Identification of a highly conserved pro-gly doublet in non-animal small heat shock proteins and characterization of its structural and functional roles in Mycobacterium tuberculosis Hsp16.3

Small heat shock proteins (sHSPs) are highly divergent in primary sequences, with short conserved motifs found in various subfamilies. Here a Pro-Gly doublet was found to be conserved in most non-animal sHSPs by sequence analysis of a total of 344 unique sHSPs (covering the subfamilies: bacterial class A, bacterial class B, archae, fungi, plant, and animal) placed in data banks. In contrast, the residues corresponding to this Pro-Gly doublet in most of animal sHSPs are often charged. Site-directed mutagenesis studies of Mycobacterium tuberculosis Hsp16.3 replacing the Gly (at position 59) residue by Cys or Trp demonstrate that this Gly is likely involved in subunit interactions, which is consistent with that in Methanococcus jannaschii Hsp16.5 and wheat Hsp16.9. Our data suggest that this Pro-Gly doublet in Hsp16.3 is not directly involved in binding of denatured substrate proteins, whereas the corresponding charged residues in bovine alpha-crystallin were instead proposed before to be involved in substrate binding. These observations indicate that the highly conserved Pro-Gly doublet is critical to discriminate between non-animal and animal sHSPs.     

Phylogenetic and Biochemical Studies Reveal a Potential Evolutionary Origin of Small Heat Shock Proteins of Animals from Bacterial Class A

Small heat shock proteins (sHSPs), as one subclass of molecular chaperones, are important for cells to protect proteins under stress conditions. Unlike the large HSPs (represented by Hsp60 and Hsp70), sHSPs are highly divergent in both primary sequences and oligomeric status, with their evolutionary relationships being unresolved. Here the phylogenetic analysis of a representative 51 sHSPs (covering the six subfamilies: bacterial class A, bacterial class B, archae, fungi, plant, and animal) reveals a close relationship between bacterial class A and animal sHSPs which form an outgroup. Accumulating data indicate that the oligomers from bacterial class A and animal sHSPs appear to exhibit polydispersity, while those from the rest exhibit monodispersity. Together, the close evolutionary relationship and the similarity in oligomeric polydispersity between bacterial class A and animal sHSPs not only suggest a potential evolutionary origin of the latter from the former, but also imply that their oligomeric polydispersity is somehow a property determined by their primary sequences. [Reviewing Editor: Dr. Martin Kreitman]     

Identification of bis-ANS binding sites in Mycobacterium tuberculosis small heat shock protein Hsp16.3: evidences for a two-step substrate-binding mechanism

Small heat shock proteins (sHSPs), as one important subclass of molecular chaperones, are able to specifically bind to denatured substrate proteins rather than to native proteins, of which their substrate-binding sites are far from clear. Our previous study showed an overlapping nature of the sites for both hydrophobic probe 1,1'-Bi(4-anilino)naphthalene-5,5'-disulfonic acid (bis-ANS) binding and substrate binding in Mycobacterium tuberculosis Hsp16.3 [X. Fu, H. Zhang, X. Zhang, Y. Cao, W. Jiao, C. Liu, Y. Song, A. Abulimiti, Z. Chang, A dual role for the N-terminal region of M. tuberculosis Hsp16.3 in self-oligomerization and binding denaturing substrate proteins, J. Biol. Chem. 280 (2005) 6337-6348]. In this work, two bis-ANS binding sites in Hsp16.3 were identified by a combined use of reverse phase HPLC, mass spectroscopy and N-terminal protein sequencing. One site is in the N-terminal region and the other one in the N-terminus of alpha-crystallin domain, both of which are similar to those identified so far in sHSPs. However, accumulating data suggest that these two sites differentially function in binding substrate proteins. With regard to this difference, we proposed a two-step mechanism by which Hsp16.3 binds substrate proteins, i.e., substrate proteins are recognized and initially captured by the N-terminal region that is exposed in the dissociated Hsp16.3 oligomers, and then the captured substrate proteins are further stabilized in the complex by the subsequent binding of the N-terminus of alpha-crystallin domain.     

Multilevel structural characteristics for the natural substrate proteins of bacterial small heat shock proteins

Small heat shock proteins (sHSPs) are ubiquitous molecular chaperones that prevent the aggregation of various non‐native proteins and play crucial roles for protein quality control in cells. It is poorly understood what natural substrate proteins, with respect to structural characteristics, are preferentially bound by sHSPs in cells. Here we compared the structural characteristics for the natural substrate proteins of Escherichia coli IbpB and Deinococcus radiodurans Hsp20.2 with the respective bacterial proteome at multiple levels, mainly by using bioinformatics analysis. Data indicate that both IbpB and Hsp20.2 preferentially bind to substrates of high molecular weight or moderate acidity. Surprisingly, their substrates contain abundant charged residues but not abundant hydrophobic residues, thus strongly indicating that ionic interactions other than hydrophobic interactions also play crucial roles for the substrate recognition and binding of sHSPs. Further, secondary structure prediction analysis indicates that the substrates of low percentage of β‐sheets or coils but high percentage of α‐helices are un‐favored by both IbpB and Hsp20.2. In addition, IbpB preferentially interacts with multi‐domain proteins but unfavorably with α + β proteins as revealed by SCOP analysis. Together, our data suggest that bacterial sHSPs, though having broad substrate spectrums, selectively bind to substrates of certain structural features. These structural characteristic elements may substantially participate in the sHSP–substrate interaction and/or increase the aggregation tendency of the substrates, thus making the substrates more preferentially bound by sHSPs.     

Characterization and function analysis of Hsp60 and Hsp10 under different acute stresses in black tiger shrimp, <Emphasis Type="Italic">Penaeus monodon</Emphasis>

Heat shock proteins (Hsps) are a class of highly conserved proteins produced in virtually all living organisms from bacteria to humans. Hsp60 and Hsp10, the most important mitochondrial chaperones, participate in environmental stress responses. In this study, the full-length complementary DNAs (cDNAs) of Hsp60 (PmHsp60) and Hsp10 (PmHsp10) were cloned from Penaeus monodon. Sequence analysis showed that PmHsp60 and PmHsp10 encoded polypeptides of 578 and 102 amino acids, respectively. The expression profiles of PmHsp60 and PmHsp10 were detected in the gills and hepatopancreas of the shrimps under pH challenge, osmotic stress, and heavy metal exposure, and results suggested that PmHsp60 and PmHsp10 were involved in the responses to these stimuli. ATPase and chaperone activity assay indicated that PmHsp60 could slow down protein denaturation and that Hsp60/Hsp10 may be combined to produce a chaperone complex with effective chaperone and ATPase activities. Overall, this study provides useful information to help further understand the functional mechanisms of the environmental stress responses of Hsp60 and Hsp10 in shrimp.     

ArHsp40, a type 1 J-domain protein<Emphasis Type="Italic">,</Emphasis> is developmentally regulated and stress inducible in post-diapause <Emphasis Type="Italic">Artemia franciscana</Emphasis>

Upon diapause termination and exposure to favorable environmental conditions, cysts of the crustacean Artemia franciscana reinitiate development, a process dependent on the resumption of metabolic activity and the maintenance of protein homeostasis. The objective of the work described herein was to characterize molecular chaperones during post-diapause growth of A. franciscana. An Hsp40 complementary DNA (cDNA) termed ArHsp40 was cloned and shown to encode a protein with an amino-terminal J-domain containing a conserved histidine, proline, and aspartic acid (HPD) motif. Following the J-domain was a Gly/Phe (G/F) rich domain, a zinc-binding domain which contained a modified CXXCXGXG motif, and the carboxyl-terminal substrate binding region, all characteristics of type I Hsp40. Multiple alignment and protein modeling showed that ArHsp40 is comparable to Hsp40s from other eukaryotes and likely to be functionally similar. qRT-PCR revealed that during post-diapause development, ArHsp40 messenger RNA (mRNA) varied slightly until the E2/E3 stage and decreased significantly upon hatching. The immunoprobing of Western blots demonstrated that ArHsp40 was also relatively constant until E2/E3 and then declined dramatically. The drop in ArHsp40 when metabolism and protein synthesis were increasing was unexpected and demonstrated developmental regulation. The reduction in ArHsp40 at such an active life history stage indicates, as one possibility, that A. franciscana possesses additional Hsp40s, one or more of which replaces ArHsp40 as development progresses. Increased synthesis upon heat shock established that in addition to being developmentally regulated, ArHsp40 is stress inducible and, because it is found in mature cysts, ArHsp40 has the potential to contribute to stress tolerance during diapause.     

The crystal structure of an esterase from the hyperthermophilic microorganism <Emphasis Type="Italic">Pyrobaculum calidifontis</Emphasis> VA1 explains its enantioselectivity

The highly thermostable esterase from the hyperthermophilic archaeon Pyrobaculum calidifontis VA1 (PestE) shows high enantioselectivity (E?>?100) in the kinetic resolution of racemic chiral carboxylic acids, but little selectivity towards acetates of tertiary alcohols (E?=?2–4). To explain these unique properties, its crystal structure has been determined at 2.0 Å resolution. The enzyme is a member of the hormone-sensitive lipase group (group H) of the esterase/lipase superfamily on the basis of the amino acid sequence identity. The PestE structure shows a canonical α/β-hydrolase fold as core domain with a cap structure at the C-terminal end of the β-sheet. A tetramer in the crystal packing is formed of two dimers; the dimeric form is observed in solution. Conserved dimers and even tetramers are found in other group H proteins. The amino acid residues Ser157, His284, and Asp254 form the catalytic triad, which is typically found in α/β-hydrolases. The oxyanion hole is composed of Gly85 and Gly86 within the conserved sequence motif HGGG(M,F,W) (amino acid residues 83–87) and Ala158. With the elucidated structure, experimental results about enantioselectivity towards the two model substrate classes (as exemplified for 3-phenylbutanoic acid ethyl ester and 1,1,1-trifluoro-2-phenylbut-3-yn-2-yl acetate) could be explained by molecular modeling. For both enantiomers of the tertiary alcohol, orientations in two binding pockets were obtained without significant energy differences corresponding to the observed low enantioselectivity due to missing steric repulsions. In contrast, for the carboxylic acid ester, two different orientations with significant energy differences for each enantiomer were found matching the high E values.     

Genomic,evolutionary and expression profile analysis of <Emphasis Type="Italic">Hsp70</Emphasis> superfamily in A and D genome of cotton (<Emphasis Type="Italic">Gossypium</Emphasis> spp.) under the challenge of <Emphasis Type="Italic">Verticillium dahliae</Emphasis>

In this study, we comparatively analyzed the 115 Hsp70 genes identified in Gossypium raimondii, Gossypium hirsutum and Gossypium arboreum genomes. Those Hsp70 genes unequally distributed among chromosomes in A and D genome of cotton (Gossypium spp.), and were classified into 29 groups according to the homology of them. Based on the localization information of the orthologs in Arabidopsis, the Hsp70 proteins were predicted to locate in cytosol, endoplasmic reticulum, mitochondrion or chloroplast. Homologous analysis indicated the evolutionary conservation of Hsp70 in cotton. In addition, those Hsp70 genes were differently expressed in Suyuan-045, Hai-7124 and TM-1, which were highly resistant, resistant, and sensitive to Verticillium dahliae respectively. The expressions of 26 Hsp70 genes were induced by Verticillium dahliae except for Hsp70-07/16/25/26, and the result suggested the potential involvement of them in responding to Verticillium wilt. Hsp70-08/30/31 was highly expressed in both Suyuan-045 and Hai-7124, and it was hypothesized that they might be involved in the resistance to the invasion of Verticillium dahliae. 144h after inoculation with Verticillium dahliae, the expression of Hsp70-13/14/15 was only up-regulated in Suyuan-045, and it was assumed that they might be involved in resistance to the extension of Verticillium dahliae. Further study on those Hsp70 genes would be valuable to reveal the role of them in Verticillium wilt resistance.     

Molecular docking of <Emphasis Type="Italic">Glycine max</Emphasis> and <Emphasis Type="Italic">Medicago truncatula</Emphasis> ureases with urea; bioinformatics approaches

Urease (EC 3.5.1.5) is a nickel-dependent metalloenzyme catalyzing the hydrolysis of urea into ammonia and carbon dioxide. It is present in many bacteria, fungi, yeasts and plants. Most species, with few exceptions, use nickel metalloenzyme urease to hydrolyze urea, which is one of the commonly used nitrogen fertilizer in plant growth thus its enzymatic hydrolysis possesses vital importance in agricultural practices. Considering the essentiality and importance of urea and urease activity in most plants, this study aimed to comparatively investigate the ureases of two important legume species such as Glycine max (soybean) and Medicago truncatula (barrel medic) from Fabaceae family. With additional plant species, primary and secondary structures of 37 plant ureases were comparatively analyzed using various bioinformatics tools. A structure based phylogeny was constructed using predicted 3D models of G. max and M. truncatula, whose crystallographic structures are not available, along with three additional solved urease structures from Canavalia ensiformis (PDB: 4GY7), Bacillus pasteurii (PDB: 4UBP) and Klebsiella aerogenes (PDB: 1FWJ). In addition, urease structures of these species were docked with urea to analyze the binding affinities, interacting amino acids and atom distances in urease-urea complexes. Furthermore, mutable amino acids which could potentially affect the protein active site, stability and flexibility as well as overall protein stability were analyzed in urease structures of G. max and M. truncatula. Plant ureases demonstrated similar physico-chemical properties with 833–878 amino acid residues and 89.39–90.91 kDa molecular weight with mainly acidic (5.15–6.10 pI) nature. Four protein domain structures such as urease gamma, urease beta, urease alpha and amidohydro 1 characterized the plant ureases. Secondary structure of plant ureases also demonstrated conserved protein architecture, with predominantly α-helix and random coil structures. In structure-based phylogeny, plant ureases from G. max, M. truncatula and C. ensiformis were clearly diverged from bacterial ureases of B. pasteurii and K. aerogenes. Glu, Thr, His and Gly were commonly found as interacting residues in most urease-urea docking complexes while Glu was available in all docked structures. Besides, Ala and Arg residues, which are reported in active-site architecture of plant and bacterial ureases were present in G. max urea-urease complex but not present in others. Moreover, Arg435 and Arg437 in M. truncatula and G. max, respectively were identified as highly mutable hotspot residues residing in amidohydro 1 domain of enzyme. In addition, a number of stabilizing residues were predicted upon mutation of these hotspot residues however Cys and Thr made strong implications since they were also found in codon-aligned sequences as substitutions of hotspot residues. Comparative analyses of primary sequence and secondary structure in 37 different plants demonstrated quite conserved natures of ureases in plant kingdom. Structure-based phylogeny indicated the presence of a possible prokaryote-eukaryote split and implicated the subjection of bacterial ureases to heavy selection in prokaryotic evolution compared to plants. Urea-urease docking complexes suggested that different species could share common interacting residues as well as may have some other uncommon residues at species-dependent way. In silico mutation analyses identified mutable amino acids, which were predicted to reside in catalytic site of enzyme therefore mutagenesis at these sites seemed to have adverse effects on enzyme efficiency or function. This study findings will become valuable preliminary resource for future studies to further understand the primary, secondary and tertiary structures of urease sequences in plants as well as it will provide insights about various binding features of urea-urease complexes.     

Over-Expression of <Emphasis Type="Italic">PmHSP17.9</Emphasis> in Transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Confers Thermotolerance

Small heat shock proteins (sHSPs) have been shown to be involved in stress tolerance. However, their functions in Prunus mume under heat treatment are poorly characterized. To improve our understanding of sHSPs, we cloned a sHSP gene, PmHSP17.9, from P. mume. Sequence alignment and phylogenetic analysis indicated that PmHSP17.9 was a member of plant cytosolic class III sHSPs. Besides heat stress, PmHSP17.9 was also upregulated by salt, dehydration, oxidative stresses and ABA treatment. Leaves of transgenic Arabidopsis thaliana that ectopically express PmHSP17.9 accumulated less O₂ ^? and H₂O₂ compared with wild type (WT) after 42 °C treatment for 6 h. Over-expression of PmHSP17.9 in transgenic Arabidopsis enhanced seedling thermotolerance by decreased relative electrolyte leakage and MDA content under heat stress treatment when compared to WT plants. In addition, the induced expression of HSP101, HSFA2, and delta 1-pyrroline-5-carboxylate synthase (P5CS) under heat stress was more pronounced in transgenic plants than in WT plants. These results support the positive role of PmHSP17.9 in response to heat stress treatment.     

Molecular characterization and homology modeling of spermidine synthase from <Emphasis Type="Italic">Synechococcus</Emphasis> sp. PCC 7942

Spermidine synthase (Spds) catalyzes the formation of spermidine by transferring the aminopropyl group from decarboxylated S-adenosylmethionine (dcSAM) to putrescine. The Synechococcus spds gene encoding Spds was expressed in Escherichia coli. The purified recombinant enzyme had a molecular mass of 33 kDa and showed optimal activity at pH 7.5, 37?°C. The enzyme had higher affinity for dcSAM (K _m, 20 µM) than for putrescine (K _m, 111 µM) and was highly specific towards the diamine putrescine with no activity observed towards longer chain diamines. The three-dimensional structural model for Synechococcus Spds revealed that most of the ligand binding residues in Spds from Synechococcus sp. PCC 7942 are identical to those of human and parasite Spds. Based on the model, the highly conserved acidic residues, Asp89, Asp159 and Asp162, are involved in the binding of substrates putrescine and dcSAM and Pro166 seems to confer substrate specificity towards putrescine.     

Residues Asn214, Gln211, Glu219 and Gln221 contained in the subfamily 3 catalytic signature of the isocitrate lyase from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> are involved in its catalytic and thermal properties

Isocitrate lyase, encoded by the aceA gene, plays an important role in the ability of Pseudomonas aeruginosa to grow on fatty acids, acetate, acyclic terpenes, and amino acids. Phylogenetic analysis indicated that the ICL superfamily is divided in two families: the ICL family, which includes five subfamilies, and the 2-methylisocitrate lyase (MICL) family. ICL from P. aeruginosa (ICL-Pa) was identified in a different ICL node (subfamily 3) than other Pseudomonas ICL enzymes (grouped in subfamily 1). Analysis also showed that psychrophilic bacteria are mainly grouped in ICL subfamily 3, whose ICL proteins contain the highly conserved catalytic pattern Q_IEN_QV_SDE_KQ_CGHQD. We performed site-directed mutagenesis, enzymatic activity, and structure modeling of conserved residues in mutated ICLs by using ICL-Pa as a model. Our results indicated that the N214 residue is essential for catalytic function, while mutating the Q211, E219, and Q221 residues impairs its catalytic and thermostability properties. Our findings suggest that conserved residues in the subfamily 3 signature of ICL-Pa play important roles in catalysis and thermostability and are likely associated with the catalytic loop structural conformation.     

Endonuclease Activity of MutL Protein of the <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> Mismatch Repair System

We have purified the MutL protein from Rhodobacter sphaeroides mismatch repair system (rsMutL) for the first time. rsMutL demonstrated endonuclease activity in vitro, as predicted by bioinformatics analysis. Based on the alignment of 1483 sequences of bacterial MutL homologs with presumed endonuclease activity, conserved functional motifs and amino acid residues in the rsMutL sequence were identified: five motifs comprising the catalytic site responsible for DNA cleavage were found in the C–terminal domain; seven conserved motifs involved in ATP binding and hydrolysis and specific to the GHKL family of ATPases were found in the N–terminal domain. rsMutL demonstrated the highest activity in the presence of Mn²⁺. The extent of plasmid DNA hydrolysis declined in the row Mn²⁺ > Co²⁺ > Mg²⁺ > Cd²⁺; Ni²⁺ and Ca²⁺ did not activate rsMutL. Divalent zinc ions inhibited rsMutL endonuclease activity in the presence of Mn²⁺ excess. ATP also suppressed plasmid DNA hydrolysis by rsMutL. Analysis of amino acid sequences and biochemical properties of five studied bacterial MutL homologs with endonuclease activity revealed that rsMutL resembles the MutL proteins from Neisseria gonorrhoeae and Pseudomonas aeruginosa.     

The N-terminal arm of small heat shock proteins is important for both chaperone activity and substrate specificity

Small heat shock proteins (sHSPs) are a ubiquitous class of molecular chaperones that interacts with substrates to prevent their irreversible insolubilization during denaturation. How sHSPs interact with substrates remains poorly defined. To investigate the role of the conserved C-terminal alpha-crystallin domain versus the variable N-terminal arm in substrate interactions, we compared two closely related dodecameric plant sHSPs, Hsp18.1 and Hsp16.9, and four chimeras of these two sHSPs, in which all or part of the N-terminal arm was switched. The efficiency of substrate protection and formation of sHSP-substrate complexes by these sHSPs with three different model substrates, firefly luciferase, citrate synthase, and malate dehydrogenase (MDH) provide new insights into sHSP/substrate interactions. Results indicate that different substrates have varying affinities for different domains of the sHSP. For luciferase and citrate synthase, the efficiency of substrate protection was determined by the identity of the N-terminal arm in the chimeric proteins. In contrast, for MDH, efficient protection clearly required interactions with the alpha-crystallin domain in addition to the N-terminal arm. Furthermore, we show that sHSP-substrate complexes with varying stability and composition can protect substrate equally, and substrate protection is not correlated with sHSP oligomeric stability for all substrates. Protection of MDH by the dimeric chimera composed of the Hsp16.9 N-terminal arm and Hsp18.1 alpha-crystallin domain supports the model that a dimeric form of the sHSP can bind and protect substrate. In total, results demonstrate that sHSP-substrate interactions are complex, likely involve multiple sites on the sHSP, and vary depending on substrate.     

<Emphasis Type="Italic">Plasmodium falciparum</Emphasis> Hsp70-z,an Hsp110 homologue,exhibits independent chaperone activity and interacts with Hsp70-1 in a nucleotide-dependent fashion

The role of molecular chaperones, among them heat shock proteins (Hsps), in the development of malaria parasites has been well documented. Hsp70s are molecular chaperones that facilitate protein folding. Hsp70 proteins are composed of an N-terminal nucleotide binding domain (NBD), which confers them with ATPase activity and a C-terminal substrate binding domain (SBD). In the ADP-bound state, Hsp70 possesses high affinity for substrate and releases the folded substrate when it is bound to ATP. The two domains are connected by a conserved linker segment. Hsp110 proteins possess an extended lid segment, a feature that distinguishes them from canonical Hsp70s. Plasmodium falciparum Hsp70-z (PfHsp70-z) is a member of the Hsp110 family of Hsp70-like proteins. PfHsp70-z is essential for survival of malaria parasites and is thought to play an important role as a molecular chaperone and nucleotide exchange factor of its cytosolic canonical Hsp70 counterpart, PfHsp70-1. Unlike PfHsp70-1 whose functions are fairly well established, the structure-function features of PfHsp70-z remain to be fully elucidated. In the current study, we established that PfHsp70-z possesses independent chaperone activity. In fact, PfHsp70-z appears to be marginally more effective in suppressing protein aggregation than its cytosol-localized partner, PfHsp70-1. Furthermore, based on coimmunoaffinity chromatography and surface plasmon resonance analyses, PfHsp70-z associated with PfHsp70-1 in a nucleotide-dependent fashion. Our findings suggest that besides serving as a molecular chaperone, PfHsp70-z could facilitate the nucleotide exchange function of PfHsp70-1. These dual functions explain why it is essential for parasite survival.     

In silico characterization of <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> purinergic receptor: a novel chemotherapeutic target

Serpentine receptors with G-protein coupled receptor like seven transmembrane (7 TM) topology are identified in Plasmodium. A class of 7 TM receptors known as purinergic receptors binds to purines such as ADP, ATP and UTP and mediates important physiological functions including regulation of calcium signaling. Here we performed in silico analysis of Plasmodium falciparum serpentine receptors and found that one of the P. falciparum serpentine receptors, PfSR12 possess nucleotide binding consensus P-loop sequence in addition to seven transmembrane domains. The presence of conserved seven transmembrane domains and a consensus nucleotide binding sequence (P-loop) suggest that PfSR12 is a putative purinergic receptor. On further analysis using docking programmes we found four active binding residues Asn149, Lys150, Asn151 and Gly152 in P-loop of PfSR12, interact with ATP. This work gives insights into the interactions between putative purinergic receptor PfSR12 and its ligand ATP which can be explored in structure based drug designing against malaria.