Coordinating subdomains of ferritin protein cages with catalysis and biomineralization viewed from the C 4 cage axes

Integrated ferritin protein cage function is the reversible synthesis of protein-caged, solid Fe₂O₃·H₂O minerals from Fe²⁺ for metabolic iron concentrates and oxidant protection; biomineral order differs in different ferritin proteins. The conserved 432 geometric symmetry of ferritin protein cages parallels the subunit dimer, trimer, and tetramer interfaces, and coincides with function at several cage axes. Multiple subdomains distributed in the self-assembling ferritin nanocages have functional relationships to cage symmetry such as Fe²⁺ transport though ion channels (threefold symmetry), biomineral nucleation/order (fourfold symmetry), and mineral dissolution (threefold symmetry) studied in ferritin variants. On the basis of the effects of natural or synthetic subunit dimer cross-links, cage subunit dimers (twofold symmetry) influence iron oxidation and mineral dissolution. 2Fe²⁺/O₂ catalysis in ferritin occurs in single subunits, but with cooperativity (n = 3) that is possibly related to the structure/function of the ion channels, which are constructed from segments of three subunits. Here, we study 2Fe²⁺ + O₂ protein catalysis (diferric peroxo formation) and dissolution of ferritin Fe₂O₃·H₂O biominerals in variants with altered subunit interfaces for trimers (ion channels), E130I, and external dimer surfaces (E88A) as controls, and altered tetramer subunit interfaces (L165I and H169F). The results extend observations on the functional importance of structure at ferritin protein twofold and threefold cage axes to show function at ferritin fourfold cage axes. Here, conserved amino acids facilitate dissolution of ferritin-protein-caged iron biominerals. Biological and nanotechnological uses of ferritin protein cage fourfold symmetry and solid-state mineral properties remain largely unexplored.     

A Novel 8-nm Protein Cage Formed by Vibrio cholerae Acylphosphatase

Here we show the formation of an ~ 8-nm cage formed by the self-assembly of acylphosphatase from Vibrio cholerae O395 (Vc-AcP). The 12-subunit cage structure forms spontaneously and is stabilized through binding of sulfate ions at its exterior face and interfacial regions. Crystal structure and studies in solutions illuminate the basis for the formation of the cage, while a single (Cys20 → Arg) mutation (Vc-AcP-C20R) transforms Vc-AcP to a potent enzyme but disrupts the assembly into a trimer.     

Crystal structures of the apo and ATP bound Mycobacterium tuberculosis nitrogen regulatory PII protein

PII constitutes a family of signal transduction proteins that act as nitrogen sensors in microorganisms and plants. Mycobacterium tuberculosis (Mtb) has a single homologue of PII whose precise role has as yet not been explored. We have solved the crystal structures of the Mtb PII protein in its apo and ATP bound forms to 1.4 and 2.4 Å resolutions, respectively. The protein forms a trimeric assembly in the crystal lattice and folds similarly to the other PII family proteins. The Mtb PII:ATP binary complex structure reveals three ATP molecules per trimer, each bound between the base of the T‐loop of one subunit and the C‐loop of the neighboring subunit. In contrast to the apo structure, at least one subunit of the binary complex structure contains a completely ordered T‐loop indicating that ATP binding plays a role in orienting this loop region towards target proteins like the ammonium transporter, AmtB. Arg38 of the T‐loop makes direct contact with the γ‐phosphate of the ATP molecule replacing the Mg²⁺ position seen in the Methanococcus jannaschii GlnK1 structure. The C‐loop of a neighboring subunit encloses the other side of the ATP molecule, placing the GlnK specific C‐terminal 3₁₀ helix in the vicinity. Homology modeling studies with the E. coli GlnK:AmtB complex reveal that Mtb PII could form a complex similar to the complex in E. coli. The structural conservation and operon organization suggests that the Mtb PII gene encodes for a GlnK protein and might play a key role in the nitrogen regulatory pathway.     

Trp RNA-Binding Attenuation Protein: Modifying Symmetry and Stability of a Circular Oligomer

Background

Subunit number is amongst the most important structural parameters that determine size, symmetry and geometry of a circular protein oligomer. The L-tryptophan biosynthesis regulator, TRAP, present in several Bacilli, is a good model system for investigating determinants of the oligomeric state. A short segment of C-terminal residues defines whether TRAP forms an 11-mer or 12-mer assembly. To understand which oligomeric state is more stable, we examine the stability of several wild type and mutant TRAP proteins.

Methodology/Principal Findings

Among the wild type B. stearothermophilus, B. halodurans and B. subtilis TRAP, we find that the former is the most stable whilst the latter is the least. Thermal stability of all TRAP is shown to increase with L-tryptophan concentration. We also find that mutant TRAP molecules that are truncated at the C-terminus - and hence induced to form 12-mers, distinct from their 11-mer wild type counterparts - have increased melting temperatures. We show that the same effect can be achieved by a point mutation S72N at a subunit interface, which leads to exclusion of C-terminal residues from the interface. Our findings are supported by dye-based scanning fluorimetry, CD spectroscopy, and by crystal structure and mass spectrometry analysis of the B. subtilis S72N TRAP.

Conclusions/Significance

We conclude that the oligomeric state of a circular protein can be changed by introducing a point mutation at a subunit interface. Exclusion (or deletion) of the C-terminus from the subunit interface has a major impact on properties of TRAP oligomers, making them more stable, and we argue that the cause of these changes is the altered oligomeric state. The more stable TRAP oligomers could be used in potential applications of TRAP in bionanotechnology.     

Role of two residues proximal to the active site of Ubc9 in substrate recognition by the Ubc9.SUMO-1 thiolester complex

The small ubiquitin-like modifier SUMO-1 is covalently attached to lysine residues on target proteins by a specific conjugation pathway involving the E1 enzyme SAE1/SAE2 and the E2 enzyme Ubc9. In an ATP-dependent manner, the C-terminus of SUMO-1 forms consecutive thiolester bonds with cysteine residues in the SAE2 subunit and Ubc9, before the Ubc9.SUMO-1 thiolester complex catalyzes the formation of an isopeptide bond between SUMO-1 and the epsilon-amino group of the target lysine residue on the protein substrate. The SUMO-1 conjugation pathway bears many similarities with that of ubiquitin and other ubiquitin-like protein modifiers (Ubls), and because of its production of a singly conjugated substrate and the lack of absolute requirement in vitro for E3 enzymes, the SUMO-1/Ubc9 system is a good model for the analysis of protein conjugation pathways that share this basic chemistry. Here we describe methods of both steady-state and half-reaction kinetic analysis of Ubc9, and use these techniques to determine the role of two residues, Asp(100) and Lys(101) of Ubc9 which are not found in E2 enzymes from other protein conjugation pathways. These residues are found close to the active site Cys in the tertiary structure of Ubc9, and although they are shown to inhibit the transesterification reaction from SAE1/SAE2, they are important for substrate recognition in the context of the thiolester complex with SUMO-1.     

Structural features and oligomeric nature of human podocin domain

Podocytes are crucial cells of the glomerular filtration unit and plays a vital role at the interface of the blood-urine barrier. Podocyte slit-diaphragm is a modified tight junction that facilitates size and charge-dependent permselectivity. Several proteins including podocin, nephrin, CD2AP, and TRPC6 form a macromolecular assembly and constitute the slit-diaphragm. Podocin is an integral membrane protein attached to the inner membrane of the podocyte via a short transmembrane region (101–125). The cytosolic N- and C-terminus help podocin to attain a hook-like structure. Podocin shares 44% homology with stomatin family proteins and similar to the stomatin proteins, podocin was shown to associate into higher-order oligomers at the site of slit-diaphragm. However, the stoichiometry of the homo-oligomers and how it partakes in the macromolecular assemblies with other slit-diaphragm proteins remains elusive. Here we investigated the oligomeric propensity of a truncated podocin construct (residues:126–350). We show that the podocin domain majorly homo-oligomerizes into a 16-mer. Circular dichroism and fluorescence spectroscopy suggest that the 16-mer oligomer has considerable secondary structure and moderate tertiary packing.     

Structure determination of archaea-specific ribosomal protein L46a reveals a novel protein fold

Three archaea-specific ribosomal proteins recently identified show no sequence homology with other known proteins. Here we determined the structure of L46a, the most conserved one among the three proteins, from Sulfolobus solfataricus P2 using NMR spectroscopy. The structure presents a twisted β-sheet formed by the N-terminal part and two helices at the C-terminus. The L46a structure has a positively charged surface which is conserved in the L46a protein family and is the potential rRNA-binding site. Searching homologous structures in Protein Data Bank revealed that the structure of L46a represents a novel protein fold. The backbone dynamics identified by NMR relaxation experiments reveal significant flexibility at the rRNA binding surface. The potential position of L46a on the ribosome was proposed by fitting the structure into a previous electron microscopy map of the ribosomal 50S subunit, which indicated that L46a contacts to domain I of 23S rRNA near a multifunctional ribosomal protein L7ae.     

NMR Structure of a Monomeric Intermediate on the Evolutionarily Optimized Assembly Pathway of a Small Trimerization Domain

Efficient formation of specific intermolecular interactions is essential for self-assembly of biological structures. The foldon domain is an evolutionarily optimized trimerization module required for assembly of the large, trimeric structural protein fibritin from phage T4. Monomers consisting of the 27 amino acids comprising a single foldon domain subunit spontaneously form a natively folded trimer. During assembly of the foldon domain, a monomeric intermediate is formed on the submillisecond time scale, which provides the basis for two consecutive very fast association reactions. Mutation of an intermolecular salt bridge leads to a monomeric protein that resembles the kinetic intermediate in its spectroscopic properties. NMR spectroscopy revealed essentially native topology of the monomeric intermediate with defined hydrogen bonds and side-chain interactions but largely reduced stability compared to the native trimer. This structural preorganization leads to an asymmetric charge distribution on the surface that can direct rapid subunit recognition. The low stability of the intermediate allows a large free-energy gain upon trimerization, which serves as driving force for rapid assembly. These results indicate different free-energy landscapes for folding of small oligomeric proteins compared to monomeric proteins, which typically avoid the transient population of intermediates.     

Interaction of the trp RNA-binding attenuation protein (TRAP) with anti-TRAP

The trp RNA-binding attenuation protein (TRAP) negatively regulates expression of the tryptophan biosynthesis genes of Bacillus subtilis. In the presence of tryptophan, TRAP is activated to bind to the 5'-leader region of the trp mRNA resulting in termination prior to the structural genes. In addition, accumulation of uncharged tRNA(Trp) induces synthesis of anti-TRAP (AT), which binds to TRAP and inhibits its function. Both of these proteins consist of oligomers of identical subunits. Here, we characterize the self-association of each of these proteins and the TRAP-AT interaction in free solution using equilibrium and velocity analytical ultracentrifugation. TRAP exists as a stable 11-mer in the absence and in the presence of tryptophan. Tryptophan binding induces a conformational change in TRAP. AT exists in a reversible equilibrium between trimer and dodecamer with an equilibrium constant of approximately 3 x 10(14)M(-3). About 20% of the trimer is incompetent to form dodecamer. The AT equilibrium is slow on the time-scale of the velocity experiment. Formation of TRAP-AT complexes occurs only in the presence of tryptophan. A complex containing one TRAP 11-mer and one AT 12-mer forms with high affinity. At higher ratios of TRAP:AT complexes containing two TRAP 11-mers and one AT 12-mer are detected. A model for the structure of the complex is proposed.     

Sequential action of mitochondrial chaperones in protein import into the matrix.

Translocation and folding of proteins imported into mitochondria are mediated by two matrix-localized chaperones, mhsp70 and hsp60. In order to investigate whether these chaperones act sequentially or in parallel, we studied their interaction with newly imported precursor proteins in isolated yeast mitochondria by coimmunoprecipitation. All precursors bound transiently to mhsp70. Release from mhsp70 required hydrolysis of ATP and did not immediately generate a tightly folded protein. For example, after imported mouse dihydrofolate reductase (a soluble monomeric enzyme) had been released from mhsp70, folding to a protease resistant conformation occurred only after a lag and was much slower than the release. Under standard import conditions, no significant association of DHFR with hsp60 could be detected. Similarly, newly imported hsp60 subunit was released from mhsp70 as an incompletely folded, unassembled intermediate which accumulated at low temperature and assembled to hsp60 14-mer at higher temperature in an ATP-dependent manner. Mas2p (the larger subunit of the MAS-encoded processing protease) first bound to mhsp70, then to hsp60, and only then assembled with its partner subunit, Mas1p. We propose that ATP-dependent release from mhsp70 is insufficient to cause folding of imported proteins and that assembly of hsp60 and Mas2p requires sequential, ATP-dependent interactions with mhsp70 and hsp60.     

Trimer-based design of pH-responsive protein cage results in soluble disassembled structures

Limited studies have been done on the interactions between subunits of self-assembling protein cages. E2 protein cage from Bacillus stearothermophilus was investigated in this work to impart pH-sensitive disassembly profile. Key amino acids were identified at the intratrimer and intertrimer interfaces, and histidine residues were introduced to these key sites to probe for their influences on the E2 assembly. We found that both the intratrimer- and the intertrimer-modified mutant proteins have the same quaternary structures as the wild type (E2-WT) at physiological pH of 7.4. At pH 5.0, the intratrimer-modified protein maintained its spherical structure. In contrast, the intertrimer modified protein lost its integrity, as observed under the electron microscope, whereas it remained soluble and nondenatured. The identified interactions between the intertrimers are critical in the formation of E2 protein cage. The pH-controlled disassembly of E2 protein cage in soluble and nondenatured form make it promising in nanoscale applications, especially for drug delivery and release in the endosomes.     

The pyruvate dehydrogenase complex from the parasitic nematode Ascaris suum: novel subunit composition and domain structure of the dihydrolipoyl transacetylase component.

The pyruvate dehydrogenase complex (PDC) from muscle of the adult parasitic nematode Ascaris suum plays a unique role in its anaerobic mitochondrial metabolism. Resolution of the intact complex in high salt dissociates the pyruvate dehydrogenase subunit but leaves the dihydrolipoyl dehydrogenase subunit (E3) and two other proteins with apparent M(r)s of 45 and 43 kDa bound to the dihydrolipoyl transacetylase (E2) core. These proteins are not observable on Coomassie brilliant blue-stained gels of other eukaryotic PDCs, but the 45-kDa protein is similar in apparent M(r), pI, and sensitivity to trypsin to the Kb subunit of the bovine kidney PDH alpha kinase. Acetylation of the ascarid PDC with [2-14C]pyruvate under conditions designed to maximize the incorporation of label into protein yielded only a single radiolabeled subunit, E2. These results confirm earlier reports that the ascarid PDC lacks protein X, an integral component recently identified in other eukaryotic PDCs. About 1.6 to 1.8 mol of 14C was incorporated/mole of E2, suggesting that the ascarid E2 contained two lipoly-bearing domains. Domain mapping of the 14C-acetylated ascarid E2 by limited tryptic digestion identified two lipoyl-bearing fragments with apparent M(r)s of 50 and 34 kDa and two core fragments with apparent M(r)s of 46 and 30 kDa. The ascarid E2 domain structure appears to be similar to that of other E2s. However, it appears that the subunit-binding domain (E2B) of the ascarid E2 may be significantly larger or be flanked by larger than normal interdomain regions. An enlarged E2B domain may be necessary to accommodate the additional binding of E3 to the E2 subunit in the ascarid complex, in the absence of protein X.     

The structures of frataxin oligomers reveal the mechanism for the delivery and detoxification of iron

Defects in the mitochondrial protein frataxin are responsible for Friedreich ataxia, a neurodegenerative and cardiac disease that affects 1:40,000 children. Here, we present the crystal structures of the iron-free and iron-loaded frataxin trimers, and a single-particle electron microscopy reconstruction of a 24 subunit oligomer. The structures reveal fundamental aspects of the frataxin mechanism. The trimer has a central channel in which one atom of iron binds. Two conformations of the channel with different metal-binding affinities suggest that a gating mechanism controls whether the bound iron is delivered to other proteins or transferred to detoxification sites. The trimer constitutes the basic structural unit of the 24 subunit oligomer. The architecture of this oligomer and several features of the trimer structure demonstrate striking similarities to the iron-storage protein ferritin. The data reveal how stepwise assembly provides frataxin with the structural flexibility to perform two functions: metal delivery and detoxification.     

Conserved amphipathic helices near the N-terminus and C-terminus of the alpha subunit of cranin (dystroglycan)

Cranin (dystroglycan) is a ubiquitously expressed extracellular matrix receptor, synthesized as a single precursor, which is cleaved into an extracellular subunit (alpha) and a transmembrane subunit (beta). The primary sequence of cranin (dystroglycan) is known from cDNA cloning, and the protein has been strongly implicated in morphogenesis, cell adhesion and human disease. Nevertheless, the domain structure of the alpha subunit has not been well studied; although the protein binds to matrix proteins, to the beta subunit, to cell surfaces, and possibly to other membrane proteins such as sarcoglycans, the domains responsible for mediating these interactions remain unknown. Here I report computer analyses that identify two distinctive amphipathic alpha-helical regions near the N-terminus and C-terminus of the alpha subunit, which are conserved in all species for which sequence information is currently available. This finding should stimulate and guide experimental studies designed to understand how the alpha subunit is associated with the cell surface and with its various ligands.     

Conserved Amphipathic Helices Near the N-Terminus and C-Terminus of the Alpha Subunit of Cranin (Dystroglycan)

Cranin (dystroglycan) is a ubiquitously expressed extracellular matrix receptor, synthesized as a single precursor, which is cleaved into an extracellular subunit (alpha) and a transmembrane subunit (beta). The primary sequence of cranin (dystroglycan) is known from cDNA cloning, and the protein has been strongly implicated in morphogenesis, cell adhesion and human disease. Nevertheless, the domain structure of the alpha subunit has not been well studied; although the protein binds to matrix proteins, to the beta subunit. to cell surfaces, and possibly to other membrane proteins such as sarcoglycans, the domains responsible for mediating these interactions remain unknown. Here I report computer analyses that identify two distinctive amphipathic alpha-helical regions near the N-terminus and C-terminus of the alpha subunit, which are conserved in all species for which sequence information is currently available. This finding should stimulate and guide experimental studies designed to understand how the alpha subunit is associated with the cell surface and with its various ligands.     

Complete hemocyanin subunit sequences of the hunting spider Cupiennius salei: recent hemocyanin remodeling in entelegyne spiders

Hemocyanins are large copper-containing respiratory proteins found in many arthropod species. Scorpions and orthognath spiders possess a highly conserved 4 x 6-mer hemocyanin that consists of at least seven distinct subunit types (termed a to g). However, many "modern" entelegyne spiders such as Cupiennius salei differ from the standard arachnid scheme and have 2 x 6-mer hemocyanins. Here we report the complete primary structure of the 2 x 6-mer hemocyanin of C. salei as deduced from cDNA sequencing, gel electrophoresis, and matrix-assisted laser desorption spectroscopy. Six distinct subunit types (1 through 6) and three additional allelic sequences were identified. Each 1 x 6-mer half-molecule most likely is composed of subunits 1-6, with subunit 1 linking the two hexamers via a disulfide bridge located in a C-terminal extension. The C. salei hemocyanin subunits all belong to the arachnid g-type, whereas the other six types (a-f) have been lost in evolution. The reconstruction of a complex hemocyanin from a single g-type subunit, which commenced about 190 million years ago and was completed about 90 million years ago, might be explained by physiological and behavioral changes that occurred during the evolution of the entelegyne spiders.     

The effect of C-terminal mutations of HSP60 on protein folding

HSP60 is an essential gene in Saccharomyces cerevisiae. The protein forms homotetradecameric double toroid complexes. The flexible C-terminal end of each subunit, which is hydrophobic in nature, protrudes inside the central cavity where protein folding occurs. In order to study the functional role of the C-terminus of Hsp60, we generated and characterized yeast strains expressing mutants of Hsp60 proteins. Most of the yeast strains expressing Hsp60 with C-terminal deletions grew normally, unless the deletion impaired the interaction between neighboring subunits. The cells carrying Hsp60 mutants with an epitope of influenza hemagglutinin (HA) and T7 alone in the C-terminal region grew normally, but the mutant containing both HA and T7 was unable to grow in nonfermentable carbon sources. In vitro biochemical assays were performed using purified Hsp60 proteins. All the mutants examined remained capable of interacting with Hsp10 in a nucleotide-dependent manner. However, binding and/or refolding of denatured rhodanese became defective in most of the hsp60 mutants. Therefore, the hydrophobic C-terminal tail of Hsp60 plays an important role in the refolding of protein substrates, although it is flexible in structure.     

Competence for natural transformation in Neisseria gonorrhoeae: components of DNA binding and uptake linked to type IV pilus expression

Catalytically active, recombinant fusion proteins of bacteriophage E endosialidase were expressed and purified from Escherichia coli. Constructs with different fusion partners added to the amino terminus of the endosialidase were enzymatically active. A post-translational proteolytic cleavage was shown to occur between serine 706 and aspartate 707 to generate the 76 kDa mature enzyme from the 90 kDa translation product. Endosialidase truncated at the C-terminus from aspartate 707 was observed to have the same 76 kDa molecular weight as wild-type enzyme using denaturing SDS-PAGE but, under native PAGE conditions, was not observed to form the approximately 250 kDa trimeric wild-type enzyme, implying that the C-terminus of the enzyme may be required for correct assembly of active trimer, rather than as part of the active site as has been previously suggested. Mutagenesis of aspartate 138 to alanine greatly reduced enzyme activity whereas conversion of other selected aspartate residues to alanine had less effect, consistent with similarities between the structure and cata-lytic mechanism of bacteriophage E endosialidase and those of exosialidases.     

The crystal structure of Mycobacterium tuberculosis alkylhydroperoxidase AhpD, a potential target for antitubercular drug design

The resistance of Mycobacterium tuberculosis to isoniazid is commonly linked to inactivation of a catalase-peroxidase, KatG, that converts isoniazid to its biologically active form. Loss of KatG is associated with elevated expression of the alkylhydroperoxidases AhpC and AhpD. AhpD has no sequence identity with AhpC or other proteins but has alkylhydroperoxidase activity and possibly additional physiological activities. The alkylhydroperoxidase activity, in the absence of KatG, provides an important antioxidant defense. We have determined the M. tuberculosis AhpD structure to a resolution of 1.9 A. The protein is a trimer in a symmetrical cloverleaf arrangement. Each subunit exhibits a new all-helical protein fold in which the two catalytic sulfhydryl groups, Cys-130 and Cys-133, are located near a central cavity in the trimer. The structure supports a mechanism for the alkylhydroperoxidase activity in which Cys-133 is deprotonated by a distant glutamic acid via the relay action of His-137 and a water molecule. The cysteine then reacts with the peroxide to give a sulfenic acid that subsequently forms a disulfide bond with Cys-130. The crystal structure of AhpD identifies a new protein fold relevant to members of this protein family in other organisms. The structural details constitute a potential platform for the design of inhibitors of potential utility as antitubercular agents and suggest that AhpD may have disulfide exchange properties of importance in other areas of M. tuberculosis biology.