Twin-arginine translocase may have a role in the chaperone function of NarJ from Escherichia coli

NarJ is a chaperone involved in folding, maturation, and molybdenum cofactor insertion of nitrate reductase A from Escherichia coli. It has also been shown that NarJ exhibits sequence homology to a family of chaperones involved in maturation and cofactor insertion of E. coli redox enzymes that are mediated by twin-arginine translocase (Tat) dependent translocation. In this study, we show that NarJ binds the N-terminal region of NarG through Far Western studies and isothermal titration calorimetry, and the binding event occurs towards a short peptide sequence that contains a homologous twin-arginine motif. Fractionation experiments also show that the interaction of NarJ to the cytoplasmic membrane exhibits Tat-dependence. Upon further investigation through Far Western blots, the interactome of NarJ also exhibits Tat-dependence. Together the data suggest that the Tat system may play a role in the maturation pathway of nitrate reductase A.     

Comparing system-specific chaperone interactions with their Tat dependent redox enzyme substrates

Redox enzyme substrates of the twin-arginine translocation (Tat) system contain a RR-motif in their leader peptide and require the assistance of chaperones, redox enzyme maturation proteins (REMPs). Here various regions of the RR-containing oxidoreductase subunit (leader peptide, full preprotein with and without a leader cleavage site, mature protein) were assayed for interaction with their REMPs. All REMPs bound their preprotein substrates independent of the cleavage site. Some showed binding to either the leader or mature region, whereas in one case only the preprotein bound its REMP. The absence of Tat also influenced the amount of chaperone-substrate interaction.

Structured summary

MINT-8047497: FdhE (uniprotkb:P13024) and FdoG (uniprotkb:P32176) physically interact (MI:0915) by two hybrid (MI:0018)MINT-8046441: HybO (uniprotkb:P69741) and HybE (uniprotkb:P0AAN1) physically interact (MI:0915) by two hybrid (MI:0018)MINT-8046375: DmsA (uniprotkb:P18775) and DmsD (uniprotkb:P69853) physically interact (MI:0915) by two hybrid (MI:0018)MINT-8046425: TorA (uniprotkb:P33225) and TorD (uniprotkb:P36662) physically interact (MI:0915) by two hybrid (MI:0018)MINT-8046393: NarJ (uniprotkb:P0AF26) and NarG (uniprotkb:P09152) physically interact (MI:0915) by two hybrid (MI:0018)MINT-8046409: NapD (uniprotkb:P0A9I5) and NapA (uniprotkb:P33937) physically interact (MI:0915) by two hybrid (MI:0018)     

DmsD,a Tat system specific chaperone,interacts with other general chaperones and proteins involved in the molybdenum cofactor biosynthesis

Many bacterial oxidoreductases depend on the Tat translocase for correct cell localization. Substrates for the Tat translocase possess twin-arginine leaders. System specific chaperones or redox enzyme maturation proteins (REMPs) are a group of proteins implicated in oxidoreductase maturation. DmsD is a REMP discovered in Escherichia coli, which interacts with the twin-arginine leader sequence of DmsA, the catalytic subunit of DMSO reductase. In this study, we identified several potential interacting partners of DmsD by using several in vitro protein–protein interaction screening approaches, including affinity chromatography, co-precipitation, and cross-linking. Candidate hits from these in vitro findings were analyzed by in vivo methods of bacterial two-hybrid (BACTH) and bimolecular fluorescence complementation (BiFC). From these data, DmsD was confirmed to interact with the general molecular chaperones DnaK, DnaJ, GrpE, GroEL, Tig and Ef-Tu. In addition, DmsD was also found to interact with proteins involved in the molybdenum cofactor biosynthesis pathway. Our data suggests that DmsD may play a role as a “node” in escorting its substrate through a cascade of chaperone assisted protein-folding maturation events.     

Conserved immunoglobulin-like features in a family of periplasmic pilus chaperones in bacteria.

Detailed structural analyses revealed a family of periplasmic chaperones in Gram-negative prokaryotes which are structurally and possibly evolutionarily related to the immunoglobulin superfamily and assist in the assembly of adhesive pili. The members of this family have similar structures consistent with the overall topology of an immunoglobulin fold. Seven pilus chaperone sequences from Escherichia coli, Haemophilus influenzae and Klebsiella pneumoniae were aligned and their consensus sequence was superimposed onto the known three-dimensional structure of PapD, a representative member of the family. The molecular details of the conserved and variable structural motifs in this family of periplasmic chaperones give important insight into their structure, function, mechanism of action and evolutionary relationship with the immunoglobulin superfamily.     

Molecular Dynamics Simulations and Structure-Guided Mutagenesis Provide Insight into the Architecture of the Catalytic Core of the Ectoine Hydroxylase

Many bacteria amass compatible solutes to fend-off the detrimental effects of high osmolarity on cellular physiology and water content. These solutes also function as stabilizers of macromolecules, a property for which they are referred to as chemical chaperones. The tetrahydropyrimidine ectoine is such a compatible solute and is widely synthesized by members of the Bacteria. Many ectoine producers also synthesize the stress protectant 5-hydroxyectoine from the precursor ectoine, a process that is catalyzed by the ectoine hydroxylase (EctD). The EctD enzyme is a member of the non-heme-containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily. A crystal structure of the EctD protein from the moderate halophile Virgibacillus salexigens has previously been reported and revealed the coordination of the iron catalyst, but it lacked the substrate ectoine and the co-substrate 2-oxoglutarate. Here we used this crystal structure as a template to assess the likely positioning of the ectoine and 2-oxoglutarate ligands within the active site by structural comparison, molecular dynamics simulations, and site-directed mutagenesis. Collectively, these approaches suggest the positioning of the iron, ectoine, and 2-oxoglutarate ligands in close proximity to each other and with a spatial orientation that will allow the region-selective and stereo-specific hydroxylation of (4S)-ectoine to (4S,5S)-5-hydroxyectoine. Our study thus provides a view into the catalytic core of the ectoine hydroxylase and suggests an intricate network of interactions between the three ligands and evolutionarily highly conserved residues in members of the EctD protein family.     

A link between sequence conservation and domain motion within the AAA+ family

The AAA+ family of proteins play fundamental roles in all three kingdoms of life. It is thought that they act as molecular chaperones in aiding the assembly or disassembly of proteins or protein complexes. Recent structural studies on a number of AAA+ family proteins have revealed that they share similar structural elements. These structures provide a possible link between nucleotide binding/hydrolysis and the conformational changes which are then amplified to generate mechanical forces for their specific functions. However, from these individual studies it is far from clear whether AAA+ proteins in general share properties in terms of nucleotide induced conformational changes. In this study, we analyze sequence conservation within the AAA+ family and identify two subfamilies, each with a distinct conserved linker sequence that may transfer conformational changes upon ATP binding/release to movements between subdomains and attached domains. To investigate the relation of these linker sequences to conformational changes, molecular dynamics (MD) simulations on X-ray structures of AAA+ proteins from each subfamily have been performed. These simulations show differences in both the N-linker peptide, subdomain motion, and cooperativity between elements of quaternary structure. Extrapolation of subdomain movements from one MD simulation enables us to produce a structure in close agreement with cryo-EM experiments.     

Myomesin 3, a novel structural component of the M-band in striated muscle

The M-band is the cytoskeletal structure that cross-links the myosin and titin filaments in the middle of the sarcomere. Apart from the myosin tails and the C-termini of titin, only two closely related structural proteins had been detected at the M-band so far, myomesin and M-protein. However, electron microscopy studies revealed structural features that do not correlate with the expression of these two proteins, indicating the presence of unknown constituents in the M-band.Using comparative sequence analysis, we have identified a third member of this gene family, myomesin 3, and characterised its biological properties. Myomesin 3 is predicted to consist of a unique head domain followed by a conserved sequence of either fibronectin- or immunoglobulin-like domains, similarly to myomesin 3 and M-protein. While all three members of the myomesin family are localised to the M-band of the sarcomere, each member shows its specific expression pattern. In contrast to myomesin, which is ubiquitously expressed in all striated muscles, and M-protein, whose expression is restricted to adult heart and fast-twitch skeletal muscle, myomesin 3 can be detected mainly in intermediate speed fibers of skeletal muscle. In analogy to myomesin, myomesin 3 targets to the M-band region of the sarcomere via its N-terminal part and forms homodimers via its C-terminal domain. However, despite the high degree of homology, no heterodimer between distinct members of the myomesin gene family can be detected. We propose that each member of the myomesin family is a component of one of the distinct ultrastructures, the M-lines, which modulate the mechanical properties of the M-bands in different muscle types.     

Chaperone networking facilitates protein targeting to the bacterial cytoplasmic membrane

Nascent polypeptides emerging from the ribosome are assisted by a pool of molecular chaperones and targeting factors, which enable them to efficiently partition as cytosolic, integral membrane or exported proteins. Extensive genetic and biochemical analyses have significantly expanded our knowledge of chaperone tasking throughout this process. In bacteria, it is known that the folding of newly-synthesized cytosolic proteins is mainly orchestrated by three highly conserved molecular chaperones, namely Trigger Factor (TF), DnaK (HSP70) and GroEL (HSP60). Yet, it has been reported that these major chaperones are strongly involved in protein translocation pathways as well. This review describes such essential molecular chaperone functions, with emphasis on both the biogenesis of inner membrane proteins and the post-translational targeting of presecretory proteins to the Sec and the twin-arginine translocation (Tat) pathways. Critical interplay between TF, DnaK, GroEL and other molecular chaperones and targeting factors, including SecB, SecA, the signal recognition particle (SRP) and the redox enzyme maturation proteins (REMPs) is also discussed. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Rapid evolution of the DNA-binding site in LAGLIDADG homing endonucleases

Sequence analysis of chloroplast and mitochondrial large subunit rRNA genes from over 75 green algae disclosed 28 new group I intron-encoded proteins carrying a single LAGLIDADG motif. These putative homing endonucleases form four subfamilies of homologous enzymes, with the members of each subfamily being encoded by introns sharing the same insertion site. We showed that four divergent endonucleases from the I-CreI subfamily cleave the same DNA substrates. Mapping of the 66 amino acids that are conserved among the members of this subfamily on the 3-dimensional structure of I-CreI bound to its recognition sequence revealed that these residues participate in protein folding, homodimerization, DNA recognition and catalysis. Surprisingly, only seven of the 21 I-CreI amino acids interacting with DNA are conserved, suggesting that I-CreI and its homologs use different subsets of residues to recognize the same DNA sequence. Our sequence comparison of all 45 single-LAGLIDADG proteins identified so far suggests that these proteins share related structures and that there is a weak pressure in each subfamily to maintain identical protein–DNA contacts. The high sequence variability we observed in the DNA-binding site of homologous LAGLIDADG endonucleases provides insight into how these proteins evolve new DNA specificity.     

Structure and variability of recently inserted Alu family members.

The HS subfamily of Alu sequences is comprised of a group of nearly identical members. Individual subfamily members share 97.7% nucleotide identity with each other and 98.9% nucleotide identity with the HS consensus sequence. Individual subfamily members are on the average 2.8 million years old, and were probably derived from a single source 'master' gene sometime after the human/great ape divergence. The recent Alu family member insertions provide a better image of the structure of Alu retroposons before they have had the opportunity to change significantly. All of the HS subfamily members are flanked by perfect direct repeats as a result of insertion at staggered nicks. The 'master' gene from which the HS subfamily members were derived had an oligo-dA rich tail at least 40 bases long. The 'master' gene is very rich in CpG dinucleotides, but nucleotide substitutions within subfamily members accumulated in a random manner typical for Alu sequence with CpG substitutions occurring 9.2 fold faster than non-CpG substitutions.     

Human relaxin-2: historical perspectives and role in cancer biology

One of the most recognised and studied family of peptide hormones is the insulin superfamily. Within this family is the relaxin subfamily which comprises seven members: relaxin-1, -2 and -3 and insulin-like peptides 3, 4, 5 and 6. Besides exhibiting sequence similarities, each member exists as an active A-B heterodimer linked by three disulfide bonds. This mini-review is divided into three broad themes: an overview of all insulin superfamily members (including structural similarities); roles of each superfamily member and finally, a focus on the pleiotropic peptide hormone, human relaxin-2. In addition to promoting vasodilatory effects leading to evaluation in Phase III clinical trials for the treatment of acute heart failure, relaxin has recently been shown to be highly expressed by cancer cells, aiding in their proliferation, invasiveness and metastasis. These contrary effects of relaxin are discussed together with current efforts in the development of relaxin antagonists that may possess future therapeutic potential for the treatment of certain cancers.     

PICKLE is a CHD subfamily II ATP-dependent chromatin remodeling factor

PICKLE plays a critical role in repression of genes that regulate development identity in Arabidopsis thaliana. PICKLE codes for a putative ATP-dependent chromatin remodeler that exhibits sequence similarity to members of subfamily II of animal CHD remodelers, which includes remodelers such as CHD3/Mi-2 that also restrict expression of developmental regulators. Whereas animal CHD3 remodelers are a component of the Mi-2/NuRD complex that promotes histone deacetylation, PICKLE promotes trimethylation of histone H3 lysine 27 suggesting that it acts via a distinct epigenetic pathway. Here, we examine whether PICKLE is also a member of a multisubunit complex and characterize the biochemical properties of recombinant PICKLE protein. Phylogenetic analysis indicates that PICKLE-related proteins in plants share a common ancestor with members of subfamily II of animal CHD remodelers. Biochemical characterization of PICKLE in planta, however, reveals that PICKLE primarily exists as a monomer. Recombinant PICKLE protein is an ATPase that is stimulated by ssDNA and mononucleosomes and binds to both naked DNA and mononucleosomes. Furthermore, recombinant PICKLE exhibits ATP-dependent chromatin remodeling activity. These studies demonstrate that subfamily II CHD proteins in plants, such as PICKLE, retain ATP-dependent chromatin remodeling activity but act through a mechanism that does not involve the ubiquitous Mi-2/NuRD complex.     

Conformational Selection Underlies Recognition of a Molybdoenzyme by Its Dedicated Chaperone

Molecular recognition is central to all biological processes. Understanding the key role played by dedicated chaperones in metalloprotein folding and assembly requires the knowledge of their conformational ensembles. In this study, the NarJ chaperone dedicated to the assembly of the membrane-bound respiratory nitrate reductase complex NarGHI, a molybdenum-iron containing metalloprotein, was taken as a model of dedicated chaperone. The combination of two techniques ie site-directed spin labeling followed by EPR spectroscopy and ion mobility mass spectrometry, was used to get information about the structure and conformational dynamics of the NarJ chaperone upon binding the N-terminus of the NarG metalloprotein partner. By the study of singly spin-labeled proteins, the E119 residue present in a conserved elongated hydrophobic groove of NarJ was shown to be part of the interaction site. Moreover, doubly spin-labeled proteins studied by pulsed double electron-electron resonance (DEER) spectroscopy revealed a large and composite distribution of inter-label distances that evolves into a single preexisting one upon complex formation. Additionally, ion mobility mass spectrometry experiments fully support these findings by revealing the existence of several conformers in equilibrium through the distinction of different drift time curves and the selection of one of them upon complex formation. Taken together our work provides a detailed view of the structural flexibility of a dedicated chaperone and suggests that the exquisite recognition and binding of the N-terminus of the metalloprotein is governed by a conformational selection mechanism.     

A conserved lysine residue in the crenarchaea-specific loop is important for the crenarchaeal splicing endonuclease activity

In Archaea, splicing endonuclease (EndA) recognizes and cleaves precursor RNAs to remove introns. Currently, EndAs are classified into three families according to their subunit structures: homotetramer, homodimer, and heterotetramer. The crenarchaeal heterotetrameric EndAs can be further classified into two subfamilies based on the size of the structural subunit. Subfamily A possesses a structural subunit similar in size to the catalytic subunit, whereas subfamily B possesses a structural subunit significantly smaller than the catalytic subunit. Previously, we solved the crystal structure of an EndA from Pyrobaculum aerophilum. The endonuclease was classified into subfamily B, and the structure revealed that the enzyme lacks an N-terminal subdomain in the structural subunit. However, no structural information is available for crenarchaeal heterotetrameric EndAs that are predicted to belong to subfamily A. Here, we report the crystal structure of the EndA from Aeropyrum pernix, which is predicted to belong to subfamily A. The enzyme possesses the N-terminal subdomain in the structural subunit, revealing that the two subfamilies of heterotetrameric EndAs are structurally distinct. EndA from A. pernix also possesses an extra loop region that is characteristic of crenarchaeal EndAs. Our mutational study revealed that the conserved lysine residue in the loop is important for endonuclease activity. Furthermore, the sequence characteristics of the loops and the positions towards the substrate RNA according to a docking model prompted us to propose that crenarchaea-specific loops and an extra amino acid sequence at the catalytic loop of nanoarchaeal EndA are derived by independent convergent evolution and function for recognizing noncanonical bulge-helix-bulge motif RNAs as substrates.     

Membrane-associated maturation of the heterotetrameric nitrate reductase of Thermus thermophilus

The nar operon, coding for the respiratory nitrate reductase of Thermus thermophilus (NRT), encodes a di-heme b-type (NarJ) and a di-heme c-type (NarC) cytochrome. The role of both cytochromes and that of a putative chaperone (NarJ) in the synthesis and maturation of NRT was studied. Mutants of T. thermophilus lacking either NarI or NarC synthesized a soluble form of NarG, suggesting that a putative NarCI complex constitutes the attachment site for the enzyme. Interestingly, the NarG protein synthesized by both mutants was inactive in nitrate reduction and misfolded, showing that membrane attachment was required for enzyme maturation. Consistent with its putative role as a specific chaperone, inactive and misfolded NarG was synthesized by narJ mutants, but in contrast to its Escherichia coli homologue, NarJ was also required for the attachment of the thermophilic enzyme to the membrane. A bacterial two-hybrid system was used to demonstrate the putative interactions between the NRT proteins suggested by the analysis of the mutants. Strong interactions were detected between NarC and NarI and between NarG and NarJ. Weaker interaction signals were detected between NarI, but not NarC, and both NarG and NarH. These results lead us to conclude that the NRT is a heterotetrameric (NarC/NarI/NarG/NarH) enzyme, and we propose a model for its synthesis and maturation that is distinct from that of E. coli. In the synthesis of NRT, a NarCI membrane complex and a soluble NarGJH complex are synthesized in a first step. In a second step, both complexes interact at the cytoplasmic face of the membrane, where the enzyme is subsequently activated with the concomitant conformational change and release of the NarJ chaperone from the mature enzyme.     

Structure of the nuclease domain of ribonuclease III from M. tuberculosis at 2.1 A

RNase III enzymes are a highly conserved family of proteins that specifically cleave double-stranded (ds)RNA. These proteins are involved in a diverse group of functions, including ribosomal RNA processing, mRNA maturation and decay, snRNA and snoRNA processing, and RNA interference. Here we report the crystal structure of the nuclease domain of RNase III from the pathogen Mycobacterium tuberculosis. Although globally similar to other RNase III folds, this structure has some features not observed in previously reported models. These include the presence of an additional metal ion near the catalytic site, as well as conserved secondary structural elements that are proposed to have functional roles in the recognition of dsRNAs.     

Structural and functional significance of the FGL sequence of the periplasmic chaperone Caf1M of Yersinia pestis

The periplasmic molecular chaperone Caf1M of Yersinia pestis is a typical representative of a subfamily of specific chaperones involved in assembly of surface adhesins with a very simple structure. One characteristic feature of this Caf1M-like subfamily is possession of an extended, variable sequence (termed FGL) between the F1 and subunit binding G1 beta-strands. In contrast, FGS subfamily members, characterized by PapD, have a short F1-G1 loop and are involved in assembly of complex pili. To elucidate the structural and functional significance of the FGL sequence, a mutant Caf1M molecule (dCaf1M), in which the 27 amino acid residues between the F1 and G1 beta-strands had been deleted, was constructed. Expression of the mutated caf1M in Escherichia coli resulted in accumulation of high levels of dCaf1M. The far-UV circular dichroism spectra of the mutant and wild-type proteins were indistinguishable and exhibited practically the same temperature and pH dependencies. Thus, the FGL sequence of Caf1M clearly does not contribute significantly to the stability of the protein conformation. Preferential cleavage of Caf1M by trypsin at Lys-119 confirmed surface exposure of this part of the FGL sequence in the isolated chaperone and periplasmic chaperone-subunit complex. There was no evidence of surface-localized Caf1 subunit in the presence of the Caf1A outer membrane protein and dCaf1M. In contrast to Caf1M, dCaf1M was not able to form a stable complex with Caf1 nor could it protect the subunit from proteolytic degradation in vivo. This demonstration that the FGL sequence is required for stable chaperone-subunit interaction, but not for folding of a stable chaperone, provides a sound basis for future detailed molecular analyses of the FGL subfamily of chaperones.     

The catalytic mechanism of the ESA1 histone acetyltransferase involves a self-acetylated intermediate

Yeast ESA1 is a member of the MYST subfamily of histone acetyltransferases (HATs), which use acetyl-coenzyme A (CoA) to acetylate specific Lys residues within histones to regulate gene expression. The structure of an ESA1-CoA complex reveals structural similarity to the catalytic core of the GCN5/PCAF subfamily of HAT proteins. Here we report additional structural and functional studies on ESA1 that demonstrate that histone acetylation proceeds through an acetyl-cysteine enzyme intermediate. This Cys residue is strictly conserved within the MYST members, suggesting a common mode of catalysis by this HAT subfamily. However, this mode of catalysis differs dramatically from the GCN5/PCAF subfamily, which mediate direct nucleophilic attack of the acetyl-CoA cofactor by the enzyme-deprotonated substrate lysine of the histone. These results demonstrate that different HAT subfamilies can use distinct catalytic mechanisms, which have implications for their distinct biological roles and for the development of HAT-specific inhibitors.