Structures of the first and second double-stranded RNA-binding domains of human TAR RNA-binding protein

The TAR RNA-binding Protein (TRBP) is a double-stranded RNA (dsRNA)-binding protein, which binds to Dicer and is required for the RNA interference pathway. TRBP consists of three dsRNA-binding domains (dsRBDs). The first and second dsRBDs (dsRBD1 and dsRBD2, respectively) have affinities for dsRNA, whereas the third dsRBD (dsRBD3) binds to Dicer. In this study, we prepared the single domain fragments of human TRBP corresponding to dsRBD1 and dsRBD2 and solved the crystal structure of dsRBD1 and the solution structure of dsRBD2. The two structures contain an α-β-β-β-α fold, which is common to the dsRBDs. The overall structures of dsRBD1 and dsRBD2 are similar to each other, except for a slight shift of the first α helix. The residues involved in dsRNA binding are conserved. We examined the small interfering RNA (siRNA)-binding properties of these dsRBDs by isothermal titration colorimetry measurements. The dsRBD1 and dsRBD2 fragments both bound to siRNA, with dissociation constants of 220 and 113 nM, respectively. In contrast, the full-length TRBP and its fragment with dsRBD1 and dsRBD2 exhibited much smaller dissociation constants (0.24 and 0.25 nM, respectively), indicating that the tandem dsRBDs bind simultaneously to one siRNA molecule. On the other hand, the loop between the first α helix and the first β strand of dsRBD2, but not dsRBD1, has a Trp residue, which forms hydrophobic and cation-π interactions with the surrounding residues. A circular dichroism analysis revealed that the thermal stability of dsRBD2 is higher than that of dsRBD1 and depends on the Trp residue.     

Crystal structure of the trimeric alpha-helical coiled-coil and the three lectin domains of human lung surfactant protein D.

BACKGROUND: Human lung surfactant protein D (hSP-D) belongs to the collectin family of C-type lectins and participates in the innate immune surveillance against microorganisms in the lung through recognition of carbohydrate ligands present on the surface of pathogens. The involvement of this protein in innate immunity and the allergic response make it the subject of much interest. RESULTS: We have determined the crystal structure of a trimeric fragment of hSP-D at 2.3 A resolution. The structure comprises an alpha-helical coiled-coil and three carbohydrate-recognition domains (CRDs). An interesting deviation from symmetry was found in the projection of a single tyrosine sidechain into the centre of the coiled-coil; the asymmetry of this residue influences the orientation of one of the adjacent CRDs. The cleft between the three CRDs presents a large positively charged surface. CONCLUSIONS: The fold of the CRD of hSP-D is similar to that of the mannan-binding protein (MBP), but its orientation relative to the alpha-helical coiled-coil region differs somewhat to that seen in the MBP structure. The novel central packing of the tyrosine sidechain within the coiled-coil and the resulting asymmetric orientation of the CRDs has unexpected functional implications. The positively charged surface might facilitate binding to negatively charged structures, such as lipopolysaccharides.     

Protein F2, a novel fibronectin-binding protein from Streptococcus pyogenes, possesses two binding domains

Binding of the group A streptococcus (GAS) to respiratory epithelium is mediated by the fibronectin (Fn)-binding adhesin, protein F1. Previous studies have suggested that certain GAS strains express Fn-binding proteins that are different from protein F1. In this study, we have cloned, sequenced, and characterized a gene ( prtF2 ) from GAS strain 100076 encoding a novel Fn-binding protein, termed protein F2. Insertional inactivation of prtF2 in strain 100076 abolishes its high-affinity Fn binding. prtF2 -related genes exist in most GAS strains that lack prtF1 (encoding protein F1) but bind Fn with high affinity. These observations suggest that protein F2 is a major Fn-binding protein in GAS. Protein F2 is highly homologous to Fn-binding proteins from Streptococcus dysgalactiae and Strep-tococcus equisimilis , particularly in its carboxy-terminal portion. Two domains are responsible for Fn binding by protein F2. One domain (FBRD) consists of three consecutive repeats, whereas the other domain (UFBD) resides on a non-repeated stretch of approximately 100 amino acids and is located 100 amino acids amino-terminal of FBRD. Each of these domains is capable of binding Fn when expressed as a separate protein. In strain 100076, protein F2 activity is regulated in response to alterations in the concentration of atmospheric oxygen.     

Two newly identified SipA domains (F1, F2) steer effector protein localization and contribute to Salmonella host cell manipulation

Salmonella Typhimurium causes bacterial enterocolitis. The type III secretion system (TTSS)-1 is a key virulence determinant of S. Typhimurium mediating host cell invasion and acute enterocolitis. The TTSS-1 effector protein SipA is transported into host cells, accumulates in characteristic foci at the bacteria-host cell interface, manipulates signalling and affects virulence. Two functional domains of SipA have previously been characterized: The N-terminal SipA region (amino acids 1-105) mediates TTSS-1 transport and the C-terminal SipA 'actin-binding' domain (ABD; amino acids 446-685) manipulates F-actin assembly. Little is known about the central region of SipA. In a deletion analysis we found that the central SipA region harbours two distinct functional domains, F1 and F2. They are involved in SipA focus formation and host manipulation. The F1 domain (amino acids 170-271) drives SipA focus formation and domain F2 (amino acids 280-394) enhances this process by mediating SipA-SipA interactions. SipA variants lacking the F1-, the F2- or the actin binding domain were attenuated in virulence assays, namely host cell invasion and/or virulence in a mouse model for enterocolitis. Our results show that the newly identified SipA domains have distinct functions. Nevertheless, cooperation between the SipA domains F1, F2 and ABD is required to promote Salmonella virulence.     

The multiple RNA-binding domains of the mRNA poly(A)-binding protein have different RNA-binding activities.

The poly(A)-binding protein (PABP) is the major mRNA-binding protein in eukaryotes, and it is essential for viability of the yeast Saccharomyces cerevisiae. The amino acid sequence of the protein indicates that it consists of four ribonucleoprotein consensus sequence-containing RNA-binding domains (RBDs I, II, III, and IV) and a proline-rich auxiliary domain at the carboxyl terminus. We produced different parts of the S. cerevisiae PABP and studied their binding to poly(A) and other ribohomopolymers in vitro. We found that none of the individual RBDs of the protein bind poly(A) specifically or efficiently. Contiguous two-domain combinations were required for efficient RNA binding, and each pairwise combination (I/II, II/III, and III/IV) had a distinct RNA-binding activity. Specific poly(A)-binding activity was found only in the two amino-terminal RBDs (I/II) which, interestingly, are dispensable for viability of yeast cells, whereas the activity that is sufficient to rescue lethality of a PABP-deleted strain is in the carboxyl-terminal RBDs (III/IV). We conclude that the PABP is a multifunctional RNA-binding protein that has at least two distinct and separable activities: RBDs I/II, which most likely function in binding the PABP to mRNA through the poly(A) tail, and RBDs III/IV, which may function through binding either to a different part of the same mRNA molecule or to other RNA(s).     

Pseudomonas aeruginosa possesses three distinct systems for sensing and using the host molecule haem

Pathogens have developed several strategies to obtain iron during infection, including the use of iron-containing molecules from the host. Haem accounts for the vast majority of the iron pool in vertebrates and thus represents an important source of iron for pathogens. Using a proteomic approach, we have identified in this work a previously uncharacterized system, which we name Hxu, that together with the known Has and Phu systems, is used by the human pathogen Pseudomonas aeruginosa to respond to haem. We show that the Has and Hxu systems are functional signal transduction pathways of the cell-surface signalling class and report the mechanism triggering the activation of these signalling systems. Both signalling cascades involve an outer membrane receptor (HasR and HxuA respectively) that upon sensing haem in the extracellular medium produces the activation of an σ^ECF factor in the cytosol. HxuA has a major role in signalling and a minor role in haem acquisition in conditions in which the HasR and PhuR receptors or other sources of iron are present. Remarkably, P. aeruginosa compensates the lack of the HasR receptor by increasing the production of HxuA, which underscores the importance of haem signalling for this pathogen.     

The human RD protein is closely related to nuclear RNA-binding proteins and has been highly conserved

We have isolated cDNA clones encoding the human RD protein, which is closely related to several known nuclear RNA-binding proteins. The RD protein contains a 60-amino acid (aa) tract almost entirely of alternating basic and acidic aa, (RD)_n, primarily arginine (Arg; R) and aspartic acid (Asp; D). The protein also contains an ‘RNP sequence domain’. Arg-rich tracts and the RNP sequence domain are both features of nuclear RNA-binding proteins. However, we have been unable to detect RNA-binding by the human RD protein. The very strong evolutionary conservation of the mammalian RD protein as sequence suggests that it plays an important role in the cell.     

Molecular characterization of mitocalcin, a novel mitochondrial Ca2+-binding protein with EF-hand and coiled-coil domains

Here we have identified and characterized a novel mitochondrial Ca2+-binding protein, mitocalcin. Western blot analysis demonstrated that mitocalcin was widely expressed in mouse tissues. The expression in brain was increased during post-natal to adult development. Further analyses were carried out in newly established neural cell lines. The protein was expressed specifically in neurons but not in glial cells. Double-labeling studies revealed that mitocalcin was colocalized with mitochondria in neurons differentiated from 2Y-3t cells. In addition, mitocalcin was enriched in the mitochondrial fraction purified from the cells. Immunohistochemical studies on mouse cerebellum revealed that the expression pattern of mitocalcin in glomeruli of the internal granular and molecular layers was well overlapped by the distribution pattern of mitochondria. Immunogold electron microscopy showed that mitocalcin was associated with mitochondrial inner membrane. Overexpression of mitocalcin in 2Y-3t cells resulted in neurite extension. Inhibition of the expression in 2Y-3t cells caused suppression of neurite outgrowth and then cell death. These findings suggest that mitocalcin may play roles in neuronal differentiation and function through the control of mitochondrial function.     

NXP-1, a human protein related to Rad21/Scc1/Mcd1, is a component of the nuclear matrix

Nuclear matrix is a complex intranuclear network supposed to be involved in the various nuclear functions. In order to identify the nuclear matrix proteins, we isolated a cDNA clone from a human placenta cDNA library. This clone was partially represented a known cDNA clone HA1237. HA1237 encoded a 631-amino-acid peptide, which we designated NXP-1. NXP-1 was related to yeast Rad21/Scc1/Mcd1, Xenopus XRAD21, and mouse PW29, and identical with HR21spA isolated from a human testis cDNA library. We developed a polyclonal antibody to the purified NXP-1 bacterially expressed as a fusion protein with GST. Western blot analysis with anti-NXP-1 polyclonal antibody showed nuclear matrix localization of NXP-1 in HeLa cells. Indirect immunofluorescence staining also showed nuclear and nuclear matrix localization of the NXP-1. Results of in vitro binding assays employing nuclear matrix preparations indicated that the N-terminal region (16-128 amino acid) of NXP-1 has an important role in nuclear matrix distribution.     

Mouse and human GTPBP2, newly identified members of the GP-1 family of GTPase

We earlier identified the GTPBP1 gene which encodes a putative GTPase structurally related to peptidyl elongation factors. This finding was the result of a search for genes, the expression of which is induced by interferon-gamma in a macrophage cell line, THP-1. In the current study, we probed the expressed sequence tag database with the deduced amino acid sequence of GTPBP1 to search for partial cDNA clones homologous to GTPBP1. We used one of the partial cDNA clones to screen a mouse brain cDNA library and identified a novel gene, mouse GTPBP2, encoding a protein consisting of 582 amino acids and carrying GTP-binding motifs. The deduced amino acid sequence of mouse GTPBP2 revealed 44.2% similarity to mouse GTPBP1. We also cloned a human homologue of this gene from a cDNA library of the human T cell line, Jurkat. GTPBP2 protein was found highly conserved between human and mouse (over 99% identical), thereby suggesting a fundamental role of this molecule across species. On Northern blot analysis of various mouse tissues, GTPBP2 mRNA was detected in brain, thymus, kidney and skeletal muscle, but was scarce in liver. Level of expression of GTPBP2 mRNA was enhanced by interferon-gamma in THP-1 cells, HeLa cells, and thioglycollate-elicited mouse peritoneal macrophages. In addition, we determined the chromosomal localization of GTPBP1 and GTPBP2 genes in human and mouse. The GTPBP1 gene was mapped to mouse chromosome 15, region E3, and human chromosome 22q12-13.1, while the GTPBP2 gene is located in mouse chromosome 17, region C-D, and human chromosome 6p21-12.     

Conserved regions in the Epstein-Barr virus leader protein define distinct domains required for nuclear localization and transcriptional cooperation with EBNA2

Epstein-Barr virus (EBV) EBNA-LP is a latent protein whose function is not fully understood. Recent studies have shown that EBNA-LP may be an important EBNA2 cofactor by enhancing EBNA2 stimulation of the latency C and LMP-1 promoters. To further our understanding of EBNA-LP function, we have introduced a series of mutations into evolutionarily conserved regions and tested the mutant proteins for the ability to enhance EBNA2 stimulation of the latency C and LMP-1 promoters. Three conserved regions (CR1 to CR3) are located in the repeat domains that are essential for the EBNA2 cooperativity function. In addition, three serine residues are also well conserved in the repeat domains. Clustered alanine mutations were introduced into CR1 to CR3, and the conserved serines were also changed to alanine residues in an EBNA-LP with two repeats, which is the minimal protein able to cooperate with EBNA2. Mutations introduced into CR1a had no effect on EBNA-LP function, while mutations introduced into CR1b resulted in EBNA-LP with slightly decreased activity. Mutations in CR1c and CR2 resulted in proteins that no longer localized exclusively to the nucleus and also had no EBNA2 cooperation activity. Mutations introduced into conserved serines S5/71 resulted in proteins with slightly higher activity, while mutations introduced into conserved serines S35/101 or in CR3 (which contains S60/126) resulted in EBNA-LP proteins with substantially reduced activity. The potential karyophilic signals within EBNA-LP CR1c and CR2 were also examined by introducing oligonucleotides encoding these positively charged amino acid groupings into a cytoplasmic test protein, herpes simplex virus DeltaIE175, and by examining the intracellular localization of the resulting proteins. This assay identified a strong nuclear localization signal between EBNA-LP amino acids 43 and 50 (109 to 117 in the second W repeat) comprising CR2, while EBNA-LP amino acids 29 to 36 (91 to 98 in the second W repeat) were unable to function independently as a nuclear localization signal. However, a combination of amino acids 29 to 50 resulted in more efficient nuclear localization than with amino acids 43 to 50 alone. These results indicate that EBNA-LP has a bipartite nuclear localization signal and that efficient nuclear localization is essential for EBNA2 cooperativity function. Interestingly, EBNA-LP with only a single repeat localized exclusively to the cytoplasm, providing an explanation for why this isoform has no activity. In addition, two conserved serine residues which are distinct from nuclear import functions are important for EBNA2 cooperativity function.     

Solution structures of the first and second RNA-binding domains of human U2 small nuclear ribonucleoprotein particle auxiliary factor (U2AF(65)).

The large subunit of the human U2 small nuclear ribonucleoprotein particle auxiliary factor (hU2AF(65)) is an essential RNA-splicing factor required for the recognition of the polypyrimidine tract immediately upstream of the 3' splice site. In the present study, we determined the solution structures of two hU2AF(65) fragments, corresponding to the first and second RNA-binding domains (RBD1 and RBD2, respectively), by nuclear magnetic resonance spectroscopy. The tertiary structure of RBD2 is similar to that of typical RNA-binding domains with the beta1-alpha1-beta2-beta3-alpha2-beta4 topology. In contrast, the hU2AF(65) RBD1 structure has unique features: (i) the alpha1 helix is elongated by one turn toward the C-terminus; (ii) the loop between alpha1 and beta2 (the alpha1/beta2 loop) is much longer and has a defined conformation; (iii) the beta2 strand is (188)AVQIN(192), which was not predicted by sequence alignments; and (iv) the beta2/beta3 loop is much shorter. Chemical shift perturbation experiments showed that the U2AF-binding RNA fragments interact with the four beta-strands of RBD2 whereas, in contrast, they interact with beta1, beta3 and beta4, but not with beta2 or the alpha1/beta2 loop, of RBD1. The characteristic alpha1-beta2 structure of the hU2AF(65) RBD1 may interact with other proteins, such as UAP56.     

The plant cyclin-dependent kinase inhibitor ICK1 has distinct functional domains for in vivo kinase inhibition,protein instability and nuclear localization

Interactor/inhibitor 1 of Cdc2 kinase (ICK1) from Arabidopsis thaliana is the first plant cyclin-dependent kinase (CDK) inhibitor, and overexpression of ICK1 inhibits CDK activity, cell division and plant growth in transgenic plants. In this study, ICK1 and deletion mutants were expressed either alone or as green fluorescent protein (GFP) fusion proteins in transgenic Arabidopsis plants. Deletion of the C-terminal 15 or 29 amino acids greatly reduced or completely abolished the effects of ICK1 on the transgenic plants, and recombinant proteins lacking the C-terminal residues lost the ability to bind to CDK complex and the kinase inhibition activity, demonstrating the role of the conserved C-terminal domain in in vivo kinase inhibition. In contrast, the mutant ICK1DeltaN108 with the N-terminal 108 residues deleted had much stronger effects on plants than the full-length ICK1. Analyses demonstrated that this effect was not because of an enhanced ability of ICK1DeltaN108 protein to inhibit CDK activity, but a result of a much higher level of ICK1DeltaN108 protein in the plants, indicating that the N-terminal domain contains a sequence or element increasing protein instability in vivo. Furthermore, GFP-ICK1 protein was restricted to the nuclei in roots of transgenic plants, even with the C-terminal or the N-terminal domain deleted, suggesting that a sequence in the central domain of ICK1 is responsible for nuclear localization. These results provide mechanistic understanding about the function and regulation of this cell cycle regulator in plants.     

Mechanism of interferon action: functionally distinct RNA-binding and catalytic domains in the interferon-inducible, double-stranded RNA-specific adenosine deaminase.

The 1,226-amino-acid sequence of the interferon-inducible double-stranded RNA-specific adenosine deaminase (dsRAD) contains three copies (RI, RII, and RIII) of the highly conserved subdomain R motif commonly found in double-stranded RNA-binding proteins. We have examined the effects of equivalent site-directed mutations in each of the three R-motif copies of dsRAD on RNA-binding activity and adenosine deaminase enzyme activity. Mutations of the R motifs were analyzed alone as single mutants and in combination with each other. The results suggest that the RIII copy is the most important of the three R motifs for dsRAD activity and that the RII copy is the least important. The RIII mutant lacked detectable enzymatic activity and displayed greatly diminished RNA-binding activity. Site-directed mutations within the highly conserved CHAE sequence of the postulated C-terminal deaminase catalytic domain destroyed enzymatic activity but did not affect RNA-binding activity. These results indicate that the three copies of the RNA-binding R subdomain are likely functionally distinct from each other and also from the catalytic domain of dsRAD.     

The amino acid sequences of the three heavy chain constant region domains of a human IgG2 myeloma protein.

The amino acid sequences of most of the CH1, CH2 and CH3 domains of IgG Zie, a myeloma protein belonging to the IgG2 subclass, are presented. These data make possible a comparison of the sequences of residues 253-446 of all four subclasses of immunoglobulins: these residues make up almost the entire Fc regions. A comparison can also be made of the CH1 domain of IgG1 Eu and the CH1 domain of IgG2 Zie. Earlier sequence analyses of the Fc regions of subclass 1 and 3 proteins, and parts of the Fc regions of subclass 2 and 4 proteins showed that about 95% of these sequences were identical. The extended comparisons made possible by the data presented here show that this very high degree of identity is maintained throughout the four subclasses. Similarly, the CH1 domains of gamma 1 and gamma 2 chains were found to have about 93% sequence identity. It is unlikely that the few single amino acid changes within the constant region domains can account for the marked differences between subclasses observed in the region domains can account for the marked differences between subclasses observed in the biological effector functions of immunoglobulin Fc regions, especially since most of the changes are highly conservative. Rather, it seems probable that these functional differences are caused by conformational differences between the subgroups, which result from sequence differences in the hinge regions.