UreG, a metallochaperone in the urease in vivo assembly B. Zambelli and S. Ciurli Laboratory of Bioinorganic Chemistry, Department of Agro-Environmental Science and Technology, University of Bologna, Viale G. Fanin 40, I-40127, Bologna, Italy Nickel is a fundamental micronutrient and an essential cofactor in the active site of important enzymes [1], but it is toxic for cellular life and carcinogenic for animals and humans [2]. Such potentially contradictory features required nickel-dependent organisms to develop efficient systems for nickel utilization and homeostasis. The preclusion of a regular intracellular nickel trafficking would result in the inactivation of the pathogenesis of medically relevant organisms, such as Helicobacter pylori and Mycobacterium tuberculosis, whose survival depends on the availability of this metal. This information can be used to develop novel strategies against nickel-dependent pathogens. Therefore, the understanding of nickel handling and detoxification mechanisms is a prerequisite in order to pinpoint specific anti-bacterial targets. The process of nickel incorporation into the active site of urease, the best characterized nickel- dependent enzyme [3,4], is a relevant example of intracellular nickel trafficking for its possible applications in medicine and agriculture. This process is based on a multi-step, tightly regulated mechanism, requiring the interplay of four accessory proteins, named UreD, UreE, UreF, UreG [5]. Among them, UreE and UreG are the best-characterized proteins at the structural and molecular level. The crystallographic structures of UreE from B. pasteurii and K. aerogenes have been determined [6,7]. UreE is responsible for nickel delivery into the apo-urease precursor through a metal site able to bind two nickel ions [8] and, in many organisms, it plays a additional role in nickel storage, through a histidine-rich C-terminal sequence [9]. UreG contains a fully conserved P-loop motif, typical of nucleotide-binding proteins, and was identified as the essential chaperone responsible for GTP hydrolysis in the process of urease active site assembly [10]. UreG from B. pasteurii (Bp) is a dimeric protein, exhibiting both zinc binding and low GTPase activities [11]. NMR spectroscopy revealed that it does not possess a rigid tertiary structure, and it exists in solution in fast equilibrium among different conformations, with large portions of unfolded backbone [11]. However, BpUreG contains a certain degree of secondary and tertiary structure, as revealed by CD and fluorescence spectroscopy, and can be classified as an intrinsically disordered protein [12]. The metal binding capability of BpUreG has been determined by metal titration analysis performed using filtration binding, followed by inductively coupled plasma (ICP), and isothermal titration calorimetry (ITC). The structural model of BpUreG, developed using threading algorithms, indicated that, in the fully folded state, this protein likely assumes an overall fold typical of a GTPase [11]. The homology among the UreG family, initially deduced on the basis of protein primary structure, was confirmed by experimental studies on UreG from M. tuberculosis (Mt) and computational analysis [13]. Mt UreG is present as a dimeric and partially unstructured protein in solution, and shows a low GTPase activity. The evidence of structural conservation among different UreG proteins was further investigated through an extensive similarity search. The identified sequences were aligned and organized in a phylogenetic tree. A structural database containing the most significant UreG proteins was built in order to compare their three-dimensional fold. The analysis of protein primary structures reveals the presence of a putative metal binding site (Glu 64 , Cys 68 , Pro 69 , His 70 in BpUreG numeration) that could reasonably be proposed as implicated in zinc binding. These fully conserved residues fall within a region of BpUreG that has been predicted to be disordered in all UreG proteins by predictors of natural disorder regions, indicating a possible structural role for zinc [11]. This hypothesis is further supported by the analysis of UreG structures as derived from molecular modeling. The backbone superimposition shows a general structural agreement except for the region that surrounds the -helix containing the putative zinc-binding site (Figure 1). The position of this protein portion indicates two possible alternative arrangements, “opening” or “closing” the hydrophilic GTP binding pocket. It is plausible that, upon zinc binding, the region folds in a more ordered conformation, contributing to UreG activation or stabilization. In order to prove this hypothesis, the residues actually involved in metal binding should be undoubtedly identified. X-ray absorption spectroscopy was applied to BpUreG, incubated in the presence of stoichiometric amount of Zn 2+ . EXAFS measurements were performed at the EMBL in DESY (Hamburg, Germany). Attempts to fit the EXAFS spectrum were carried out using all the possible binding configuration 449