Mutational and structural analysis of the binding interface between type I interferons and their receptor Ifnar2.

Type I interferons (IFN) exert pleiotropic activities through binding to two cell surface receptors, ifnar1 and ifnar2. We are investigating the biophysical basis of IFN signaling by characterizing the complex of the extra-cellular domain of ifnar2 (ifnar2-EC) with IFNs on the level of purified recombinant proteins in vitro. Here, we present a detailed mutational study on the functional epitopes on both IFN and ifnar2. Kinetic and thermodynamic parameters were determined by label-free heterogeneous phase detection. On IFNalpha2, a relatively small functional epitope comprising ten amino acid residues was localized, which is nearly entirely formed by residues on the AB loop. Two hot-spot residues, L30 and R33, account for two-thirds of the total interaction energy. Comparing the anti-viral potency of the various mutants to the binding affinity towards ifnar2 revealed a proportional correlation between the two, suggesting a rate-limiting role of ifnar2 binding in IFN signaling. On ifnar2, residues T46, I47 and M48 were identified as hot-spots in the interaction with IFNalpha2. For another ten residues on ifnar2, significant contribution of interaction energy was determined. Based on these data, the functional epitope on ifnar2 was defined according to a homology model based on other members of the class II hCR family in good agreement with the complementary functional epitope on IFNalpha2. Although IFNalpha2 and IFNbeta bind competitively to the same functional epitope, mutational analysis revealed distinct centers of binding for these IFNs on ifnar2. This small shift of the binding site may result in different angular orientation, which can be critically coupled to cytoplasmic signaling.     

Identification of a functional site on the type I TGF-beta receptor by mutational analysis of its ectodomain

Six charged amino acid residues located in the ectodomain of the full-length type I transforming growth factor (TGF)-beta receptor were individually mutated to alanine. Mutation of residues D47, D98, K102 and E104 resulted in functionally impaired receptors as demonstrated by a marked decrease in ligand-dependent signaling and ligand internalization relative to the wild-type receptor. The other two mutants (K39A and K87A) exhibited wild-type-like activity. Molecular modeling indicates that the four functionally important residues are located on the convex face of the ectodomain structure. Since mutation of these four residues affects signaling and ligand internalization but not ligand binding, we propose that this functional site is an interacting site between type I and II receptors.     

Mitochondrial Complex I: structural and functional aspects

This review examines two aspects of the structure and function of mitochondrial Complex I (NADH Coenzyme Q oxidoreductase) that have become matter of recent debate. The supramolecular organization of Complex I and its structural relation with the remainder of the respiratory chain are uncertain. Although the random diffusion model [C.R. Hackenbrock, B. Chazotte, S.S. Gupte, The random collision model and a critical assessment of diffusion and collision in mitochondrial electron transport, J. Bioenerg. Biomembranes 18 (1986) 331-368] has been widely accepted, recent evidence suggests the presence of supramolecular aggregates. In particular, evidence for a Complex I-Complex III supercomplex stems from both structural and kinetic studies. Electron transfer in the supercomplex may occur by electron channelling through bound Coenzyme Q in equilibrium with the pool in the membrane lipids. The amount and nature of the lipids modify the aggregation state and there is evidence that lipid peroxidation induces supercomplex disaggregation. Another important aspect in Complex I is its capacity to reduce oxygen with formation of superoxide anion. The site of escape of the single electron is debated and either FMN, iron-sulphur clusters, and ubisemiquinone have been suggested. The finding in our laboratory that two classes of hydrophobic inhibitors have opposite effects on superoxide production favours an iron-sulphur cluster (presumably N2) is the direct oxygen reductant. The implications in human pathology of better knowledge on these aspects of Complex I structure and function are briefly discussed.     

A novel mechanism of TRAF signaling revealed by structural and functional analyses of the TRADD-TRAF2 interaction

TRAF proteins are major mediators for the cell activation, cell survival, and antiapoptotic functions of the TNF receptor superfamily. They can be recruited to activated TNF receptors either by direct interactions with the receptors or indirectly via the adaptor protein TRADD. We now report the structure of the TRADD-TRAF2 complex, which is highly distinct from receptor-TRAF2 interactions. This interaction is significantly stronger and we show by an in vivo signaling assay that TRAF2 signaling is more readily initiated by TRADD than by direct receptor-TRAF2 interactions. TRADD is specific for TRAF1 and TRAF2, which ensures the recruitment of clAPs for the direct inhibition of caspase activation in the signaling complex. The stronger affinity and unique specificity of the TRADD-TRAF2 interaction are crucial for the suppression of apoptosis and provide a mechanistic basis for the perturbation of TRAF recruitment in sensitizing cell death induction.     

Mechanistic aspects of DnaA-RepA interaction as revealed by yeast forward and reverse two-hybrid analysis

Using yeast forward and reverse two-hybrid analysis and biochemical techniques, we present novel and definitive in vivo and in vitro evidence that both the N-terminal domain I and C-terminal domain IV of the host-encoded DnaA initiator protein of Escherichia coli interact physically with plasmid-encoded RepA initiator of pSC101. The N-terminal, but not the C-terminal, region of RepA interacted with DnaA in vitro. These protein-protein interactions are critical for two very early steps of replication initiation, namely origin unwinding and helicase loading. Neither domain I nor IV of DnaA could individually collaborate with RepA to promote pSC101 replication. However, when the two domains are co-expressed within a common cell milieu and allowed to associate non-covalently with each other via a pair of leucine zippers, replication of the plasmid was supported in vivo. Thus, the result shows that physical tethering, either non-covalent or covalent, of domain I and IV of DnaA and interaction of both domains with RepA, are critical for replication initiation. The results also provide the molecular basis for a novel, potential, replication-based bacterial two-hybrid system.     

Methylation of class I translation termination factors: structural and functional aspects

During protein synthesis, release of polypeptide from the ribosome occurs when an in frame termination codon is encountered. Contrary to sense codons, which are decoded by tRNAs, stop codons present in the A-site are recognized by proteins named class I release factors, leading to the release of newly synthesized proteins. Structures of these factors bound to termination ribosomal complexes have recently been obtained, and lead to a better understanding of stop codon recognition and its coordination with peptidyl-tRNA hydrolysis in bacteria. Release factors contain a universally conserved GGQ motif which interacts with the peptidyl-transferase centre to allow peptide release. The Gln side chain from this motif is methylated, a feature conserved from bacteria to man, suggesting an important biological role. However, methylation is catalysed by completely unrelated enzymes. The function of this motif and its post-translational modification will be discussed in the context of recent structural and functional studies.     

Evolutionary,structural and functional relationships revealed by comparative analysis of syntenic genes in Rhizobiales

Background  

Comparative genomics has provided valuable insights into the nature of gene sequence variation and chromosomal organization of closely related bacterial species. However, questions about the biological significance of gene order conservation, or synteny, remain open. Moreover, few comprehensive studies have been reported for rhizobial genomes.     

Sequence and structural aspects of functional diversification in class I alpha-mannosidase evolution.

MOTIVATION: Class I alpha-mannosidases comprise a homologous and functionally diverse family of glycoside hydrolases. Phylogenetic analysis based on an amino acid sequence alignment of the catalytic domain of class I alpha-mannosidases reveals four well-supported phylogenetic groups within this family. These groups include a number of paralogous members generated by gene duplications that occurred as far back as the initial divergence of the crown-group of eukaryotes. Three of the four phylogenetic groups consist of enzymes that have group-specific biochemical specificity and/or sites of activity. An attempt has been made to uncover the role that natural selection played in the sequence and structural divergence between the phylogenetically and functionally distinct Endoplasmic Reticulum (ER) and Golgi apparatus groups. RESULTS: Comparison of site-specific amino acid variability profiles for the ER and Golgi groups revealed statistically significant evidence for functional diversification at the sequence level and indicated a number of residues that are most likely to have played a role in the functional divergence between the two groups. The majority of these sites appear to contain residues that have been fixed within one organelle-specific group by positive selection. Somewhat surprisingly these selected residues map to the periphery of the alpha-mannosidase catalytic domain tertiary structure. Changes in these peripherally located residues would not seem to have a gross effect on protein function. Thus diversifying selection between the two groups may have acted in a gradual manner consistent with the Darwinian model of natural selection. CONTACT: bishogr@millsaps.edu.     

New structural proteins of Halobacterium salinarum gas vesicle revealed by comparative proteomics analysis

The Halobacterium salinarum gas vesicle (GV) is an extremely stable intracellular organelle with air trapped inside a proteinaceous membrane. Reported here is a comparative proteomics analysis of GV and GV depleted lysate (GVD) to reveal the membrane structural proteins. Ten proteins encoded by gvp-1 (gvpMLKJIHGFED-1 and gvpACNO-1) and five proteins encoded by gvp-2 (gvpMLKJIHGFED-2 and gvpACNO-2) gene clusters for the biogenesis of spindle- and cylindrical-, respectively, shaped GV were identified by LC-MS/MS. The peptides of GvpA1, I1, J1, A2, and J2 were exclusively identified in purified GV, GvpD1, H1, L1, and F2 only in GVD, and GvpC1, N1, O1, F1, H2, and O2 in both samples. The identification of GvpA1, C1, F1, J1, and A2 in GV is in agreement with their previously known structural function. In addition, the detection of GvpI1, N1, O1, H2, J2, and O2 in GV suggested they are new structural proteins. Among these, the structural role of GvpI1 and N1 in GV was further validated by immuno-detection of protein A-tagged GvpI1 and N1 fusion proteins in purified GV. Thus, LC-MS/MS could reveal at least a half dozen gas vesicle structural proteins in the predominant spindle-shaped GV that may be helpful for studying its biogenesis.     

New functional and structural insights from updated mutational databases for complement factor H,Factor I,membrane cofactor protein and C3

aHUS (atypical haemolytic uraemic syndrome), AMD (age-related macular degeneration) and other diseases are associated with defective AP (alternative pathway) regulation. CFH (complement factor H), CFI (complement factor I), MCP (membrane cofactor protein) and C3 exhibited the most disease-associated genetic alterations in the AP. Our interactive structural database for these was updated with a total of 324 genetic alterations. A consensus structure for the SCR (short complement regulator) domain showed that the majority (37%) of SCR mutations occurred at its hypervariable loop and its four conserved Cys residues. Mapping 113 missense mutations onto the CFH structure showed that over half occurred in the C-terminal domains SCR-15 to -20. In particular, SCR-20 with the highest total of affected residues is associated with binding to C3d and heparin-like oligosaccharides. No clustering of 49 missense mutations in CFI was seen. In MCP, SCR-3 was the most affected by 23 missense mutations. In C3, the neighbouring thioester and MG (macroglobulin) domains exhibited most of 47 missense mutations. The mutations in the regulators CFH, CFI and MCP involve loss-of-function, whereas those for C3 involve gain-of-function. This combined update emphasizes the importance of the complement AP in inflammatory disease, clarifies the functionally important regions in these proteins, and will facilitate diagnosis and therapy.     

Sequence-dependent structural variations of DNA revealed by DNase I.

Two global helix parameters important for DNA-DNase I interaction are the geometry of the minor groove and the DNA stiffness that resists bending toward major groove. Thus, local averaging of P-O3' bonds cutting frequencies (InP) reflects global helix parameters revealed by DNase I. Using the approximation that locally averaged InP values depend only on the type of the dinucleotide steps involved in the region of interaction, we calculated the collective contribution (sigma Dd) for ten different dinucleotide steps. Our results suggest that, at the first approximation, global varying helix parameters revealed by DNase I, might be predicted from sequence. Obtained sigma Dd function can be used as a sequence-dependent measure of protein-induced DNA flexure in the direction towards the major groove, which is usually connected to widening of the minor groove. In the course of analysis of Mg2+ and Mn2+ dependent DNase I digestions, no significant difference was found, in spite of the supposed differences in enzyme activity. These results suggest that if the second Mn2(+)-dependent active site exists, its activity is lower than that of the first one.     

Functional domains of the Escherichia coli dnaK heat shock protein as revealed by mutational analysis

The employment of a set of truncated dnaK peptides produced by deletion and insertion mutations in the Escherichia coli dnaK gene allowed us to define regions of the dnaK protein which are involved in particular enzymatic functions. The results obtained suggest that the dnaK polypeptide is organized into at least two distinct functional domains. The highly conserved amino-terminal portion is required for the ATPase activity. The carboxyl-terminal portion, characterized by relatively low similarity among species, is responsible for the autophosphorylating activity. The mutant dnaK protein C[74], which lacks amino acid sequences at the extreme carboxyl-terminal portion of the protein, retains both the ATPase and the autophosphorylating activities. The results obtained with the full-length (70-kDa) dnaK756 protein suggest that the thermolabile defect of the dnaK756 mutation affects directly or indirectly the ATPase active site of the enzyme. The autophosphorylating activity of the dnaK+, dnaK756, and C[74] polypeptides was activated at least 10-fold by the addition of CaCl2.     

Catalytic center of an archaeal type 2 ribonuclease H as revealed by X-ray crystallographic and mutational analyses

The catalytic center of an archaeal Type 2 RNase H has been identified by a combination of X-ray crystallographic and mutational analyses. The crystal structure of the Type 2 RNase H from Thermococcus kodakaraensis KOD1 has revealed that the N-terminal major domain adopts the RNase H fold, despite the poor sequence similarity to the Type 1 RNase H. Mutational analyses showed that the catalytic reaction requires four acidic residues, which are well conserved in the Type 1 RNase H and the members of the polynucleotidyl transferase family. Thus, the Type 1 and Type 2 RNases H seem to share a common catalytic mechanism, except for the requirement of histidine as a general base in the former enzyme. Combined with the results from deletion mutant analyses, the structure suggests that the C-terminal domain of the Type 2 RNase H is involved in the interaction with the DNA/RNA hybrid.     

Actinorhodin biosynthesis: structural requirements for post-PKS tailoring intermediates revealed by functional analysis of ActVI-ORF1 reductase

Actinorhodin (ACT) produced by Streptomyces coelicolor A3(2) is an aromatic polyketide antibiotic, whose basic carbon skeleton is derived from type II polyketide synthase (PKS). Although an acyl carrier protein (ACP) serves as an anchor of nascent intermediates during chain elongation in the type II PKS complex, it generally remains unknown when an ACP-free intermediate is released from the complex to post-PKS modification ("tailoring") steps. In ACT biosynthesis, a stereospecific ketoreductase (RED1) encoded by actVI-ORF1 reduces the 3beta-keto group of a proposed bicyclic intermediate to an (S) secondary alcohol. The bicyclic intermediate is formed from the steps of PKS and its closely associated enzymes and lies at the interface toward ACT-tailoring steps. To clarify whether RED1 recognizes the ACP-bound bicyclic intermediate or the ACP-free bicyclic intermediate, recombinant RED1 was purified for enzymatic characterization. RED1 was heterologously expressed in Escherichia coli and purified using Ni-chelate and gel filtration column chromatographies to homogeneity in soluble form. Enzymatic studies in vitro on RED1 with synthetic analogues, in place of an unstable bicyclic intermediate, showed that RED1 recognizes 3-oxo-4-naphthylbutyric acid (ONBA) as a preferred substrate and not its N-acetylcysteamine thioester. This strongly suggests that RED1 recognizes ACP-free bicyclic beta-keto acid as the first committed intermediate of tailoring steps. Kinetic studies of RED1 showed high affinity with ONBA, consistent with the requirement for an efficient reduction of a labile beta-keto carboxylic acid. Interestingly, the methyl ester of ONBA acted as a competitive inhibitor of RED1, indicating the presence of strict substrate recognition toward the terminal acid functionality.     

Probing protein stability and proteolytic resistance by loop scanning: a comprehensive mutational analysis

Improvement in protein thermostability was often found to be associated with increase in its proteolytic resistance as revealed by comparative studies of homologous proteins from extremophiles or mutational studies. Structural elements of protein responsible for this association are not firmly established although loops are implicated indirectly due to their structural role in protein stability. To get a better insight, a detailed study of protein wide mutants and their influence on stability and proteolytic resistance would be helpful. To generate such a data set, a model protein, Bacillus subtilis lipase was subjected to loop scanning site-saturation mutagenesis on 86 positions spanning all loops including termini. Upon screening of ∼16,000 clones, 17 single mutants with improved thermostability were identified with increment in apparent melting temperature (Tm_app) by 1–6°C resulting in an increase in free energy of unfolding (ΔG_unf) by 0.04–1.16 kcal/mol. Proteolytic resistance of all single mutants upon incubation with nonspecific protease, Subtilisin A, was determined. Upon comparison, post-proteolysis residual activities as well as kinetics of proteolysis of mutants showed excellent correlation with ΔG_unf, (r > 0.9), suggesting that proteolysis was strongly correlated with the global stability of this protein. This significant correlation in this set, with least possible sequence changes (single aa substitution), while covering >60% of protein surface strongly argues for the covariance of these two variables. Compared to studies from extremophiles, with large sequence heterogeneity, the observed correlation in such a narrow sequence space (ΔΔG_unf = 1.57 kcal⁻¹) justifies the robustness of this relation.     

New findings on phosphodiesterases,MoPdeH and MoPdeL,in Magnaporthe oryzae revealed by structural analysis

The cyclic adenosine monophosphate (cAMP) signalling pathway mediates signal communication and sensing during infection‐related morphogenesis in eukaryotes. Many studies have implicated cAMP as a critical mediator of appressorium development in the rice blast fungus, Magnaporthe oryzae. The cAMP phosphodiesterases, MoPdeH and MoPdeL, as key regulators of intracellular cAMP levels, play pleiotropic roles in cell wall integrity, cellular morphology, appressorium formation and infectious growth in M. oryzae. Here, we analysed the roles of domains of MoPdeH and MoPdeL separately or in chimeras. The results indicated that the HD and EAL domains of MoPdeH are indispensable for its phosphodiesterase activity and function. Replacement of the MoPdeH HD domain with the L1 and L2 domains of MoPdeL, either singly or together, resulted in decreased cAMP hydrolysis activity of MoPdeH. All of the transformants exhibited phenotypes similar to that of the ΔMopdeH mutant, but also revealed that EAL and L1 play additional roles in conidiation, and that L1 is involved in infectious growth. We further found that the intracellular cAMP level is important for surface signal recognition and hyphal autolysis. The intracellular cAMP level negatively regulates Mps1‐MAPK and positively regulates Pmk1‐MAPK in the rice blast fungus. Our results provide new information to better understand the cAMP signalling pathway in the development, differentiation and plant infection of the fungus.     

ARTS, the unusual septin: structural and functional aspects

The human Septin 4 gene (Sept4) encodes two major protein isoforms; Sept4_i1 (H5/PNUTL2) and Sept4_i2/ARTS. Septins have been traditionally studied for their role in cytokinesis and their filament-forming abilities, but subsequently have been implicated in diverse functions, including membrane dynamics, cytoskeletal reorganization, vesicle trafficking, and tumorigenesis. ARTS is localized at mitochondria and promotes programmed cell death (apoptosis). These features distinguish ARTS from any other known human septin family member. This review compares the structural and functional properties of ARTS with other septins. In addition, it describes how a combination of two distinct promoters, differential splicing, and intron retention leads to the generation of two different Sept4 variants with diverse biological activity.