Multi-site Directed Mutagenesis Research Articles

RankProt: A multi criteria-ranking platform to attain protein thermostabilizing mutations and its in vitro applications - Attribute based prediction method on the principles of Analytical Hierarchical Process.

Attaining recombinant thermostable proteins is still a challenge for protein engineering. The complexity is the length of time and enormous efforts required to achieve the desired results. Present work proposes a novel and economic strategy of attaining protein thermostability by predicting site-specific mutations at the shortest possible time. The success of the approach can be attributed to Analytical Hierarchical Process and the outcome was a rationalized thermostable mutation(s) prediction tool- RankProt. Briefly the method involved ranking of 17 biophysical protein features as class predictors, derived from 127 pairs of thermostable and mesostable proteins. Among the 17 predictors, ionic interactions and main-chain to main-chain hydrogen bonds were the highest ranked features with eigen value of 0.091. The success of the tool was judged by multi-fold in silico validation tests and it achieved the prediction accuracy of 91% with AUC 0.927. Further, in vitro validation was carried out by predicting thermostabilizing mutations for mesostable Bacillus subtilis lipase and performing the predicted mutations by multi-site directed mutagenesis. The rationalized method was successful to render the lipase thermostable with optimum temperature stability and Tm increase by 20°C and 7°C respectively. Conclusively it can be said that it was the minimum number of mutations in comparison to the number of mutations incorporated to render Bacillus subtilis lipase thermostable, by directed evolution techniques. The present work shows that protein stabilizing mutations can be rationally designed by balancing the biophysical pleiotropy of proteins, in accordance to the selection pressure.

Improved insecticidal activities of Novel Bacillus thuringiensis Cry1-type genes

Recently, Bacillus thuringiensis (Bt) cry genes encoding insecticidal Cry proteins have been widely applied for the construction of transgenic crops resistant to insect pests. This study aimed to construct novel mutant cry1Ac genes for genetically modified crops with enhanced insecticidal activities. Using multi-site directed mutagenesis, 34 mutant cry1Ac genes were synthesized and converted at 24 amino acid residues, located on domain I (8 residues) and domain II (16 residues). These mutant genes were expressed as a fusion protein with polyhedrin using the baculovirus expression system. The expressed proteins were occluded into polyhedra and activated stably to 65kDa by trypsin. Among these, Mut-N04, N06 and N16 showed high levels of insecticidal activities against larvae of Plutella xylostella, Spodoptera exigua, and Ostrinia furnacalis. Mut-N16, which showed the highest insecticidal activity, is expected to be a desirable cry gene for introduction into transgenic crops. This study could provide useful means to construct mutant cry genes with improved insecticidal activities and expand host spectra for transgenic crops.

Sumoylation of Kif18A plays a role in regulating mitotic progression.

BackgroundKif18A, the kinesin-8 motor protein, plays an essential role in regulating alignment of bi-oriented chromosomes at the midzone during mitosis. Kinesin proteins, including Kif18A, are often deregulated in many types of cancers and are thought to play a critical role in cancer progression. However, little is known about the post-translational modifications of Kif18A and their effects on its biological activity.MethodsKif18A was identified to be a SUMO2 acceptor by using Ni-IDA resin to precipitate proteins from cells stably expressing His6-SUMO2. To identify the potential lysine residues, multi-site directed mutagenesis together with transient transfection and Ni-IDA pull-down assay were carried out. The confocal time-lapse imaging and immunofluorescent staining were used to study the roles of SUMO2 modification on Kif18A’s activity during the cell cycle.ResultsKif18A is covalently modified by SUMO2 during the cell cycle, and its sumoylation peaks at metaphase and then rapidly decreases upon anaphase onset. Mutational analysis identifies multiple lysine residues (K148, K442, K533, K660 and K683) as potential SUMO acceptors. The functional studies reveal that sumoylation of Kif18A has little effect on protein stability and subcellular localization. However, compared with the wild-type control, ectopic expression of SUMO-resistant mutants of Kif18A results in a significant delay of mitotic exit. Confocal microscopy shows that cells expressing SUMO-resistant Kif18A display a compromised dissociation of BubR1 from kinetochores after anaphase onset.ConclusionsOur studies reveal that sumoylation functions as an unidentified form of post-translational modification that regulates Kif18A activity during mitotic progression.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1226-9) contains supplementary material, which is available to authorized users.

Asymmetrical Fc Engineering Greatly Enhances Antibody-dependent Cellular Cytotoxicity (ADCC) Effector Function and Stability of the Modified Antibodies

Antibody-dependent cellular cytotoxicity (ADCC) is mediated through the engagement of the Fc segment of antibodies with Fcγ receptors (FcγRs) on immune cells upon binding of tumor or viral antigen. The co-crystal structure of FcγRIII in complex with Fc revealed that Fc binds to FcγRIII asymmetrically with two Fc chains contacting separate regions of the FcγRIII by utilizing different residues. To fully explore this asymmetrical nature of the Fc-FcγR interaction, we screened more than 9,000 individual clones in Fc heterodimer format in which different mutations were introduced at the same position of two Fc chains using a high throughput competition AlphaLISA® assay. To this end, we have identified a panel of novel Fc variants with significant binding improvement to FcγRIIIA (both Phe-158 and Val-158 allotypes), increased ADCC activity in vitro, and strong tumor growth inhibition in mice xenograft human tumor models. Compared with previously identified Fc variants in conventional IgG format, Fc heterodimers with asymmetrical mutations can achieve similar or superior potency in ADCC-mediated tumor cell killing and demonstrate improved stability in the CH2 domain. Fc heterodimers also allow more selectivity toward activating FcγRIIA than inhibitory FcγRIIB. Afucosylation of Fc variants further increases the affinity of Fc to FcγRIIIA, leading to much higher ADCC activity. The discovery of these Fc variants will potentially open up new opportunities of building the next generation of therapeutic antibodies with enhanced ADCC effector function for the treatment of cancers and infectious diseases.

DNA Damage Processing by Human 8-Oxoguanine-DNA Glycosylase Mutants with the Occluded Active Site

8-Oxoguanine-DNA glycosylase (OGG1) removes premutagenic lesion 8-oxoguanine (8-oxo-G) from DNA and then nicks the nascent abasic (apurinic/apyrimidinic) site by β-elimination. Although the structure of OGG1 bound to damaged DNA is known, the dynamic aspects of 8-oxo-G recognition are not well understood. To comprehend the mechanisms of substrate recognition and processing, we have constructed OGG1 mutants with the active site occluded by replacement of Cys-253, which forms a wall of the base-binding pocket, with bulky leucine or isoleucine. The conformational dynamics of OGG1 mutants were characterized by single-turnover kinetics and stopped-flow kinetics with fluorescent detection. Additionally, the conformational mobility of wild type and the mutant OGG1 substrate complex was assessed using molecular dynamics simulations. Although pocket occlusion distorted the active site and greatly decreased the catalytic activity of OGG1, it did not fully prevent processing of 8-oxo-G and apurinic/apyrimidinic sites. Both mutants were notably stimulated in the presence of free 8-bromoguanine, indicating that this base can bind to the distorted OGG1 and facilitate β-elimination. The results agree with the concept of enzyme plasticity, suggesting that the active site of OGG1 is flexible enough to compensate partially for distortions caused by mutation.

Mutation of Light-dependent Phosphorylation Sites of the Drosophila Transient Receptor Potential-like (TRPL) Ion Channel Affects Its Subcellular Localization and Stability

The Drosophila phototransduction cascade terminates in the opening of the ion channel transient receptor potential (TRP) and TRP-like (TRPL). Contrary to TRP, TRPL undergoes light-dependent subcellular trafficking between rhabdomeric photoreceptor membranes and an intracellular storage compartment, resulting in long term light adaptation. Here, we identified in vivo phosphorylation sites of TRPL that affect TRPL stability and localization. Quantitative mass spectrometry revealed a light-dependent change in the TRPL phosphorylation pattern. Mutation of eight C-terminal phosphorylation sites neither affected multimerization of the channels nor the electrophysiological response of flies expressing the mutated channels. However, these mutations resulted in mislocalization and enhanced degradation of TRPL after prolonged dark-adaptation. Mutation of subsets of the eight C-terminal phosphorylation sites also led to a reduction of TRPL content and partial mislocalization in the dark. This suggests that a light-dependent switch in the phosphorylation pattern of the TRPL channel mediates stable expression of TRPL in the rhabdomeres upon prolonged dark-adaptation.

Quantitative Characterization of the Activation Steps of Mannan-binding Lectin (MBL)-associated Serine Proteases (MASPs) Points to the Central Role of MASP-1 in the Initiation of the Complement Lectin Pathway

Mannan-binding lectin (MBL)-associated serine proteases, MASP-1 and MASP-2, have been thought to autoactivate when MBL/ficolin·MASP complexes bind to pathogens triggering the complement lectin pathway. Autoactivation of MASPs occurs in two steps: 1) zymogen autoactivation, when one proenzyme cleaves another proenzyme molecule of the same protease, and 2) autocatalytic activation, when the activated protease cleaves its own zymogen. Using recombinant catalytic fragments, we demonstrated that a stable proenzyme MASP-1 variant (R448Q) cleaved the inactive, catalytic site Ser-to-Ala variant (S646A). The autoactivation steps of MASP-1 were separately quantified using these mutants and the wild type enzyme. Analogous mutants were made for MASP-2, and rate constants of the autoactivation steps as well as the possible cross-activation steps between MASP-1 and MASP-2 were determined. Based on the rate constants, a kinetic model of lectin pathway activation was outlined. The zymogen autoactivation rate of MASP-1 is ∼3000-fold higher, and the autocatalytic activation of MASP-1 is about 140-fold faster than those of MASP-2. Moreover, both activated and proenzyme MASP-1 can effectively cleave proenzyme MASP-2. MASP-3, which does not autoactivate, is also cleaved by MASP-1 quite efficiently. The structure of the catalytic region of proenzyme MASP-1 R448Q was solved at 2.5 Å. Proenzyme MASP-1 R448Q readily cleaves synthetic substrates, and it is inhibited by a specific canonical inhibitor developed against active MASP-1, indicating that zymogen MASP-1 fluctuates between an inactive and an active-like conformation. The determined structure provides a feasible explanation for this phenomenon. In summary, autoactivation of MASP-1 is crucial for the activation of MBL/ficolin·MASP complexes, and in the proenzymic phase zymogen MASP-1 controls the process.

S-Nitrosylation Inhibits Pannexin 1 Channel Function

S-nitrosylation is a post-translational modification on cysteine(s) that can regulate protein function, and pannexin 1 (Panx1) channels are present in the vasculature, a tissue rich in nitric oxide (NO) species. Therefore, we investigated whether Panx1 can be S-nitrosylated and whether this modification can affect channel activity. Using the biotin switch assay, we found that application of the NO donor S-nitrosoglutathione (GSNO) or diethylammonium (Z)-1-1(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA NONOate) to human embryonic kidney (HEK) 293T cells expressing wild type (WT) Panx1 and mouse aortic endothelial cells induced Panx1 S-nitrosylation. Functionally, GSNO and DEA NONOate attenuated Panx1 currents; consistent with a role for S-nitrosylation, current inhibition was reversed by the reducing agent dithiothreitol and unaffected by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a blocker of guanylate cyclase activity. In addition, ATP release was significantly inhibited by treatment with both NO donors. To identify which cysteine residue(s) was S-nitrosylated, we made single cysteine-to-alanine substitutions in Panx1 (Panx1(C40A), Panx1(C346A), and Panx1(C426A)). Mutation of these single cysteines did not prevent Panx1 S-nitrosylation; however, mutation of either Cys-40 or Cys-346 prevented Panx1 current inhibition and ATP release by GSNO. This observation suggested that multiple cysteines may be S-nitrosylated to regulate Panx1 channel function. Indeed, we found that mutation of both Cys-40 and Cys-346 (Panx1(C40A/C346A)) prevented Panx1 S-nitrosylation by GSNO as well as the GSNO-mediated inhibition of Panx1 current and ATP release. Taken together, these results indicate that S-nitrosylation of Panx1 at Cys-40 and Cys-346 inhibits Panx1 channel currents and ATP release.

Investigating the Role of the Nematode‐Specific FAR Family of Proteins

Nematode genomes encode for proteins specific to the phylum Nematoda, which bear crucial importance for understanding nematode biology. Proteins specific to the phylum Nematoda are ideal targets for drugs with low toxicity to the host and the environment. Despite the importance of nematode‐specific proteins and protein families, few have been identified and very little information regarding them is available on public databases. Our work involves a novel, nematode‐specific family of proteins, the fatty acid and retinol binding (FAR) proteins of the phylum Nematoda. It was recently discovered that parasitic nematodes of plants, animals and humans secrete these structurally novel FAR proteins into the tissues they occupy. This family of proteins has been shown to be highly conserved across both plant and animal parasitic nematodes, yet there is limited information available regarding this family of proteins and their in vivo function(s) are poorly understood. Our specific research goals are to evaluate the ligand binding properties of FAR protein mutants created via multi site‐directed mutagenesis (MSDM). Constructs that do not bind the most commonly screened ligands for FAR proteins will be used to ascertain data on sequence variation and amino acids imperative for ligand binding. To date, we have created approximately 50 mutants containing two or more mutations. We expect that our research on this family of proteins may be significant for a broad range of scientists and agriculturalists in understanding plant and animal parasitism, nematode evolution and parasite‐control methods.

Evidence for Irreversible Inhibition of Glycogen Synthase Kinase-3β by Tideglusib

Tideglusib is a GSK-3 inhibitor currently in phase II clinical trials for the treatment of Alzheimer disease and progressive supranuclear palsy. Sustained oral administration of the compound to a variety of animal models decreases Tau hyperphosphorylation, lowers brain amyloid plaque load, improves learning and memory, and prevents neuronal loss. We report here that tideglusib inhibits GSK-3β irreversibly, as demonstrated by the lack of recovery in enzyme function after the unbound drug has been removed from the reaction medium and the fact that its dissociation rate constant is non-significantly different from zero. Such irreversibility may explain the non-competitive inhibition pattern with respect to ATP shown by tideglusib and perhaps other structurally related compounds. The replacement of Cys-199 by an Ala residue in the enzyme seems to increase the dissociation rate, although the drug retains its inhibitory activity with decreased potency and long residence time. In addition, tideglusib failed to inhibit a series of kinases that contain a Cys homologous to Cys-199 in their active site, suggesting that its inhibition of GSK-3β obeys to a specific mechanism and is not a consequence of nonspecific reactivity. Results obtained with [(35)S]tideglusib do not support unequivocally the existence of a covalent bond between the drug and GSK-3β. The irreversibility of the inhibition and the very low protein turnover rate observed for the enzyme are particularly relevant from a pharmacological perspective and could have significant implications on its therapeutic potential.

Multi Site-Directed Mutagenesis of Human Flavin-Containing Monooxygenase 5 (FMO5) Produces a Mutant with 20-fold Higher Affinity for Methimazole: Evidence that Ile61 is Crucial for Catalytic Activity

Multi Site-Directed Mutagenesis of Human Flavin-Containing Monooxygenase 5 (FMO5) Produces a Mutant with 20-fold Higher Affinity for Methimazole: Evidence that Ile61 is Crucial for Catalytic Activity

Consensus Substrate Sequence for Protein-tyrosine Phosphatase Receptor Type Z

Protein-tyrosine phosphatase receptor type Z (Ptprz) has multiple substrate proteins, including G protein-coupled receptor kinase-interactor 1 (Git1), membrane-associated guanylate kinase, WW and PDZ domain-containing 1 (Magi1), and GTPase-activating protein for Rho GTPase (p190RhoGAP). We have identified a dephosphorylation site at Tyr-1105 of p190RhoGAP; however, the structural determinants employed for substrate recognition of Ptprz have not been fully defined. In the present study, we revealed that Ptprz selectively dephosphorylates Git1 at Tyr-554, and Magi1 at Tyr-373 and Tyr-858 by in vitro and cell-based assays. Of note, the dephosphorylation of the Magi1 Tyr-858 site required PDZ domain-mediated interaction between Magi1 and Ptprz in the cellular context. Alignment of the primary sequences surrounding the target phosphotyrosine residue in these three substrates showed considerable similarity, suggesting a consensus motif for recognition by Ptprz. We then estimated the contribution of surrounding individual amino acid side chains to the catalytic efficiency by using fluorescent peptides based on the Git1 Tyr-554 sequence in vitro. The typical substrate motif for the catalytic domain of Ptprz was deduced to be Glu/Asp-Glu/Asp-Glu/Asp-Xaa-Ile/Val-Tyr(P)-Xaa (Xaa is not an acidic residue). Intriguingly, a G854D substitution of the Magi1 Tyr-858 site matching better to the motif sequence turned this site to be susceptible to dephosphorylation by Ptprz independent of the PDZ domain-mediated interaction in cells. Furthermore, we found by database screening that the substrate motif is present in several proteins, including paxillin at Tyr-118, its major phosphorylation site. Expectedly, we verified that Ptprz efficiently dephosphorylates paxillin at this site in cells. Our study thus provides key insights into the molecular basis for the substrate recognition of Ptprz.

Fission Yeast Swi1-Swi3 Complex Facilitates DNA Binding of Mrc1

Replication fork protection complex Swi1-Swi3 and replication checkpoint mediator Mrc1 are required for maintenance of replication fork integrity during the course of DNA replication in the fission yeast Schizosaccharomyces pombe. These proteins play crucial roles in stabilizing stalled forks and activating replication checkpoint signaling pathways. Although they are conserved replication fork components, precise biochemical roles of these proteins are not known. Here we purified Mrc1 and Swi1-Swi3 proteins and show that these proteins bind to DNA independently but synergistically in vitro. Mrc1 binds preferentially to arrested fork or D-loop-like structures, although the affinity is relatively low, whereas the Swi1-Swi3 complex binds to double-stranded DNA with higher affinity. In the presence of a low concentration of Swi1-Swi3, Mrc1 generates a novel ternary complex and binds to various types of DNA with higher affinity. Moreover, purified Mrc1 and Swi1-Swi3 physically interact with each other, and this interaction is lost by mutations in the known DNA binding domain of Mrc1 (K235E,K236E). The interaction is also lost in a mutant form of Swi1 (E662K) that is specifically defective in polar fork arrest at a site called RTS1 and causes sensitivity to genotoxic agents, although the DNA binding affinity of Swi1-Swi3 is not affected by this mutation. As expected, the synergistic effect of the Swi1-Swi3 on DNA binding of Mrc1 is also lost by these mutations affecting the interaction between Mrc1 and Swi1-Swi3. Our results reveal an aspect of molecular interactions that may play an important role in replication pausing and fork stabilization.

Ubiquitin-independent Proteasomal Degradation of Endoplasmic Reticulum-localized Connexin43 Mediated by CIP75

Connexin43 (Cx43) is a transmembrane protein that forms gap junction channels. Regulation of Cx43 turnover is one mechanism to control the level of intercellular communication that occurs through gap junction channels. Proteasomal degradation of Cx43 is regulated in part through CIP75, a ubiquitin-like and ubiquitin-associated domain containing protein. CIP75 interacts with endoplasmic reticulum-localized Cx43, as demonstrated through co-immunoprecipitation and immunofluorescence microscopy experiments. CIP75 also binds to free monoubiquitin and lysine 48-linked tetraubiquitin chains in vitro and binds to ubiquitinated proteins in cellular lysates. However, analysis of Cx43 that immunoprecipitated with CIP75 demonstrated that the Cx43 associated with CIP75 was not ubiquitinated, and a mutant form of Cx43 that lacked lysines capable of ubiquitination retained the capacity to interact with CIP75. These results suggest that although CIP75 can interact with ubiquitinated cellular proteins, its interaction with Cx43 and stimulation of Cx43 proteasomal degradation does not require the ubiquitination of Cx43.

Elucidation of a Novel Extracellular Calcium-binding Site on Metabotropic Glutamate Receptor 1α (mGluR1α) That Controls Receptor Activation*

Metabotropic glutamate receptor 1α (mGluR1α) exerts important effects on numerous neurological processes. Although mGluR1α is known to respond to extracellular Ca(2+) ([Ca(2+)](o)) and the crystal structures of the extracellular domains (ECDs) of several mGluRs have been determined, the calcium-binding site(s) and structural determinants of Ca(2+)-modulated signaling in the Glu receptor family remain elusive. Here, we identify a novel Ca(2+)-binding site in the mGluR1α ECD using a recently developed computational algorithm. This predicted site (comprising Asp-318, Glu-325, and Asp-322 and the carboxylate side chain of the receptor agonist, Glu) is situated in the hinge region in the ECD of mGluR1α adjacent to the reported Glu-binding site, with Asp-318 involved in both Glu and calcium binding. Mutagenesis studies indicated that binding of Glu and Ca(2+) to their distinct but partially overlapping binding sites synergistically modulated mGluR1α activation of intracellular Ca(2+) ([Ca(2+)](i)) signaling. Mutating the Glu-binding site completely abolished Glu signaling while leaving its Ca(2+)-sensing capability largely intact. Mutating the predicted Ca(2+)-binding residues abolished or significantly reduced the sensitivity of mGluR1α not only to [Ca(2+)](o) and [Gd(3+)](o) but also, in some cases, to Glu. The dual activation of mGluR1α by [Ca(2+)](o) and Glu has important implications for the activation of other mGluR subtypes and related receptors. It also opens up new avenues for developing allosteric modulators of mGluR function that target specific human diseases.

Structural and Enzymatic Insights into the ATP Binding and Autophosphorylation Mechanism of a Sensor Histidine Kinase

DesK is a sensor histidine kinase (HK) that allows Bacillus subtilis to respond to cold shock, triggering the adaptation of membrane fluidity via transcriptional control of a fatty acid desaturase. It belongs to the HK family HPK7, which includes the nitrogen metabolism regulators NarX/Q and the antibiotic sensor LiaS among other important sensor kinases. Structural information on different HK families is still scarce and several questions remain, particularly concerning the molecular features that determine HK specificity during its catalytic autophosphorylation and subsequent response-regulator phosphotransfer reactions. To analyze the ATP-binding features of HPK7 HKs and dissect their mechanism of autophosphorylation at the molecular level, we have studied DesK in complex with ATP using high resolution structural approaches in combination with biochemical studies. We report the first crystal structure of an HK in complex with its natural nucleotidic substrate. The general fold of the ATP-binding domain of DesK is conserved, compared with well studied members of other families. Yet, DesK displays a far more compact structure at the ATP-binding pocket: the ATP lid loop is much shorter with no secondary structural organization and becomes ordered upon ATP loading. Sequence conservation mapping onto the molecular surface, semi-flexible protein-protein docking simulations, and structure-based point mutagenesis allow us to propose a specific domain-domain geometry during autophosphorylation catalysis. Supporting our hypotheses, we have been able to trap an autophosphorylating intermediate state, by protein engineering at the predicted domain-domain interaction surface.

Mechanism of Action of an Imidopiperidine Inhibitor of Human Polynucleotide Kinase/Phosphatase

The small molecule, 2-(1-hydroxyundecyl)-1-(4-nitrophenylamino)-6-phenyl-6,7a-dihydro-1H-pyrrolo[3,4-b]pyridine-5,7(2H,4aH)-dione (A12B4C3), is a potent inhibitor of the phosphatase activity of human polynucleotide kinase/phosphatase (PNKP) in vitro. Kinetic analysis revealed that A12B4C3 acts as a noncompetitive inhibitor, and this was confirmed by fluorescence quenching, which showed that the inhibitor can form a ternary complex with PNKP and a DNA substrate, i.e. A12B4C3 does not prevent DNA from binding to the phosphatase DNA binding site. Conformational analysis using circular dichroism, UV difference spectroscopy, and fluorescence resonance energy transfer all indicate that A12B4C3 disrupts the secondary structure of PNKP. Investigation of the potential site of binding of A12B4C3 to PNKP using site-directed mutagenesis pointed to interaction between Trp(402) of PNKP and the inhibitor. Cellular studies revealed that A12B4C3 sensitizes A549 human lung cancer cells to the topoisomerase I poison, camptothecin, but not the topoisomerase II poison, etoposide, in a manner similar to small interfering RNA against PNKP. A12B4C3 also inhibits the repair of DNA single and double strand breaks following exposure of cells to ionizing radiation, but does not inhibit two other key strand-break repair enzymes, DNA polymerase beta or DNA ligase III, providing additional evidence that PNKP is the cellular target of the inhibitor.

The Processivity of Kinesin-2 Motors Suggests Diminished Front-Head Gating

Kinesin-2 motors, which are involved in intraflagellar transport and cargo transport along cytoplasmic microtubules, differ from motors in the canonical kinesin-1 family by having a heterodimeric rather than homodimeric structure and possessing a three amino acid insertion in their neck linker domain. To determine how these structural features alter the chemomechanical coupling in kinesin-2, we used single-molecule bead experiments to measure the processivity and velocity of mouse kinesin-2 heterodimer (KIF3A/B) and the engineered homodimers KIF3A/A and KIF3B/B and compared their behavior to Drosophila kinesin-1 heavy chain (KHC). Single-motor run lengths of kinesin-2 were 4-fold shorter than those of kinesin-1. Extending the kinesin-1 neck linker by three amino acids led to a similar reduction in processivity. Furthermore, kinesin-2 processivity varied inversely with ATP concentration. Stochastic simulations of the kinesin-1 and kinesin-2 hydrolysis cycles suggest that "front-head gating," in which rearward tension prevents ATP binding to the front head when both heads are bound to the microtubule, is diminished in kinesin-2. Because the mechanical tension that underlies front-head gating must be transmitted through the neck linker domains, we propose that the diminished coordination in kinesin-2 is a result of its longer and, hence, more compliant neck linker element.

CAMP-dependent Protein Kinase A (PKA) Signaling Induces TNFR1 Exosome-like Vesicle Release via Anchoring of PKA Regulatory Subunit RIIβ to BIG2

The 55-kDa TNFR1 (type I tumor necrosis factor receptor) can be released to the extracellular space by two mechanisms, the proteolytic cleavage and shedding of soluble receptor ectodomains and the release of full-length receptors within exosome-like vesicles. We have shown that the brefeldin A-inhibited guanine nucleotide exchange protein BIG2 associates with TNFR1 and selectively modulates the release of TNFR1 exosome-like vesicles via an ARF1- and ARF3-dependent mechanism. Here, we assessed the role of BIG2 A kinase-anchoring protein (AKAP) domains in the regulation of TNFR1 exosome-like vesicle release from human vascular endothelial cells. We show that 8-bromo-cyclic AMP induced the release of full-length, 55-kDa TNFR1 within exosome-like vesicles via a protein kinase A (PKA)-dependent mechanism. Using RNA interference to decrease specifically the levels of individual PKA regulatory subunits, we demonstrate that RIIbeta modulates both the constitutive and cAMP-induced release of TNFR1 exosome-like vesicles. Consistent with its AKAP function, BIG2 was required for the cAMP-induced PKA-dependent release of TNFR1 exosome-like vesicles via a mechanism that involved the binding of RIIbeta to BIG2 AKAP domains B and C. We conclude that both the constitutive and cAMP-induced release of TNFR1 exosome-like vesicles occur via PKA-dependent pathways that are regulated by the anchoring of RIIbeta to BIG2 via AKAP domains B and C. Thus, BIG2 regulates TNFR1 exosome-like vesicle release by two distinct mechanisms, as a guanine nucleotide exchange protein that activates class I ADP-ribosylation factors and as an AKAP for RIIbeta that localizes PKA signaling within cellular TNFR1 trafficking pathways.

Mutagenesis of Bacillus thuringiensiscry1Ac gene and its insecticidal activity against Plutella xylostella and Ostrinia furnacalis

The cry gene, encoding the Cry protein, has been recently introduced into crops to generate transgenic plants that are resistant to pest insects. In this study, we produced various Cry mutant proteins by converting cry1Ac gene to a novel cry1-44 gene, improving the insecticidal activity and enlarging the host spectrum by multi site-directed mutagenesis. For this mutagenesis, 1857 bp of the modified cry1Ac gene ( mod-cry1Ac), which was extensively changed in plant codon usage, was used as a template and amplified by polymerase chain reaction with 20 mutagenic primers, based on cry1-44 sequences. In total, 16 mutant cry genes ( mut-cry1Ac) were generated and converted at 32 amino acid residues, located on domain I (1 residue) and domain II (31 residues). Ten mutants were expressed, as a polyhedrin fusion form, using a baculovirus expression system. The expressed proteins were 95 kDa and the trypsin-activated mutant proteins were all stable at 65 kDa. In the bioassay of Plutella xylostella third-instar larvae, LC 50 of Mut-2 was 1.5-fold lower than that of Mod-Cry1Ac. In the neonate bioassay of Ostrinia furnacalis, LC 50 values of Mut-4, Mut-6, and Mut-8 were 10- to 13-fold lower than that of Mod-Cry1Ac, and even 12- to 15-fold lower than that of Mut-2, with the highest insecticidal activity against P. xylostella. As a result, several mutant cry genes demonstrated higher insecticidal activities, such as Mut-2 to P. xylostella and Mut-4, Mut-6, and Mut-8 to O. furnacalis, which might be expected of desirable cry genes for introduction into transgenic crops.