酰胺酶（BcII）的圆二色光谱显示，无金属酶和锌离子结合酶之间存在明显差异，这表明锌离子影响蛋白质的折叠，使α-螺旋含量增加。为了确定蛋白质中参与这种锌离子诱导变化的区域，我们对无金属的BcII以及锌离子结合量为1价和2价的BcII进行了氢氘交换研究，并结合基质辅助激光解吸电离串联飞行时间质谱分析。跨越金属结合金属β-内酰胺酶（MBL）超家族共有序列His-X-His-X-Asp基序以及连接蛋白质N端和C端结构域的环的区域，在本质无序和有序状态之间发生了锌离子依赖性的结构变化。这种固有的灵活性甚至似乎允许形成金属离子桥连的蛋白质-蛋白质复合物，这可能解释了在改变锌/蛋白质比例时获得的电喷雾电离质谱结果，以及199mHg扰动γ射线角关联光谱数据的化学计量比依赖性变化。我们认为，存在于所有所有MBL亚类中的这种灵活的“锌臂”基序，在无金属的MBL中是无序的，并且可能在酶活性的“按需激活”调节中参与从不同于MBL的载锌分子获取金属离子。
