Structural and functional analysis of the PDZ domains of human HtrA1 and HtrA3

High-temperature requirement A (HtrA) and its homologs contain a serine protease domain followed by one or two PDZ domains. Bacterial HtrA proteins and the mitochondrial protein HtrA2/Omi maintain cell function by acting as both molecular chaperones and proteases to manage misfolded proteins. The biological roles of the mammalian family members HtrA1 and HtrA3 are less clear. We report a detailed structural and functional analysis of the PDZ domains of human HtrA1 and HtrA3 using peptide libraries and affinity assays to define specificity, structural studies to view the molecular details of ligand recognition, and alanine scanning mutagenesis to investigate the energetic contributions of individual residues to ligand binding. In common with HtrA2/Omi, we show that the PDZ domains of HtrA1 and HtrA3 recognize hydrophobic polypeptides, and while C-terminal sequences are preferred, internal sequences are also recognized. However, the details of the interactions differ, as different domains rely on interactions with different residues within the ligand to achieve high affinity binding. The results suggest that mammalian HtrA PDZ domains interact with a broad range of hydrophobic binding partners. This promiscuous specificity resembles that of bacterial HtrA family members and suggests a similar function for recognizing misfolded polypeptides with exposed hydrophobic sequences. Our results support a common activation mechanism for the HtrA family, whereby hydrophobic peptides bind to the PDZ domain and induce conformational changes that activate the protease. Such a mechanism is well suited to proteases evolved for the recognition and degradation of misfolded proteins.     

Structural and functional analysis of the three MIF4G domains of nonsense-mediated decay factor UPF2

Nonsense-mediated decay (NMD) is a eukaryotic quality control pathway, involving conserved proteins UPF1, UPF2 and UPF3b, which detects and degrades mRNAs with premature stop codons. Human UPF2 comprises three tandem MIF4G domains and a C-terminal UPF1 binding region. MIF4G-3 binds UPF3b, but the specific functions of MIF4G-1 and MIF4G-2 are unknown. Crystal structures show that both MIF4G-1 and MIF4G-2 contain N-terminal capping helices essential for stabilization of the 10-helix MIF4G core and that MIF4G-2 interacts with MIF4G-3, forming a rigid assembly. The UPF2/UPF3b/SMG1 complex is thought to activate the kinase SMG1 to phosphorylate UPF1 in vivo. We identify MIF4G-3 as the binding site and in vitro substrate of SMG1 kinase and show that a ternary UPF2 MIF4G-3/UPF3b/SMG1 complex can form in vitro. Whereas in vivo complementation assays show that MIF4G-1 and MIF4G-2 are essential for NMD, tethering assays reveal that UPF2 truncated to only MIF4G-3 and the UPF1-binding region can still partially accomplish NMD. Thus UPF2 MIF4G-1 and MIF4G-2 appear to have a crucial scaffolding role, while MIF4G-3 is the key module required for triggering NMD.     

Dissection of functional domains of the polar localization factor PodJ in Caulobacter crescentus

The polar organelle development protein, PodJ, is important for proper establishment of polarity in Caulobacter crescentus. podJ null mutants are unable to form holdfast or pili, have reduced swarming motility, and have difficulty ejecting the flagellum during the swarmer to stalked cell transition. In this study, we create a series of truncation mutants to investigate functional domains of PodJ. We show that PodJ has a transmembrane domain between amino acids 600 and 670. We identify a periplasmic region important for pili production and a cytoplasmic region required for holdfast formation and swarming motility, and establish that PleC localization is not required for holdfast formation and motility in soft agar. Analysis of the mutants reveals that the last 54 amino acids of the protein negatively regulate processing of the full-length form of the protein, PodJ(L), to a shorter form, PodJ(S). Finally, we identify a cytoplasmic region of PodJ involved in targeting it to the flagellar pole, and a periplasmic region required for localization of PleC.     

Structural and functional domains of E coli initiation factor IF2.

Initiation of translation in prokaryotes requires the participation of at least three soluble proteins: the initiation factors IF1, IF2 and IF3. Initiation factor 2, which is one of the largest proteins involved in translation (97.3 kDa) has been shown to stimulate in vitro the binding of fMet-tRNA(fMet) to the 30S ribosomal subunit. After formation of 70S translation initiation complex, IF2 is believed to participate in GTP hydrolysis, thereby promoting its own release. Here we review evidence which indicates the functional importance of the different structural domains of IF2, emphasizing new information obtained by in vivo experiments.     

Structural and functional domains of tubulin

The molecular aspects of the microtubule system is a research area that has developed very rapidly during the past decade. Research on the assembly mechanisms and chemistry of tubulin and the molecular biology of microtubules have advanced our understanding of microtubule formation and its regulation. The emerging view of tubulin is of a macromolecule containing spatially discrete sequences that constitute functionally different domains with respect to self-association, interactions with microtubule associated proteins (MAPs) and specific ligands. Recent studies point to the role of the carboxyl-terminal moiety of tubulin subunits in regulating its assembly into microtubules. These investigations combined with further studies on the spatial relationships between tubulin domains should provide new insights into the detailed structural basis of microtubule assembly.     

Rhesus cytomegalovirus particles prevent activation of interferon regulatory factor 3

One of the most important innate host defense mechanisms against viral infection is the induction of interferon (IFN)-stimulated genes (ISGs). Immediately upon entry, viruses activate interferon-regulatory factor 3 (IRF3), as well as nuclear factor kappaB (NF-kappaB), which transactivate a subset of ISGs, proinflammatory genes, as well as IFN genes. Most large DNA viruses exhibit countermeasures against induction of this response. However, whereas human cytomegalovirus (HCMV) inhibits IFN-dependent induction of ISGs, IFN-independent induction of ISGs is observed both in the presence and, even moreso, in the absence of viral gene expression. Rhesus CMV (RhCMV) is an emerging animal model for HCMV sharing important similarities in primary structure, epidemiology, and pathogenesis. To determine whether RhCMV would similarly induce ISGs, we performed DNA microarray and quantitative PCR analysis of ISG expression in rhesus fibroblasts infected with RhCMV or HCMV. In contrast to HCMV, however, RhCMV did not induce expression of ISGs or proinflammatory genes at any time after infection. Moreover, dimerization and nuclear accumulation of IRF3, readily observed in HCMV-infected cells, was absent from RhCMV-infected cells, whereas neither virus seemed to activate NFkappaB. RhCMV also blocked IRF3 activation by live or UV-inactivated HCMV, suggesting that RhCMV inhibits viral IRF3 activation and the resultant ISG induction with extraordinary efficiency. Since infection during inhibition of protein expression by cycloheximide or inactivation of viral gene expression by UV treatment did not trigger IRF3 activation or ISG expression by RhCMV, we conclude that RhCMV virions contain a novel inhibitor of IFN-independent viral induction of ISG expression by IRF3.     

Structural and functional domains on actin

Actin plays several essential roles in cellular processes and is a vital component in the contractile apparatus. To accomplish its many cellular tasks, actin must interact with a wide range of other proteins in addition to self-assembling into filaments. Characterization of these functional domains and localized binding regions on the actin monomer is therefore an important undertaking. Strategies for elucidating the many interaction sites include X-ray diffraction, NMR and fluorescence spectroscopy, chemical modification, chemical cross-linking, protein cleavage, and the study of sequence homologies between the many isotypes of actin. Based on these varied data, we discuss the possible spatial relationships between the interaction sites.     

Stability and DNA binding ability of the DNA binding domains of interferon regulatory factors 1 and 3

The thermodynamic properties and DNA binding ability of the N-terminal DNA binding domains of interferon regulatory factors IRF-1 (DBD1) and IRF-3 (DBD3) were studied using microcalorimetric and optical methods. DBD3 is significantly more stable than DBD1: at 20 degrees C the Gibbs energy of unfolding of DBD3 is -28.6 kJ/mol, which is 2 times larger than that of DBD1, -14.9 kJ/mol. Fluorescence anisotropy titration experiments showed that at this temperature the association constants with the PRDI binding site are 1.1 x 10(6) M(-)(1) for DBD1 and 3.6 x 10(6) M(-)(1) for DBD3, corresponding to Gibbs energies of association of -34 and -37 kJ/mol, respectively. However, the larger binding energy of DBD3 is due to its larger electrostatic component, while its nonelectrostatic component is smaller than that of DBD1. Therefore, DBD1 appears to have more sequence specificity than DBD3. Binding of DBD1 to target DNA is characterized by a substantially larger negative enthalpy than binding of DBD3, implying that the more flexible structure of DBD1 forms tighter contacts with DNA than the more rigid structure of DBD3. Thus, the strength of the DBDs' specific association with DNA is inversely related to the stability of the free DBDs.     

Identification of novel alternative splicing variants of interferon regulatory factor 3

Interferon regulatory factor 3 (IRF-3) plays a crucial role in host defense against viral and microbial infection as well as in cell growth regulation. IRF-3a is the only structurally and functionally characterized IRF-3 splicing variant and has been established to antagonize IRF-3 activity. Here, five novel splicing variants of IRF-3, referred to as IRF-3b, -3c, -3d, -3e, and -3f, were identified and shown to be generated by deletion of exons 2, 3, or 6 or some combination thereof. RT-PCR examination revealed that these novel splicing variants were more frequently expressed in human liver, esophagus, and cervical tumor tissues than in their normal counterparts. Additionally, electrophoretic mobility shift assay and subcellular localization showed only IRF-3 and IRF-3e were capable of binding the PRDI/III element of interferon-beta (IFNβ) promoter in vitro and underwent cytoplasm-to-nucleus translocation following Poly(I:C) stimulation. Coimmunoprecipitation assay revealed that only IRF-3c (3f) of novel splicing variants associated with IRF-3 in vivo. Further luciferase assay showed IRF-3c (3f) and IRF-3e failed to transactivate PRDI/III-containing promoter but appeared to inhibit transactivation potential of IRF-3 to varying degrees. Taken together, our findings suggest novel splicing variants may function as negative modulators of IRF-3 and may be correlated with pathogenesis of human tumors.     

Molecular characterization and association analysis of porcine interferon regulatory factor 1 gene

Interferon regulatory factor 1 (IRF1) is a member of IRF-family that was discovered to activate promoters in type I interferon (IFN) genes. It is shown to play functionally diverse role in the regulation of the immune system. In this report, the porcine IRF1 cDNA were cloned and a 7500 bp genomic DNA structure was identified. The putative IRF1 protein included 322 amino acids. Alignment and phylogenetic analysis of the predicted porcine IRF1 amino acids sequence with its homologies of other species show high identity (over 88%). Tissues expression of IRF1 mRNA was observed by RT-PCR, the results revealed IRF1 gene expressed widely in all analyzed tissues. Using the radiation hybrid panel, the porcine IRF1 gene was mapped to porcine chromosome 2 and closely linked to the locus IL4 (LOD = 7.09, 57cR). A SNP in exon2 of porcine IRF1 gene was demonstrated by sequencing and PCR–RFLP analysis. The further association analysis indicated that the SNP was significant associate with level of IFN-γ (day 20) in serum (P = 0.0001) and the ratio of IFN-γ to IL10 (day 20; day 35) in serum (P = 0.0165; P = 0.0095). The results suggested that the porcine IRF1 gene is strong candidate gene for these immune traits in pig.