Calmodulin in complex with the first IQ motif of myosin-5a functions as an intact calcium sensor.

The motor function of vertebrate myosin-5a is inhibited by its tail in a Ca2+-dependent manner. We previously demonstrated that the calmodulin (CaM) bound to the first isoleucine-glutamine (IQ) motif (IQ1) of myosin-5a is responsible for the Ca2+-dependent regulation of myosin-5a. We have solved the crystal structure of a truncated myosin-5a containing the motor domain and IQ1 (MD-IQ1) complexed with Ca2+-bound CaM (Ca2+-CaM) at 2.5-Å resolution. Compared with the structure of the MD-IQ1 complexed with essential light chain (an equivalent of apo-CaM), MD-IQ1/Ca2+-CaM displays large conformational differences in IQ1/CaM and little difference in the motor domain. In the MD-IQ1/Ca2+-CaM structure, the N-lobe and the C-lobe of Ca2+-CaM adopt an open conformation and grip the C-terminal and the N-terminal portions of the IQ1, respectively. Remarkably, the interlobe linker of CaM in IQ1/Ca2+-CaM is in a position opposite that in IQ1/apo-CaM, suggesting that CaM flip-flops relative to the IQ1 during the Ca2+ transition. We demonstrated that CaM continuously associates with the IQ1 during the Ca2+ transition and that the binding of CaM to IQ1 increases Ca2+ affinity and substantially changes the kinetics of the Ca2+ transition, suggesting that the IQ1/CaM complex functions as an intact Ca2+ sensor responding to distinct calcium signals.

Identification of the Isoform-specific Interactions between the Tail and the Head of Class V Myosin.

Vertebrates have three isoforms of class V myosin (Myo5), Myo5a, Myo5b, and Myo5c, which are involved in transport of multiple cargoes. It is well established that the motor functions of Myo5a and Myo5b are regulated by a tail inhibition mechanism. Here we found that the motor function of Myo5c was also inhibited by its globular tail domain (GTD), and this inhibition was abolished by high Ca(2+), indicating that the tail inhibition mechanism is conserved in vertebrate Myo5. Interestingly, we found that Myo5a-GTD and Myo5c-GTD were not interchangeable in terms of inhibition of motor function, indicating isoform-specific interactions between the GTD and the head of Myo5. To identify the isoform-specific interactions, we produced a number of Myo5 chimeras by swapping the corresponding regions of Myo5a and Myo5c. We found that Myo5a-GTD, with its H11-H12 loop being substituted with that of Myo5c, was able to inhibit the ATPase activity of Myo5c and that Myo5a-GTD was able to inhibit the ATPase activity of Myo5c-S1 and Myo5c-HMM only when their IQ1 motif was substituted with that of Myo5a. Those results indicate that the H11-H12 loop in the GTD and the IQ1 motif in the head dictate the isoform-specific interactions between the GTD and head of Myo5. Because the IQ1 motif is wrapped by calmodulin, whose conformation is influenced by the sequence of the IQ1 motif, we proposed that the calmodulin bound to the IQ1 motif interacts with the H11-H12 loop of the GTD in the inhibited state of Myo5.

Mouse myosin-19 is a plus-end-directed, high-duty ratio molecular motor.

Class XIX myosin (Myo19) is a vertebrate-specific unconventional myosin, responsible for the transport of mitochondria. To characterize biochemical properties of Myo19, we prepared recombinant mouse Myo19-truncated constructs containing the motor domain and the IQ motifs using the baculovirus/Sf9 expression system. We identified regulatory light chain (RLC) of smooth muscle/non-muscle myosin-2 as the light chain of Myo19. The actin-activated ATPase activity and the actin-gliding velocity of Myo19-truncated constructs were about one-third and one-sixth as those of myosin-5a, respectively. The apparent affinity of Myo19 to actin was about the same as that of myosin-5a. The RLCs bound to Myo19 could be phosphorylated by myosin light chain kinase, but this phosphorylation had little effect on the actin-activated ATPase activity and the actin-gliding activity of Myo19-truncated constructs. Using dual fluorescence-labeled actin filaments, we determined that Myo19 is a plus-end-directed molecular motor. We found that, similar to that of the high-duty ratio myosin, such as myosin-5a, ADP release rate was comparable with the maximal actin-activated ATPase activity of Myo19, indicating that ADP release is a rate-limiting step for the ATPase cycle of acto-Myo19. ADP strongly inhibited the actin-activated ATPase activity and actin-gliding activity of Myo19-truncated constructs. Based on the above results, we concluded that Myo19 is a high-duty ratio molecular motor moving to the plus-end of the actin filament.

Cooperation between the two heads of smooth muscle myosin is essential for full activation of the motor function by phosphorylation.

The motor function of smooth muscle myosin (SmM) is regulated by phosphorylation of the regulatory light chain (RLC) bound to the neck region of the SmM heavy chain. It is generally accepted that unphosphorylated RLC induces interactions between the two heads and between the head and the tail, thus inhibiting the motor activity of SmM, whereas phosphorylation of RLC interrupts those interactions, thus reversing the inhibition and restoring the motor activity to the maximal value. One assumption of this model is that single-headed SmM is fully active regardless of phosphorylation. To re-evaluate this model, we produced a number of SmM constructs with coiled coils of various lengths and examined their structure and regulation. With these constructs we identified the segment in the coiled-coil key for the formation of a stable double-headed structure. In agreement with the current model, we found that the actin-activated ATPase activity of unphosphorylated SmM increased with shortening of the coiled-coil. However, contrary to the current model, we found that the actin-activated ATPase activity of phosphorylated SmM decreased with shortening coiled-coil and only the stable double-headed SmM was fully activated by phosphorylation. These results indicate that single-headed SmM is neither fully active nor fully inhibited. Based on our findings, we propose that cooperation between the two heads is essential, not only for the inhibition of unphosphorylated SmM, but also for the activation of phosphorylated SmM.

Calmodulin bound to the first IQ motif is responsible for calcium-dependent regulation of myosin 5a.

Myosin 5a is as yet the best-characterized unconventional myosin motor involved in transport of organelles along actin filaments. It is well-established that myosin 5a is regulated by its tail in a Ca(2+)-dependent manner. The fact that the actin-activated ATPase activity of myosin 5a is stimulated by micromolar concentrations of Ca(2+) and that calmodulin (CaM) binds to IQ motifs of the myosin 5a heavy chain indicates that Ca(2+) regulates myosin 5a function via bound CaM. However, it is not known which IQ motif and bound CaM are responsible for the Ca(2+)-dependent regulation and how the head-tail interaction is affected by Ca(2+). Here, we found that the CaM in the first IQ motif (IQ1) is responsible for Ca(2+) regulation of myosin 5a. In addition, we demonstrate that the C-lobe fragment of CaM in IQ1 is necessary for mediating Ca(2+) regulation of myosin 5a, suggesting that the C-lobe fragment of CaM in IQ1 participates in the interaction between the head and the tail. We propose that Ca(2+) induces a conformational change of the C-lobe of CaM in IQ1 and prevents interaction between the head and the tail, thus activating motor function.

Roles of the different components of magnesium chelatase in abscisic acid signal transduction.

The H subunit of Mg-chelatase (CHLH) was shown to regulate abscisic acid (ABA) signaling and the I subunit (CHLI) was also reported to modulate ABA signaling in guard cells. However, it remains essentially unknown whether and how the Mg-chelatase-catalyzed Mg-protoporphyrin IX-production differs from ABA signaling. Using a newly-developed surface plasmon resonance system, we showed that ABA binds to CHLH, but not to the other Mg-chelatase components/subunits CHLI, CHLD (D subunit) and GUN4. A new rtl1 mutant allele of the CHLH gene in Arabidopsis thaliana showed ABA-insensitive phenotypes in both stomatal movement and seed germination. Upregulation of CHLI1 resulted in ABA hypersensitivity in seed germination, while downregulation of CHLI conferred ABA insensitivity in stomatal response in Arabidopsis. We showed that CHLH and CHLI, but not CHLD, regulate stomatal sensitivity to ABA in tobacco (Nicotiana benthamiana). The overexpression lines of the CHLD gene showed wild-type ABA sensitivity in Arabidopsis. Both the GUN4-RNA interference and overexpression lines of Arabidopsis showed wild-type phenotypes in the major ABA responses. These findings provide clear evidence that the Mg-chelatase-catalyzed Mg-ProtoIX production is distinct from ABA signaling, giving information to understand the mechanism by which the two cellular processes differs at the molecular level.

Antimicrobial peptides from the venoms of Vespa bicolor Fabricius.

Hornets possess highly toxic venoms, which are rich in toxins, enzymes and biologically active peptides. Many bioactive substances have been identified from wasp venoms. Vespa mastoparan (MP-VBs) and Vespa chemotatic peptide presenting antimicrobial action (VESP-VBs) were purified and characterized from the venom of the wasp, Vespa bicolor Fabricius. The precursors encoding VESP-VBs and MP-VBs were cloned from the cDNA library of the venomous glands. Analyzed by FAB-MS, the amino acid sequence and molecular mass for VESP-VB1 were FMPIIGRLMSGSL and 1420.6, for MP-VB1 were INMKASAAVAKKLL and 1456.5, respectively. The primary structures of these peptides are homologous to those of chemotactic peptides and mastoparans isolated from other vespid venoms. These peptides showed strong antimicrobial activities against bacteria and fungi and induced mast cell degranulation, but displayed almost no hemolytic activity towards human blood red cells.

Cuticular hydrocarbon chemistry, an important factor shaping the current distribution pattern of the imported fire ants in the USA.

Two sibling species, Solenopsis richteri and S. invicta, were both introduced into the southern USA from South America in the early 20th century. Today, S. richteri occupies higher latitudes and colder areas, while S. invicta occupies lower latitudes. Between the distributions of the two species, there is a large area of viable hybrid (S. richteri × S. invicta) populations. This study aimed to characterize the forces driving this distribution pattern and the underlying mechanisms. Cuticular hydrocarbons (CHCs) of freshly killed workers of S. invicta, hybrids, and S. richteri were removed using hexane. Both intact and CHCs-extracted workers were subjected to a constant rate of increasing temperature from 10 to 60 °C to obtain relative water loss and the water loss transition temperature (Tc-ant). Mass loss and Tc-ant were both significantly increased with CHCs removal. We then examined the CHC composition of three species. CHC profiles of S. richteri are characterized by significant amounts of short-chain (C23-C27) saturated and unsaturated hydrocarbons. In contrast, profiles of S. invicta consist primarily of long-chain (C27-C29) saturated hydrocarbons; unsaturated alkenes are completely lacking. Hybrid fire ants show intermediate profiles of the two parent species. We measured the melting point (Tm) and water-loss transition temperature of CHC blends (Tc-CHC) of different ant species colonies using differential scanning calorimetry (DSC) and an artificial membrane system, respectively. There were 3-5 Tms of each CHCs sample of different ant colonies due to their complex chemistry. The highest Tms (Tm-maxs) of CHCs samples from S. invicta and the hybrid were significantly higher than that from S. richteri. The correlation between Tc-CHC and Tm-max obtained from the same CHCs sample was highly significant. These results reveal that species having higher Tc and Tm-max retain more water under relatively higher temperature, and consequently are able to occupy warmer environments. We conclude that CHC chemistry plays a role in shaping current distribution patterns of S. richteri, S. invicta and their hybrid in the United States.

Molecular cloning and characterization of novel cathelicidin-derived myeloid antimicrobial peptide from Phasianus colchicus.

Cathelicidins were initially characterized as a family of antimicrobial peptides. Now it is clear that they fulfill several immune functions in addition to their antimicrobial activity. In the current work, three cDNA sequences encoding pheasant cathelicidins were cloned from a constructed bone marrow cDNA library of Phasianus colchicus, using a nested-PCR-based cloning strategy. The three deduced mature antimicrobial peptides, Pc-CATH1, 2 and 3 are composed of 26, 32, and 29 amino acid residues, respectively. Unlike the mammalian cathelicidins that are highly divergent even within the same genus, Pc-CATHs are remarkably conserved with chicken fowlicidins with only a few of residues mutated according to the phylogenetic analysis result. Synthetic Pc-CATH1 exerted strong antimicrobial activity against most of bacteria and fungi tested, including the clinically isolated (IS) drug-resistant strains. Most MIC values against Gram-positive bacteria were in the range of 0.09-2.95 µM in the presence of 100mM NaCl. Pc-CATH1 displayed a negligible hemolytic activity against human erythrocytes, lysing 3.6% of erythrocytes at 3.15 µM (10 µg/ml), significantly higher than the corresponding MIC. Pc-CATH1 was stable in the human serum for up to 72 h, revealing its extraordinary serum stability. These specific features of Pc-CATH1 may make its applications much wider given the potency and breadth of the peptide's bacteriocidal capacity and its resistance towards serum and high-salt environments.

A novel defensin-like peptide from salivary glands of the hard tick, Haemaphysalis longicornis.

A novel defensin-like antimicrobial peptide named longicornsin was isolated from the salivary glands of the hard tick, Haemaphysalis longicornis, using a 10-kDa cut-off Centriprep filter and reversed-phase high-performance liquid chromatography (RP-HPLC). Its amino acid sequence was determined as DFGCGQGMIFMCQRRCMRLYPGSTGFCRGFRCMCDTHIPLRPPFMVG by Edman degradation. The cDNA encoding longicornsin was cloned by cDNA library screening. The predicted protein from the cDNA sequence was composed of 78 amino acids including a mature longicornsin. It showed similarity with defensin-like peptides from other ticks by BLAST search. Different from most other tick defensin-like peptides, longicornsin had a C-terminal extension. Purified longicornsin exerted potent antimicrobial activities against bacteria and fungi. Interestingly, it even showed strong antimicrobial ability against drug-resistant microorganisms and Helicobacter pylori. The results of this study indicated that longicornsin is a potential candidate for novel antimicrobial drug design.

Novel families of antimicrobial peptides with multiple functions from skin of Xizang plateau frog, Nanorana parkeri.

Xizang plateau frog (Nanorana parkeri) captured in Lhasa, Tibet, China, solely lives in the subtropical plateau, where there is strong ultraviolet radiation and long duration of sunshine. Considering its harsh living environment, the frog's innate defense against microbes and environmental stress was investigated. In current study, three antimicrobial peptides (AMPs) were purified and characterized from the skin secretion of N. parkeri. The coding cDNA sequences were also cloned from the skin cDNA library of N. parkeri. By structural characterization, two peptides were identified belonging to Japonicin-1 family, and named as Japonicin-1Npa (FLLFPLMCKIQGKC) and Japonicin-1Npb (FVLPLVMCKILRKC). The third peptide isolated named Parkerin with a unique sequence of GWANTLKNVAGGLCKITGAA did not show similarity to any known amphibian AMPs. Multi-functions of three AMPs were examined (antioxidant, MCD, hemolytic etc). Their solution structures determined by CD and antimicrobial mechanisms investigated by SEM are very well consistent with their functional characters. Current result suggests that these novel multi-functional AMPs could play an important role in defending N. parkeri against environmental oxidative stress and pathogenic microorganisms, which may partially reveal the ecological adaptation of these plateau-living amphibians.

Novel cathelicidin-derived antimicrobial peptides from Equus asinus.

In the present study, EA-CATH1 and EA-CATH2 were identified from a constructed lung cDNA library of donkey (Equus asinus) as members of cathelicidin-derived antimicrobial peptides, using a nested PCR-based cloning strategy. Composed of 25 and 26 residues, respectively, EA-CATH1 and EA-CATH2 are smaller than most other cathelicidins and have no sequence homology to other cathelicidins identified to date. Chemically synthesized EA-CATH1 exerted potent antimicrobial activity against most of the 32 strains of bacteria and fungi tested, especially the clinically isolated drug-resistant strains, and minimal inhibitory concentration values against Gram-positive bacteria were mostly in the range of 0.3-2.4 microg mL(-1). EA-CATH1 showed an extraordinary serum stability and no haemolytic activity against human erythrocytes in a dose up to 20 microg mL(-1). CD spectra showed that EA-CATH1 mainly adopts an alpha-helical conformation in a 50% trifluoroethanol/water solution, but a random coil in aqueous solution. Scanning electron microscope observations of Staphylococcus aureus (ATCC2592) treated with EA-CATH1 demonstrated that EA-CATH could cause rapid disruption of the bacterial membrane, and in turn lead to cell lysis. This might explain the much faster killing kinetics of EA-CATH1 than conventional antibiotics revealed by killing kinetics data. In the presence of CaCl(2), EA-CATH1 exerted haemagglutination activity, which might potentiate an inhibition against the bacterial polyprotein interaction with the host erythrocyte surface, thereby possibly restricting bacterial colonization and spread.

A novel serine protease inhibitor from the venom of Vespa bicolor Fabricius.

Hornets possess highly toxic venoms, which are rich in toxin, enzymes and biologically active peptides. Many bioactive substances have been identified from wasp venoms but only a few serine protease inhibitors have been identified from two kinds of wasp venoms. In this work, a serine protease inhibitor named bicolin was purified and characterized from the venom of the wasp, Vespa bicolor Fabricius. The precursor encoding bicolin was cloned from the cDNA library of the venomous glands. It is a cysteine-rich small protein containing 54 amino acid residues including 6 half-cysteines. The peptide is homologous to serine protease inhibitors isolated from venoms of Anoplius samariensis and Pimpla hypochondriaca. Bicolin showed inhibitory ability against trypsin and thrombin.