Bacterial 5S rRNA-binding proteins of the CTC family

The presence of CTC family proteins is a unique feature of bacterial cells. In the CTC family, there are true ribosomal proteins (found in ribosomes of exponentially growing cells), and at the same time there are also proteins temporarily associated with the ribosome (they are produced by the cells under stress only and incorporate into the ribosome). One feature is common for these proteins — they specifically bind to 5S rRNA. In this review, the history of investigations of the best known representatives of this family is described briefly. Structural organization of the CTC family proteins and their occurrence among known taxonomic bacterial groups are discussed. Structural features of 5S rRNA and CTC protein are described that predetermine their specific interaction. Taking into account the position of a CTC protein and its intermolecular contacts in the ribosome, a possible role of its complex with 5S rRNA in ribosome functioning is discussed.     

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.     

The asymmetric trimeric architecture of [2Fe-2S] IscU: implications for its scaffolding during iron-sulfur cluster biosynthesis

IscU is a key component of the ISC machinery and is involved in the biogenesis of iron-sulfur (Fe-S) proteins. IscU serves as a scaffold for assembly of a nascent Fe-S cluster prior to its delivery to an apo protein. Here, we report the first crystal structure of IscU with a bound [2Fe-2S] cluster from the hyperthermophilic bacterium Aquifex aeolicus, determined at a resolution of 2.3 Å, using multiwavelength anomalous diffraction of the cluster. The holo IscU formed a novel asymmetric trimer that harbored only one [2Fe-2S] cluster. One iron atom of the cluster was coordinated by the S^γ atom of Cys36 and the N^ε atom of His106, and the other was coordinated by the S^γ atoms of Cys63 and Cys107 on the surface of just one of the protomers. However, the cluster was buried inside the trimer between the neighboring protomers. The three protomers were conformationally distinct from one another and associated around a noncrystallographic pseudo-3-fold axis. The three flexible loop regions carrying the ligand-binding residues (Cys36, Cys63, His106 and Cys107) and the N-terminal α1 helices were positioned at the interfaces and underwent substantial conformational rearrangement, which stabilized the association of the asymmetric trimer. This unique trimeric A. aeolicus holo-IscU architecture was clearly distinct from other known monomeric apo-IscU/SufU structures, indicating that asymmetric trimer organization, as well as its association/dissociation, would be involved in the scaffolding function of IscU.     

General Stress Protein CTC from Bacillus subtilis Specifically Binds to Ribosomal 5S RNA

Two recombinant proteins of the CTC family were prepared: the general stress protein CTC from Bacillus subtilis and its homolog from Aquifex aeolicus. The general stress protein CTC from B. subtilis forms a specific complex with 5S rRNA and its stable fragment of 60 nucleotides, which contains internal loop E. The ribosomal protein TL5 from Thermus thermophilus, which binds with high affinity to 5S rRNA in the loop E region, was also shown to replace the CTC protein from B. subtilis in its complexes with 5S rRNA and its fragment. The findings suggest that the protein CTC from B. subtilis binds to the same site on 5S rRNA as the protein TL5. The protein CTC from A. aeolicus, which is 50 amino acid residues shorter from the N-terminus than the proteins TL5 from T. thermophilus and CTC from B. subtilis, does not interact with 5S rRNA.     

FlgM proteins from different bacteria exhibit different structural characteristics

Intrinsically disordered proteins (IDPs) are a unique class of proteins that do not require a stable structure for function. The importance of IDPs in many biological processes has been established but there remain unanswered questions about their evolution and conservation of their disordered state within a protein family. Our group has been studying the structural similarities among orthologous FlgM proteins, a model class of IDPs. We have previously shown that the FlgM protein from the thermophile Aquifex aeolicus has more structure at A. aeolicus' physiological temperature (85 °C) than is observed for the Salmonella typhimurium FlgM, suggesting that the disordered nature of FlgM varies among organisms and is not universally conserved. In this work, we extend these studies to the FlgM proteins from Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, and Bacillus subtilis. We demonstrate that the B. subtilis, E. coli, and S. typhimurium FlgMs exist in a premolten globule-like conformation, though the B. subtilis FlgM is in a more compacted conformation than the other two. The P. aeruginosa and P. mirabilis FlgM proteins exist in a currently unknown conformation that is not either coil-like or premolten globule-like. The P. aeruginosa FlgM appears to contain more weak intramolecular contacts given its more compacted state than the P. mirabilis FlgM. These results provide experimental evidence that members of the same protein family can exhibit different degrees of disorder, though understanding how different disordered states evolve in the same protein family will require more study.     

Phylogenetic analyses provide the first insights into the evolution of OVATE family proteins in land plants

Background and Aims

The OVATE gene encodes a nuclear-localized regulatory protein belonging to a distinct family of plant-specific proteins known as the OVATE family proteins (OFPs). OVATE was first identified as a key regulator of fruit shape in tomato, with nonsense mutants displaying pear-shaped fruits. However, the role of OFPs in plant development has been poorly characterized.

Methods

Public databases were searched and a total of 265 putative OVATE protein sequences were identified from 13 sequenced plant genomes that represent the major evolutionary lineages of land plants. A phylogenetic analysis was conducted based on the alignment of the conserved OVATE domain from these 13 selected plant genomes. The expression patterns of tomato SlOFP genes were analysed via quantitative real-time PCR. The pattern of OVATE gene duplication resulting in the expansion of the gene family was determined in arabidopsis, rice and tomato.

Key Results

Genes for OFPs were found to be present in all the sampled land plant genomes, including the early-diverged lineages, mosses and lycophytes. Phylogenetic analysis based on the amino acid sequences of the conserved OVATE domain defined 11 sub-groups of OFPs in angiosperms. Different evolutionary mechanisms are proposed for OVATE family evolution, namely conserved evolution and divergent expansion. Characterization of the AtOFP family in arabidopsis, the OsOFP family in rice and the SlOFP family in tomato provided further details regarding the evolutionary framework and revealed a major contribution of tandem and segmental duplications towards expansion of the OVATE gene family.

Conclusions

This first genome-wide survey on OFPs provides new insights into the evolution of the OVATE protein family and establishes a solid base for future functional genomics studies on this important but poorly characterized regulatory protein family in plants.     

Crystal structure of prephenate dehydrogenase from Streptococcus mutans

Prephenate dehydrogenase (PDH) is a bacterial enzyme that catalyzes conversion of prephenate to 4-hydroxyphenylpyruvate through the oxidative decarboxylation pathway for tyrosine biosynthesis. This enzymatic pathway exists in prokaryotes but is absent in mammals, indicating that it is a potential target for the development of new antibiotics. The crystal structure of PDH from Streptococcus mutans in a complex with NAD⁺ shows that the enzyme exists as a homo-dimer, each monomer consisting of two domains, a modified nucleotide binding N-terminal domain and a helical prephenate C-terminal binding domain. The latter is the dimerization domain. A structural comparison of PDHs from mesophilic S. mutans and thermophilic Aquifex aeolicus showed differences in the long loop between β6 and β7, which may be a reason for the high K_m values of PDH from Streptococcus mutans.     

Structural and thermodynamic characterization of the recognition of the S100-binding peptides TRTK12 and p53 by calmodulin

Calmodulin (CaM) is a multifunctional messenger protein that activates a wide variety of signaling pathways in eukaryotic cells in a calcium-dependent manner. CaM has been proposed to be functionally distinct from the S100 proteins, a related family of eukaryotic calcium-binding proteins. Previously, it was demonstrated that peptides derived from the actin-capping protein, TRTK12, and the tumor-suppressor protein, p53, interact with multiple members of the S100 proteins. To test the specificity of these peptides, they were screened using isothermal titration calorimetry against 16 members of the human S100 protein family, as well as CaM, which served as a negative control. Interestingly, both the TRTK12 and p53 peptides were found to interact with CaM. These interactions were further confirmed by both fluorescence and nuclear magnetic resonance spectroscopies. These peptides have distinct sequences from the known CaM target sequences. The TRTK12 peptide was found to independently interact with both CaM domains and bind with a stoichiometry of 2:1 and dissociations constants K_d,C-term = 2 ± 1 µM and K_d,N-term = 14 ± 1 µM. In contrast, the p53 peptide was found to interact only with the C-terminal domain of CaM, K_d,C-term =2 ± 1 µM, 25°C. Using NMR spectroscopy, the locations of the peptide binding sites were mapped onto the structure of CaM. The binding sites for both peptides were found to overlap with the binding interface for previously identified targets on both domains of CaM. This study demonstrates the plasticity of CaM in target binding and may suggest a possible overlap in target specificity between CaM and the S100 proteins.     

An Ancient Autoproteolytic Domain Found in GAIN,ZU5 and Nucleoporin98

A large family of G protein-coupled receptors (GPCRs) involved in cell adhesion has a characteristic autoproteolysis motif of HLT/S known as the GPCR proteolysis site (GPS). GPS is also shared by polycystic kidney disease proteins and it precedes the first transmembrane segment in both families. Recent structural studies have elucidated the GPS to be part of a larger domain named GPCR autoproteolysis inducing (GAIN) domain. Here we demonstrate the remote homology relationships of GAIN domain to ZU5 domain and Nucleoporin98 (Nup98) C-terminal domain by structural and sequence analysis. Sequence homology searches were performed to extend ZU5-like domains to bacteria and archaea, as well as new eukaryotic families. We found that the consecutive ZU5-UPA-death domain domain organization is commonly used in human cytoplasmic proteins with ZU5 domains, including CARD8 (caspase recruitment domain-containing protein 8) and NLRP1 (NACHT, LRR and PYD domain-containing protein 1) from the FIIND (Function to Find) family. Another divergent family of extracellular ZU5-like domains was identified in cartilage intermediate layer proteins and FAM171 proteins. Current diverse families of GAIN domain subdomain B, ZU5 and Nup98 C-terminal domain likely evolved from an ancient autoproteolytic domain with an HFS motif. The autoproteolytic site was kept intact in Nup98, p53-induced protein with a death domain and UNC5C-like, deteriorated in many ZU5 domains and changed in GAIN and FIIND. Deletion of the strand after the cleavage site was observed in zonula occluden-1 and some Nup98 homologs. These findings link several autoproteolytic domains, extend our understanding of GAIN domain origination in adhesion GPCRs and provide insights into the evolution of an ancient autoproteolytic domain.     

Analysis of the interaction of 16S rRNA and cytoplasmic membrane with the C-terminal part of the Streptococcus pneumoniae Era GTPase.

Era, an essential GTPase, plays a regulatory role in several cellular processes. The Era protein of Streptococcus pneumoniae has recently been shown to bind to 16S rRNA and the cytoplasmic membrane. However, exact locations of Era responsible for RNA- and membrane-binding were unknown. To identify the regions in Era that interact with the RNA and membrane, the C-terminal part of S. pneumoniae Era was systematically deleted while the N-terminal part, responsible for the GTPase activity of the protein, was kept intact. The resulting truncated Era proteins were purified and characterized. The C-terminal deletion of 9 or 19 amino-acid residues did not affect 16S rRNA-binding activity while further deletions of the C-terminus (29-114 amino-acid residues) abolished the activity. These results indicate that the integrity of the putative KH domain of Era, spanning the amino-acid residues between approximately 22-83 from the C-terminus, is required for 16S rRNA-binding. Furthermore, the Era proteins with a deletion up to 45 residues from the C-terminus retained membrane-binding activity, but longer deletions significantly reduced the activity. These results indicate that part of the putative KH domain is also required for membrane-binding. Thus, these results indicate for the first time that the regions critical for the membrane- and 16S rRNA-binding activities of Era overlap. The era gene with a deletion of 9 or 19 codons from its 3' terminus complemented an Escherichia coli mutant strain deficient in Era production whereas the genes with longer deletions failed to do so, thereby indicating that the KH domain is essential for Era function. Taken together, the results of this study indicate that the putative KH domain is required for 16S rRNA-binding activity and that part of the KH domain is also required for membrane-binding activity. The results also suggest that the interaction between Era and 16S rRNA is essential for bacterial growth.     

News from an ancient world: two novel astacin metalloproteases from the horseshoe crab

In this work, we report the cloning, heterologous expression, and characterization of two novel astacin proteases from the chelicerate Limulus polyphemus (horseshoe crab), designated as LAST (Limulus astacin) and LAST_MAM (Limulus astacin containing a MAM domain), respectively. The expression pattern showed ubiquitous occurrence of LAST_MAM, while LAST was predominantly restricted to the eyes and brain, indicating a function in the nervous system. Both enzymes contain the characteristic metzincin-type zinc-binding region and Met turn. While LAST is made up only of the typical prodomain and astacin-like protease domain, LAST_MAM contains an additional MAM (meprin A5 protein tyrosine phosphatase μ) domain, which so far only has been found in few astacins such as the vertebrate meprin Hydra and squid enzymes, and in a number of other extracellular proteins such as A5 protein and tyrosine phosphatase μ. These gave rise to the designation MAM for this protein module. MAM domains have been shown to be responsible for protein oligomerization in meprin proteases and tyrosine phosphatase μ. Since the horseshoe crab has kept its body plan for almost half a billion years, it is therefore a privileged organism for the study of protease evolution. In this context, we could show by phylogenetic analysis that this protease is not related to the other MAM-domain-containing astacins indicating different evolutionary origins of these proteins. Moreover, we clearly demonstrated the divergent evolvement of the MAM module itself, and not only with regard to proteases. However, there are some unique functional features that are not shared by other members of this protein family. For example, LAST_MAM is the only astacin protease known so far that is active in its zymogen form, indicating that the presence of the N-terminal propeptide does not prevent proteolytic activity.     

Production of fully assembled and active Aquifex aeolicus F1FO ATP synthase in Escherichia coli

Background

F₁F_O ATP synthases catalyze the synthesis of ATP from ADP and inorganic phosphate driven by ion motive forces across the membrane. A number of ATP synthases have been characterized to date. The one from the hyperthermophilic bacterium Aquifex aeolicus presents unique features, i.e. a putative heterodimeric stalk. To complement previous work on the native form of this enzyme, we produced it heterologously in Escherichia coli.

Methods

We designed an artificial operon combining the nine genes of A. aeolicus ATP synthase, which are split into four clusters in the A. aeolicus genome. We expressed the genes and purified the enzyme complex by affinity and size-exclusion chromatography. We characterized the complex by native gel electrophoresis, Western blot, and mass spectrometry. We studied its activity by enzymatic assays and we visualized its structure by single-particle electron microscopy.

Results

We show that the heterologously produced complex has the same enzymatic activity and the same structure as the native ATP synthase complex extracted from A. aeolicus cells. We used our expression system to confirm that A. aeolicus ATP synthase possesses a heterodimeric peripheral stalk unique among non-photosynthetic bacterial F₁F_O ATP synthases.

Conclusions

Our system now allows performing previously impossible structural and functional studies on A. aeolicus F₁F_O ATP synthase.

General significance

More broadly, our work provides a valuable platform to characterize many other membrane protein complexes with complicated stoichiometry, i.e. other respiratory complexes, the nuclear pore complex, or transporter systems.     

A ribosomal protein from Thermus thermophilus is homologous to a general shock protein

The gene encoding the ribosomal protein from Thermus thermophilus, TL5, which binds to the 5S rRNA, has been cloned and sequenced. The codon usage shows a clear preference for G/C rich codons that is characteristic for many genes in thermophilic bacteria. The deduced amino acid sequence consists of 206 residues. The sequence of TL5 shows a strong similarity to a general shock protein from Bacillus subtilis, named CTC. The protein CTC is homologous in its N-terminal part to the 5S rRNA binding protein, L25, from E coli. An alignment of the TL5, CTC and L25 sequences displays a number of residues that are totally conserved. No clear sequence similarity was found between TL5 and other proteins which are known to bind to 5S rRNA. The evolutionary relationship of a heat shock protein in mesophiles and a ribosomal protein in thermophilic bacteria as well as a possible role of TL5 in the ribosome are discussed.     

Tandem duplications of a degenerated GTP-binding domain at the origin of GTPase receptors Toc159 and thylakoidal SRP

The evolutionary origin of some nuclear encoded proteins that translocate proteins across the chloroplast envelope remains unknown. Therefore, sequences of GTPase proteins constituting the Arabidopsis thaliana translocon at the outer membrane of chloroplast (atToc) complexes were analyzed by means of HCA. In particular, atToc159 and related proteins (atToc132, atToc120, and atToc90) do not have proven homologues of prokaryotic or eukaryotic ancestry. We established that the three domains commonly referred to as A, G, and M originate from the GTPase G domain, tandemly repeated, and probably evolving toward an unstructured conformation in the case of the A domain. It resulted from this study a putative common ancestor for these proteins and a new domain definition, in particular the splitting of A into three domains (A1, A2, and A3), has been proposed. The family of Toc159, previously containing A. thaliana and Pisum sativum, has been extended to Medicago truncatula and Populus trichocarpa and it has been revised for Oryza sativa. They have also been compared to GTPase subunits involved in the cpSRP system. A distant homology has been revealed among Toc and cpSRP GTP-hydrolyzing proteins of A. thaliana, and repetitions of a GTPase domain were also found in cpSRP protein receptors, by means of HCA analysis.     

Structural features and chaperone activity of the NudC protein family

The NudC family consists of four conserved proteins with representatives in all eukaryotes. The archetypal nudC gene from Aspergillus nidulans is a member of the nud gene family that is involved in the maintenance of nuclear migration. This family also includes nudF, whose human orthologue, Lis1, codes for a protein essential for brain cortex development. Three paralogues of NudC are known in vertebrates: NudC, NudC-like (NudCL), and NudC-like 2 (NudCL2). The fourth distantly related member of the family, CML66, contains a NudC-like domain. The three principal NudC proteins have no catalytic activity but appear to play as yet poorly defined roles in proliferating and dividing cells. We present crystallographic and NMR studies of the human NudC protein and discuss the results in the context of structures recently deposited by structural genomics centers (i.e., NudCL and mouse NudCL2). All proteins share the same core CS domain characteristic of proteins acting either as cochaperones of Hsp90 or as independent small heat shock proteins. However, while NudC and NudCL dimerize via an N-terminally located coiled coil, the smaller NudCL2 lacks this motif and instead dimerizes as a result of unique domain swapping. We show that NudC and NudCL, but not NudCL2, inhibit the aggregation of several target proteins, consistent with an Hsp90-independent heat shock protein function. Importantly, and in contrast to several previous reports, none of the three proteins is able to form binary complexes with Lis1. The availability of structural information will be of help in further studies on the cellular functions of the NudC family.