Structural and biological analysis of the metal sites of Escherichia coli hydrogenase accessory protein HypB.

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.

The peptidyl-prolyl isomerase activity of SlyD is not required for maturation of Escherichia coli hydrogenase.

Escherichia coli SlyD, which is involved in the biosynthesis of the metal cluster in the [NiFe]-hydrogenase enzymes, exhibits several activities including that of a peptidyl-prolyl isomerase (PPIase). Mutations that result in deficient PPIase activity do not produce corresponding decreases in the other activities of SlyD in vitro or in hydrogenase production levels in vivo.

The role of complex formation between the Escherichia coli hydrogenase accessory factors HypB and SlyD.

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.

Metallocenter assembly of the hydrogenase enzymes.

The biosynthesis of the [NiFe]- and [FeFe]-hydrogenase enzymes requires the activities of multiple proteins that assemble the intricate metallocenters on the enzyme precursor proteins in an energy-dependent process. These accessory proteins include enzymes that synthesize the non-protein iron ligands as well as metallochaperones for the delivery of nickel to the [NiFe]-hydrogenase. Over the past few years many of these proteins have been examined in vitro. The biochemical properties, in the context of the earlier genetic studies, provide a basis for assigning function to the individual accessory proteins and mapping out the sequential steps of the metallocenter assembly pathways. This framework will serve as a foundation for detailed mechanistic analysis of these complex biomolecular factories.

Metal binding activity of the Escherichia coli hydrogenase maturation factor HypB.

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.

In vitro and in vivo assays to assess the functions of calnexin and calreticulin in ER protein folding and quality control.

Newly synthesized polypeptides entering the endoplasmic reticulum (ER) encounter a large array of molecular chaperones and folding factors that facilitate proper folding as well as assess folding status, retaining non-native proteins within the ER. Calnexin (CNX), an ER membrane protein, and its soluble homologue, calreticulin (CRT), are two important molecular chaperones that contribute to both processes. They are highly unusual chaperones in that they act as lectins, binding the Asn-linked oligosaccharides of newly synthesized glycoproteins, as well as recognizing the polypeptide segments of glycoproteins. Furthermore, they associate with ERp57, a thiol oxidoreductase, that is thought to enhance the oxidative folding of glycoproteins bound to CNX/CRT. These characteristics of CNX and CRT as well as their mode of action have been elucidated though the use of multiple in vitro and in vivo approaches. This chapter will focus on the description of a number of in vitro assays that have been used to characterize the lectin and ERp57-binding functions of CNX/CRT and also their abilities to act as molecular chaperones to suppress protein aggregation. In addition, we will describe insect and mammalian expression systems in which major histocompatibility complex class I molecules are used as model glycoprotein substrates for CNX and CRT. These systems have been valuable in assessing folding and quality control events in vivo that are influenced by CNX or CRT as well as in characterizing the spectrum of substrates that are recognized by these chaperones.

Lectin-deficient calnexin is capable of binding class I histocompatibility molecules in vivo and preventing their degradation.

Calnexin is a membrane-bound lectin of the endoplasmic reticulum (ER) that binds transiently to newly synthesized glycoproteins. By interacting with oligosaccharides of the form Glc(1)Man(9)GlcNAc(2), calnexin enhances the folding of glycoprotein substrates, retains misfolded variants in the ER, and in some cases participates in their degradation. Calnexin has also been shown to bind polypeptides in vivo that do not possess a glycan of this form and to function in vitro as a molecular chaperone for nonglycosylated proteins. To test the relative importance of the lectin site compared with the polypeptide-binding site, we have generated six calnexin mutants defective in oligosaccharide binding using site-directed mutagenesis. Expressed as glutathione S-transferase fusions, these mutants were still capable of binding ERp57, a thiol oxidoreductase, and preventing the aggregation of a nonglycosylated substrate, citrate synthase. They were, however, unable to bind Glc(1) Man(9)GlcNAc(2) oligosaccharide and were compromised in preventing the aggregation of the monoglucosylated substrate jack bean alpha-mannosidase. Two of these mutants were then engineered into full-length calnexin for heterologous expression in Drosophila cells along with the murine class I histocompatibility molecules K(b) and D(b) as model glycoproteins. In this system, lectin site-defective calnexin was able to replace wild type calnexin in forming a complex with K(b) and D(b) heavy chains and preventing their degradation. Thus, at least for class I molecules, the lectin site of calnexin is dispensable for some of its chaperone functions.

Localization of the lectin, ERp57 binding, and polypeptide binding sites of calnexin and calreticulin.

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 approximately 50% 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.

Synthesis of a molecular mimic of the Glc1Man9 oligoside as potential inhibitor of calnexin binding to DeltaF508 CFTR protein.

Deletion of phenylalanine at position 508 of the CFTR protein is associated with a severe form of cystic fibrosis. Biosynthetic arrest of the misfolded DeltaF508 CFTR protein in the endoplasmic reticulum is due to prolonged interaction with protein chaperones. In order to overcome this retention and thereby restore the delivery of the protein to the plasma membrane, a molecular mimic of the glycoprotein oligoside moiety has been designed and synthesized. Ability of this mimic to inhibit the binding of the natural Glc1Man9GlcNAc oligoside to calnexin has been measured.