The formation of the [NiFe] metallocenter of Escherichia coli hydrogenase 3 requires the participation of proteins encoded by the hydrogenase pleiotropy operon hypABCDEF. The insertion of Ni(II) into the precursor enzyme follows the incorporation of the iron center and is the function of HypA, a Zn(II)-binding protein, and HypB, a GTPase. The Ni(II) donor and the mechanism of transfer of Ni(II) into the hydrogenase precursor protein are not known. In this study, we demonstrate that HypB is a nickel-binding protein capable of binding 1 equiv of Ni(II) with a K(d) in the sub-picomolar range. In addition, HypB has a weaker metal-binding site that is not specific for Ni(II) over Zn(II). Examination of the isolated C-terminal GTPase domain revealed that the high-affinity metal binding capability was severely abrogated but the low-affinity site was intact. By mutating conserved cysteine and histidine residues in E. coli HypB, we have localized the high-affinity Ni(II)-binding site to an N-terminal CXXCGC motif and the low-affinity metal-binding site to the GTPase domain. A model for the function of HypB during the Ni(II) loading of hydrogenase is proposed.

Calnexin and calreticulin are membrane-bound and soluble chaperones, respectively, of the endoplasmic reticulum (ER) which interact transiently with a broad spectrum of newly synthesized glycoproteins. In addition to sharing substantial sequence identity, both calnexin and calreticulin bind to monoglucosylated oligosaccharides of the form Glc 1 Man 5-9 GlcNAc 2 , interact with the thiol oxidoreductase, ERp57, and are capable of acting as chaperones in vitro to suppress the aggregation of non-native proteins. To understand how these diverse functions are coordinated, we have localized the lectin, ERp57 binding, and polypeptide binding sites of calnexin and calreticulin. Recent structural studies suggest that both proteins consist of a globular domain and an extended arm domain comprised of two sequence motifs repeated in tandem. Our results indicate that the primary lectin site of calnexin and calreticulin resides within the globular domain, but the results also point to a much weaker secondary site within the arm domain which lacks specificity for monoglucosylated oligosaccharides. For both proteins, a site of interaction with ERp57 is centered on the arm domain, which retains ϳ50% of binding compared with full-length controls. This site is in addition to a Zn 2؉ -dependent site located within the globular domain of both proteins. Finally, calnexin and calreticulin suppress the aggregation of unfolded proteins via a polypeptide binding site located within their globular domains but require the arm domain for full chaperone function. These findings are integrated into a model that describes the interaction of glycoprotein folding intermediates with calnexin and calreticulin.As the site of synthesis of proteins destined for secretion, cell surface expression, and residency in the secretory pathway, the endoplasmic reticulum (ER) 1 contains an array of folding enzymes and molecular chaperones that facilitate the folding of newly synthesized proteins. Peptidylprolyl cis-trans-isomerase and members of the protein disulfide isomerase family enzymatically catalyze rate-limiting steps in the folding pathway of polypeptides, whereas molecular chaperones such as Grp94 and BiP function by preventing aggregation through cycles of binding and release of unfolded polypeptides. Another set of chaperones present in the ER, calnexin (CNX) and calreticulin (CRT), interact preferentially with glycoproteins that bear Asn-linked oligosaccharides, enhancing their folding and subunit assembly (1-4). This preferential binding is caused by the presence within CNX and CRT of a lectin site with specificity for the oligosaccharideprocessing intermediate, Glc 1 Man 9 GlcNAc 2 (5-8). However, oligosaccharide binding is not an absolute requirement for their association with diverse glycoproteins that transit the ER. Both molecules have been shown to bind in vitro and in vivo to nonglycosylated proteins and peptides as well as to glycoproteins lacking the Glc 1 Man 9 GlcNAc 2 oligosaccharide (9 -22). CNX, a type I transmembrane protein, and it...

The Escherichia coli protein SlyD is a member of the FK-506-binding protein family of peptidylprolyl isomerases. In addition to its peptidylprolyl isomerase domain, SlyD is composed of a molecular chaperone domain and a C-terminal tail rich in potential metal-binding residues. SlyD interacts with the [NiFe]-hydrogenase accessory protein HypB and contributes to nickel insertion during biosynthesis of the hydrogenase metallocenter. This study examines the HypB-SlyD complex and its significance in hydrogenase activation. Protein variants were prepared to delineate the interface between HypB and SlyD. Complex formation requires the HypB linker region located between the high affinity N-terminal Ni(II) site and the GTPase domain of the protein. In the case of SlyD, the deletion of a short loop in the chaperone domain abrogates the interaction with HypB. Mutations in either protein that disrupt complex formation in vitro also result in deficient hydrogenase production in vivo, indicating that the contact between HypB and SlyD is important for hydrogenase maturation. Surprisingly, SlyD stimulates release of nickel from the high affinity Ni(II)-binding site of HypB, an activity that is also disrupted by mutations that affect complex formation. Furthermore, a SlyD truncation lacking the C-terminal metal-binding tail still interacts with HypB but is deficient in stimulating metal release and is not functional in vivo. These results suggest that SlyD could activate metal release from HypB during metallation of the [NiFe] hydrogenase.The assembly of the [NiFe] metallocenter of Escherichia coli hydrogenase 3 requires the participation of proteins encoded by the hyp (hydrogenase pleiotropy) genes hypAB-CDEF (reviewed in Refs. 1-3). HypA and HypC are replaced by the homologous HybF and HybG proteins, respectively, for the assembly of hydrogenases 1 and 2 (1, 2). HypC, HypD, HypE, and HypF participate in the biosynthesis of the Fe(CN) 2 (CO) cluster and delivery to the hydrogenase precursor protein (4 -6). The subsequent incorporation of nickel (7,8) requires the GTPase HypB and HypA. These proteins were initially implicated in the nickel insertion step by genetic studies in which the hydrogenase deficiency resulting from chromosomal mutations was at least partially restored by growing the bacteria in excess nickel (9 -13). E. coli HypB binds one nickel ion with a K d value in the picomolar range to the cysteines in the N-terminal CXXCGC motif (referred to as the "high affinity site," see Fig. 1 for domain architecture) (14). In addition, both HypB and HypA bind a nickel ion with micromolar affinity (14 -17) as follows: HypA at a site that includes the conserved second residue His-2 (15, 16), and HypB to several conserved amino acids in the GTPase domain (referred to as the "low affinity site") (14, 18). Whether one or a combination of these metal sites serve as a source of nickel for the hydrogenase enzyme has not yet been determined.Upon searching for additional hydrogenase biosynthetic factors in E. coli, a protein called Sl...

The [NiFe]-hydrogenase protein produced by many types of bacteria contains a dinuclear metal center that is required for enzymatic activity. Assembly of this metal cluster involves the coordinated activity of a number of helper proteins including the accessory protein, HypB, which is necessary for Ni(II) incorporation into the hydrogenase proteins. The HypB protein from Escherichia coli has two metal-binding sites, a high-affinity Ni(II) site that includes ligands from the N-terminal domain and a low-affinity metal site located within the C-terminal GTPase domain. In order to determine the physiological relevance of the two separate sites, hydrogenase production was assessed in strains of E. coli expressing wild-type HypB, the isolated GTPase domain, or site-directed mutants of metal-binding residues. These experiments demonstrate that both metal sites of HypB are critical for the maturation of the hydrogenase enzymes in E. coli. X-ray absorption spectroscopy of purified proteins was used to examine the detailed coordination spheres of each nickel-loaded site. In addition, because the low-affinity metal site has a stronger preference for Zn(II) than Ni(II), the ligands and geometry for this metal were also resolved. The results from these experiments are discussed in the context of a mechanism for Ni(II) insertion into the hydrogenase protein.