Crystal structure of HydF scaffold protein provides insights into [FeFe]-hydrogenase maturation

[FeFe]-hydrogenases catalyze the reversible production of H₂ in some bacteria and unicellular eukaryotes. These enzymes require ancillary proteins to assemble the unique active site H-cluster, a complex structure composed of a 2Fe center bridged to a [4Fe-4S] cubane. The first crystal structure of a key factor in the maturation process, HydF, has been determined at 3 Å resolution. The protein monomer present in the asymmetric unit of the crystal comprises three domains: a GTP-binding domain, a dimerization domain, and a metal cluster-binding domain, all characterized by similar folding motifs. Two monomers dimerize, giving rise to a stable dimer, held together mainly by the formation of a continuous β-sheet comprising eight β-strands from two monomers. Moreover, in the structure presented, two dimers aggregate to form a supramolecular organization that represents an inactivated form of the HydF maturase. The crystal structure of the latter furnishes several clues about the events necessary for cluster generation/transfer and provides an excellent model to begin elucidating the structure/function of HydF in [FeFe]-hydrogenase maturation.     

[FeFe]-Hydrogenase models assembled into vesicular structures

Compartmentalization is a major prerequisite for the origin of life on earth according to Wächtershäuser “Iron-Sulfur-World”. The hypothesis is mainly based on an autocatalytic inorganic energy reproducing redox system consisting of iron and sulfur as requirement for the subsequent synthesis of complex organic structures. Here, we modified [FeFe]-hydrogenase models by means of covalent coupling to either oleic acid or the amphiphilic block copolymer polybutadiene-polyethyleneoxide (PB-PEO) and incorporated those into the membranes of vesicles composed of phospholipids (liposomes) or the unmodified amphiphilic polymer (polymersomes). We employed a [2Fe-2S] cluster as a hydrogenase model, since these structures are known to be suitable catalysts for the generation of H₂ in the presence of weak acids. Successful incorporation was confirmed by spectrophotometric iron quantification and the vesicles formed were characterized by size determination (photon correlation spectroscopy (PCS)), and zeta potential as well as by cryo-transmission electron microscopy (Cryo-TEM). The modified models could be incorporated into liposomes or polymersomes up to molar proportions of 3.15% and 28%, respectively. Due to the immobilization in vesicular bilayers the [FeFe]-hydrogenase models can even exhibit catalytic action under the particular conditions of the intravesicular microenvironment. Our results suggest that the vesicular systems described may be applied as a nanoreactor for the reduction of encapsulated substances by generating hydrogen and thus as a minimal cell model.     

Identification and Biochemical Characterization of Molybdenum Cofactor-binding Proteins from Arabidopsis thaliana

The molybdenum cofactor (Moco) forms part of the catalytic center in all eukaryotic molybdenum enzymes and is synthesized in a highly conserved pathway. Among eukaryotes, very little is known about the processes taking place subsequent to Moco biosynthesis, i.e. Moco transfer, allocation, and insertion into molybdenum enzymes. In the model plant Arabidopsis thaliana, we identified a novel protein family consisting of nine members that after recombinant expression are able to bind Moco with K_D values in the low micromolar range and are therefore named Moco-binding proteins (MoBP). For two of the nine proteins atomic structures are available in the Protein Data Bank. Surprisingly, both crystal structures lack electron density for the C terminus, which may indicate a high flexibility of this part of the protein. C-terminal truncated MoBPs showed significantly decreased Moco binding stoichiometries. Experiments where the MoBP C termini were exchanged among MoBPs converted a weak Moco-binding MoBP into a strong binding MoBP, thus indicating that the MoBP C terminus, which is encoded by a separate exon, is involved in Moco binding. MoBPs were able to enhance Moco transfer to apo-nitrate reductase in the Moco-free Neurospora crassa mutant nit-1. Furthermore, we show that the MoBPs are localized in the cytosol and undergo protein-protein contact with both the Moco donor protein Cnx1 and the Moco acceptor protein nitrate reductase under in vivo conditions, thus indicating for the MoBPs a function in Arabidopsis cellular Moco distribution.     

The Mammalian Proteins MMS19, MIP18, and ANT2 Are Involved in Cytoplasmic Iron-Sulfur Cluster Protein Assembly

Iron-sulfur (Fe-S) clusters are essential cofactors of proteins with a wide range of biological functions. A dedicated cytosolic Fe-S cluster assembly (CIA) system is required to assemble Fe-S clusters into cytosolic and nuclear proteins. Here, we show that the mammalian nucleotide excision repair protein homolog MMS19 can simultaneously bind probable cytosolic iron-sulfur protein assembly protein CIAO1 and Fe-S proteins, confirming that MMS19 is a central protein of the CIA machinery that brings Fe-S cluster donor proteins and the receiving apoproteins into proximity. In addition, we show that mitotic spindle-associated MMXD complex subunit MIP18 also interacts with both CIAO1 and Fe-S proteins. Specifically, it binds the Fe-S cluster coordinating regions in Fe-S proteins. Furthermore, we show that ADP/ATP translocase 2 (ANT2) interacts with Fe-S apoproteins and MMS19 in the CIA complex but not with the individual proteins. Together, these results elucidate the composition and interactions within the late CIA complex.     

Importance of the protein framework for catalytic activity of [FeFe]-hydrogenases

The active center (H-cluster) of [FeFe]-hydrogenases is embedded into a hydrophobic pocket within the protein. We analyzed several amino acids, located in the vicinity of this niche, by site-directed mutagenesis of the [FeFe]-hydrogenases from Clostridium pasteurianum (CpI) and Chlamydomonas reinhardtii (CrHydA1). These amino acids are highly conserved and predicted to be involved in H-cluster coordination. Characterization of two hydrogenase variants confirmed this hypothesis. The exchange of residues CrHydA1Met(415) and CrHydA1Lys(228) resulted in inactive proteins, which, according to EPR and FTIR analyses, contain no intact H-cluster. However, [FeFe]-hydrogenases in which CpIMet(353) (CrHydA1Met(223)) and CpICys(299) (CrHydA1Cys(169)) were exchanged to leucine and serine, respectively, showed a structurally intact H-cluster with catalytic activity either absent (CpIC299S) or strongly diminished (CpIM353L). In the case of CrHydA1C169S, the H-cluster was trapped in an inactive state exhibiting g values and vibrational frequencies that resembled the H(trans) state of DdH from Desulfovibrio desulfuricans. This cysteine residue, interacting with the bridge head nitrogen of the di(methyl)amine ligand, seems therefore to represent an essential contribution of the immediate protein environment to the reaction mechanism. Exchanging methionine CpIM(353) (CrHydA1M(223)) to leucine led to a strong decrease in turnover without affecting the K(m) value of the electron donor. We suggest that this methionine constitutes a "fine-tuning" element of hydrogenase activity.     

Relationship between Ni(II) and Zn(II) Coordination and Nucleotide Binding by the Helicobacter pylori [NiFe]-Hydrogenase and Urease Maturation Factor HypB

The pathogen Helicobacter pylori requires two nickel-containing enzymes, urease and [NiFe]-hydrogenase, for efficient colonization of the human gastric mucosa. These enzymes possess complex metallocenters that are assembled by teams of proteins in multistep pathways. One essential accessory protein is the GTPase HypB, which is required for Ni(II) delivery to [NiFe]-hydrogenase and participates in urease maturation. Ni(II) or Zn(II) binding to a site embedded in the GTPase domain of HypB modulates the enzymatic activity, suggesting a mechanism of regulation. In this study, biochemical and structural analyses of H. pylori HypB (HpHypB) revealed an intricate link between nucleotide and metal binding. HpHypB nickel coordination, stoichiometry, and affinity were modulated by GTP and GDP, an effect not observed for zinc, and biochemical evidence suggests that His-107 coordination to nickel toggles on and off in a nucleotide-dependent manner. These results are consistent with the crystal structure of HpHypB loaded with Ni(II), GDP, and P_i, which reveals a nickel site distinct from that of zinc-loaded Methanocaldococcus jannaschii HypB as well as subtle changes to the protein structure. Furthermore, Cys-142, a metal ligand from the Switch II GTPase motif, was identified as a key component of the signal transduction between metal binding and the enzymatic activity. Finally, potassium accelerated the enzymatic activity of HpHypB but had no effect on the other biochemical properties of the protein. Altogether, this molecular level information about HpHypB provides insight into its cellular function and illuminates a possible mechanism of metal ion discrimination.     

Structure-function relationships in [FeFe]-hydrogenase active site maturation

Since the discovery that, despite the active site complexity, only three gene products suffice to obtain active recombinant [FeFe]-hydrogenase, significant light has been shed on this process. Both the source of the CO and CN(-) ligands to iron and the assembly site of the catalytic subcluster are known, and an apo structure of HydF has been published recently. However, the nature of the substrate(s) for the synthesis of the bridging dithiolate ligand to the subcluster remains to be established. From both spectroscopy and model chemistry, it is predicted that an amine function in this ligand plays a central role in catalysis, acting as a base in the heterolytic cleavage of hydrogen.     

Control by Potassium of the Size Distribution of Escherichia coli FtsZ Polymers Is Independent of GTPase Activity

The influence of potassium content (at neutral pH and millimolar Mg²⁺) on the size distribution of FtsZ polymers formed in the presence of constantly replenished GTP under steady-state conditions was studied by a combination of biophysical methods. The size of the GTP-FtsZ polymers decreased with lower potassium concentration, in contrast with the increase in the mass of the GDP-FtsZ oligomers, whereas no effect was observed on FtsZ GTPase activity and critical concentration of polymerization. Remarkably, the concerted formation of a narrow size distribution of GTP-FtsZ polymers previously observed at high salt concentration was maintained in all KCl concentrations tested. Polymers induced with guanosine 5′-(α,β-methylene)triphosphate, a slowly hydrolyzable analog of GTP, became larger and polydisperse as the potassium concentration was decreased. Our results suggest that the potassium dependence of the GTP-FtsZ polymer size may be related to changes in the subunit turnover rate that are independent of the GTP hydrolysis rate. The formation of a narrow size distribution of FtsZ polymers under very different solution conditions indicates that it is an inherent feature of FtsZ, not observed in other filament-forming proteins, with potential implications in the structural organization of the functional Z-ring.     

An Intrinsically Disordered Photosystem II Subunit,PsbO, Provides a Structural Template and a Sensor of the Hydrogen-bonding Network in Photosynthetic Water Oxidation

Photosystem II (PSII) is a membrane-bound enzyme that utilizes solar energy to catalyze the photooxidation of water. Molecular oxygen is evolved after four sequential light-driven oxidation reactions at the Mn₄CaO₅ oxygen-evolving complex, producing five sequentially oxidized states, S_n. PSII is composed of 17 membrane-spanning subunits and three extrinsic subunits, PsbP, PsbQ, and PsbO. PsbO is intrinsically disordered and plays a role in facilitation of the water oxidizing cycle. Native PsbO can be removed and substituted with recombinant PsbO, thereby restoring steady-state activity. In this report, we used reaction-induced Fourier transform infrared spectroscopy to obtain information concerning the role of PsbP, PsbQ, and PsbO during the S state cycle. Light-minus-dark difference spectra were acquired, monitoring structural changes associated with each accessible flash-induced S state transition in a highly purified plant PSII preparation (Triton X-100, octylthioglucoside). A comparison of S₂ minus S₁ spectra revealed that removal of PsbP and PsbQ had no significant effect on the data, whereas amide frequency and intensity changes were associated with PsbO removal. These data suggest that PsbO acts as an organizational template for the PSII reaction center. To identify any coupled conformational changes arising directly from PsbO, global ¹³C-PsbO isotope editing was employed. The reaction-induced Fourier transform infrared spectra of accessible S states provide evidence that PsbO spectral contributions are temperature (263 and 277 K) and S state dependent. These experiments show that PsbO undergoes catalytically relevant structural dynamics, which are coupled over long distance to hydrogen-bonding changes at the Mn₄CaO₅ cluster.     

Direct Interactions of Intraflagellar Transport Complex B Proteins IFT88, IFT52, and IFT46

Intraflagellar transport (IFT) particles of Chlamydomonas reinhardtii contain two distinct protein complexes, A and B, composed of at least 6 and 15 protein subunits, respectively. As isolated from C. reinhardtii flagella, IFT complex B can be further reduced to a ∼500-kDa core that contains IFT88, 2× IFT81, 2× IFT74/72, IFT52, IFT46, IFT27, IFT25, and IFT22. In this study, yeast-based two-hybrid analysis was combined with bacterial coexpression to show that three of the core B subunits, IFT88, IFT52, and IFT46, interact directly with each other and, together, are capable of forming a ternary complex. Chemical cross-linking results support the IFT52-IFT88 interaction and provide additional evidence of an association between IFT27 and IFT81. With previous studies showing that IFT81 and IFT74/72 interact to form a (IFT81)₂(IFT74/72)₂ heterotetramer and that IFT27 and IFT25 form a heterodimer, the architecture of complex B is revealing itself. Last, electroporation of recombinant IFT46 was used to rescue flagellar assembly of a newly identified ift46 mutant and to monitor in vivo localization and movement of the IFT particles.     

Application of gene-shuffling for the rapid generation of novel [FeFe]-hydrogenase libraries

A gene-shuffling technique was identified, optimized and used to generate diverse libraries of recombinant [FeFe]-hydrogenases. Six native [FeFe]-hydrogenase genes from species of Clostridia were first cloned and separately expressed in Escherichia coli concomitantly with the assembly proteins required for [FeFe]-hydrogenase maturation. All enzymes, with the exception of C. thermocellum HydA, exhibited significant activity when expressed. Single-stranded DNA fragments from genes encoding the two most active [FeFe]-hydrogenases were used to optimize a gene-shuffling protocol and generate recombinant enzyme libraries. Random sampling demonstrates that several shuffled products are active. This represents the first successful application of gene-shuffling using hydrogenases. Moreover, we demonstrate that a single set of [FeFe]-hydrogenase maturation proteins is sufficient for the heterologous assembly of the bioinorganic active site of several native and shuffled [FeFe]-hydrogenases.     

The role of the maturase HydG in [FeFe]-hydrogenase active site synthesis and assembly

[FeFe]-hydrogenases catalyze the protons/hydrogen interconversion through a unique di-iron active site consisting of three CO and two CN ligands, and a non-protein SCH₂XCH₂S (X = N or O) dithiolate bridge. Site assembly requires two “Radical-S-adenosylmethionine (SAM or AdoMet)” iron-sulfur enzymes, HydE and HydG, and one GTPase, HydF. The sequence homology between HydG and ThiH, a Radical-SAM enzyme which cleaves tyrosine into p-cresol and dehydroglycine, and the finding of a similar cleavage reaction catalyzed by HydG suggests a mechanism for hydrogenase maturation. Here we propose that HydG is specifically involved in the synthesis of the dithiolate ligand, with two tyrosine-derived dehydroglycines as precursors along with an [FeS] cluster of HydG functioning both as electron shuttle and source of the sulfur atoms.     

Phytoplasma-induced Changes in the Acetylome and Succinylome of Paulownia tomentosa Provide Evidence for Involvement of Acetylated Proteins in Witches' Broom Disease

1. Download : Download high-res image (89KB)
2. Download : Download full-size image

Highlights

• •Quantitative global proteome, acetylome and succinylome of phytoplasma-infected Paulownia tomentosa seedlings.
• •Acetylation may be more important than succinylation in response to phytoplasma infection.
• •Acetylation modified the activities of POR and RuBisCO.
• •Possible model to elucidate the molecular mechanism responses to PaWB from proteome and PTMs.

     

Investigation of amino acid containing [FeFe] hydrogenase models concerning pendant base effects

The present investigations deal with the modeling of the peptide surrounding of [FeFe] hydrogenase using amine containing disulphides to simulate possible influences of the amino acid lysine (K237) on the electrochemical and electrocatalytic properties of biomimetic compounds based on [Fe2S2] moieties. Fe₃(CO)₁₂ was reacted with Boc-4-amino-1,2-dithiolane, Boc-Adt-OMe (Adt = 4-amino-1,2-dithiolane-4-carboxylic acid, Boc = tert-butoxycarbonyl) and Boc-Adp tert-butyl ester (Adp = (S)-2-amino-3-(1,2-dithiolan-4-yl)propionic acid) to elongate the FeN distance in comparison to the well known [Fe₂{(SCH₂)₂NR}(CO)₆] model complexes. Efforts to deprotect the complexes containing Boc-4-amino-1,2-dithiolane with trifluoroacetic acid result in the formation of [Fe₃(μ³-O)(μ-O₂C₂F₃)₆(OC₄H₈)₂(H₂O)]. The novel [2Fe2S] complexes are characterized using spectroscopic, electrochemical techniques and X-ray diffraction studies.     

Protein interactions and localization of the Escherichia coli accessory protein HypA during nickel insertion to [NiFe] hydrogenase

Nickel delivery during maturation of Escherichia coli [NiFe] hydrogenase 3 includes the accessory proteins HypA, HypB, and SlyD. Although the isolated proteins have been characterized, little is known about how they interact with each other and the hydrogenase 3 large subunit, HycE. In this study the complexes of HypA and HycE were investigated after modification with the Strep-tag II. Multiprotein complexes containing HypA, HypB, SlyD, and HycE were observed, consistent with the assembly of a single nickel insertion cluster. An interaction between HypA and HycE did not require the other nickel insertion proteins, but HypB was not found with the large subunit in the absence of HypA. The HypA-HycE complex was not detected in the absence of the HypC or HypD proteins, involved in the preceding iron insertion step, and this interaction is enhanced by nickel brought into the cell by the NikABCDE membrane transporter. Furthermore, without the hydrogenase 1, 2, and 3 large subunits, complexes between HypA, HypB, and SlyD were observed. These results support the hypothesis that HypA acts as a scaffold for assembly of the nickel insertion proteins with the hydrogenase precursor protein after delivery of the iron center. At different stages of the hydrogenase maturation process, HypA was observed at or near the cell membrane by using fluorescence confocal microscopy, as was HycE, suggesting membrane localization of the nickel insertion event.     

The Molecular Basis of Iron-induced Oligomerization of Frataxin and the Role of the Ferroxidation Reaction in Oligomerization

The role of the mitochondrial protein frataxin in iron storage and detoxification, iron delivery to iron-sulfur cluster biosynthesis, heme biosynthesis, and aconitase repair has been extensively studied during the last decade. However, still no general consensus exists on the details of the mechanism of frataxin function and oligomerization. Here, using small-angle x-ray scattering and x-ray crystallography, we describe the solution structure of the oligomers formed during the iron-dependent assembly of yeast (Yfh1) and Escherichia coli (CyaY) frataxin. At an iron-to-protein ratio of 2, the initially monomeric Yfh1 is converted to a trimeric form in solution. The trimer in turn serves as the assembly unit for higher order oligomers induced at higher iron-to-protein ratios. The x-ray crystallographic structure obtained from iron-soaked crystals demonstrates that iron binds at the trimer-trimer interaction sites, presumably contributing to oligomer stabilization. For the ferroxidation-deficient D79A/D82A variant of Yfh1, iron-dependent oligomerization may still take place, although >50% of the protein is found in the monomeric state at the highest iron-to-protein ratio used. This demonstrates that the ferroxidation reaction controls frataxin assembly and presumably the iron chaperone function of frataxin and its interactions with target proteins. For E. coli CyaY, the assembly unit of higher order oligomers is a tetramer, which could be an effect of the much shorter N-terminal region of this protein. The results show that understanding of the mechanistic features of frataxin function requires detailed knowledge of the interplay between the ferroxidation reaction, iron-induced oligomerization, and the structure of oligomers formed during assembly.     

Molecular basis of the interaction between proapoptotic truncated BID (tBID) protein and mitochondrial carrier homologue 2 (MTCH2) protein: key players in mitochondrial death pathway

The molecular basis of the interaction between mitochondrial carrier homologue 2 (MTCH2) and truncated BID (tBID) was characterized. These proteins participate in the apoptotic pathway, and the interaction between them may serve as a target for anticancer lead compounds. In response to apoptotic signals, MTCH2 recruits tBID to the mitochondria, where it activates apoptosis. A combination of peptide arrays screening with biochemical and biophysical techniques was used to characterize the mechanism of the interaction between tBID and MTCH2 at the structural and molecular levels. The regions that mediate the interaction between the proteins were identified. The two specific binding sites between the proteins were determined to be tBID residues 59-73 that bind MTCH2 residues 140-161, and tBID residues 111-125 that bind MTCH2 residues 240-290. Peptides derived from tBID residues 111-125 and 59-73 induced cell death in osteosarcoma cells. These peptides may serve as lead compounds for anticancer drugs that act by targeting the tBID-MTCH2 interaction.     

Identification of Molecular Switch Regulating Interactions of Janus Kinase 3 with Cytoskeletal Proteins

Janus kinase 3 (Jak3) is a nonreceptor tyrosine kinase expressed in both hematopoietic and nonhematopoietic cells. Although mutations that abrogate Jak3 functions cause different immunological disorders, its constitutive activation leads to various types of cancer. Previously, we demonstrated that Jak3 interacted with actin-binding protein villin, thereby facilitating cytoskeletal remodeling and wound repair. In this study, we characterize the structural determinants that regulate the interactions between Jak3 and cytoskeletal proteins of the villin/gelsolin family. Functional reconstitution of kinase activity by recombinant full-length (wt) Jak3 using Jak3-wt or villin/gelsolin-wt as substrate showed that Jak3 autophosphorylation was the rate-limiting step during interactions between Jak3 and cytoskeletal proteins. Determination of kinetic parameters showed that phosphorylated (P) Jak3-wt binds to P-villin-wt with a dissociation constant (K_d) of 23 nm and a Hill''s coefficient of 3.7. Pairwise binding between Jak3 mutants and P-villin-wt showed that the FERM domain of Jak3 was sufficient for binding to P-villin-wt with a K_d of 40.0 nm. However, the SH2 domain of Jak3 prevented P-villin-wt from binding to the FERM domain of nonphosphorylated protein. We demonstrate that the intramolecular interaction between the FERM and SH2 domains of nonphosphorylated Jak3 prevented Jak3 from binding to villin and that tyrosine autophosphorylation of Jak3 at the SH2 domain decreased these intramolecular interactions and facilitated binding of the FERM domain to villin. Thus we demonstrate the molecular mechanism of interactions between Jak3 and cytoskeletal proteins where tyrosine phosphorylation of the SH2 domain acted as an intramolecular switch for the interactions between Jak3 and cytoskeletal proteins.     

Biochemical characterization of Pumilio1 and Pumilio2 in Xenopus oocytes

Precise control of the timing of translational activation of dormant mRNAs stored in oocytes is required for normal progression of oocyte maturation. We previously showed that Pumilio1 (Pum1) is specifically involved in the translational control of cyclin B1 mRNA during Xenopus oocyte maturation, in cooperation with cytoplasmic polyadenylation element-binding protein (CPEB). It was reported that another Pumilio, Pumilio2 (Pum2), exists in Xenopus oocytes and that this protein regulates the translation of RINGO mRNA, together with Deleted in Azoospermia-like protein (DAZL). In this study, we characterized Pum1 and Pum2 biochemically by using newly produced antibodies that discriminate between them. Pum1 and Pum2 are bound to several key proteins involved in translational control of dormant mRNAs, including CPEB and DAZL, in immature oocytes. However, Pum1 and Pum2 themselves have no physical interaction. Injection of anti-Pum1 or anti-Pum2 antibody accelerated CPEB phosphorylation, cyclin B1 translation, and oocyte maturation. Pum1 phosphorylation coincides with the dissociation of CPEB from Pum1 and the translational activation of cyclin B1 mRNA, a target of Pum1, whereas Pum2 phosphorylation occurred at timing earlier than that for Pum1. Some, but not all, of cyclin B1 mRNAs release the deadenylase PARN during oocyte maturation, whereas Pum1 remains associated with the mRNA. On the basis of these findings, we discuss the functions of Pum1 and Pum2 in translational control of mRNAs during oocyte maturation.