AAA+ Ring and linker swing mechanism in the dynein motor.

Dynein ATPases power diverse microtubule-based motilities. Each dynein motor domain comprises a ring-like head containing six AAA+ modules and N- and C-terminal regions, together with a stalk that binds microtubules. How these subdomains are arranged and generate force remains poorly understood. Here, using electron microscopy and image processing of tagged and truncated Dictyostelium cytoplasmic dynein constructs, we show that the heart of the motor is a hexameric ring of AAA+ modules, with the stalk emerging opposite the primary ATPase site (AAA1). The C-terminal region is not an integral part of the ring but spans between AAA6 and near the stalk base. The N-terminal region includes a lever-like linker whose N terminus swings by approximately 17 nm during the ATPase cycle between AAA2 and the stalk base. Together with evidence of stalk tilting, which may communicate changes in microtubule binding affinity, these findings suggest a model for dynein's structure and mechanism.

Isolation and grouping of RNA phages by Itaru Watanabe et al. (1967).

I. Watanabe et al. isolated approximately 30 strains of RNA phages from various parts of Japan. To isolate RNA phages, they assessed the infection specificity of male Escherichia coli and RNase sensitivity. They found that the isolated strains of RNA phages could be serologically separated into three groups. Furthermore, most of them were serologically related, and the antiphage rabbit serum prepared by one of these phages neutralized most of the other phages. The only serologically unrelated phage was the RNA phage Qß, which was isolated at the Institute for Virus Research, Kyoto University, in 1961.

Structural basis of Sec-independent membrane protein insertion by YidC.

Newly synthesized membrane proteins must be accurately inserted into the membrane, folded and assembled for proper functioning. The protein YidC inserts its substrates into the membrane, thereby facilitating membrane protein assembly in bacteria; the homologous proteins Oxa1 and Alb3 have the same function in mitochondria and chloroplasts, respectively. In the bacterial cytoplasmic membrane, YidC functions as an independent insertase and a membrane chaperone in cooperation with the translocon SecYEG. Here we present the crystal structure of YidC from Bacillus halodurans, at 2.4 Å resolution. The structure reveals a novel fold, in which five conserved transmembrane helices form a positively charged hydrophilic groove that is open towards both the lipid bilayer and the cytoplasm but closed on the extracellular side. Structure-based in vivo analyses reveal that a conserved arginine residue in the groove is important for the insertion of membrane proteins by YidC. We propose an insertion mechanism for single-spanning membrane proteins, in which the hydrophilic environment generated by the groove recruits the extracellular regions of substrates into the low-dielectric environment of the membrane.

Comparison of composition-gradient sedimentation equilibrium and composition-gradient static light scattering as techniques for quantitative characterization of biomolecular interactions: A case study.

The equilibrium hetero-association of NADH oxidase and peroxiredoxin was characterized by means of independently conducted measurements of composition-gradient sedimentation equilibrium and composition-gradient static light scattering. Results obtained from both experiments were quantitatively accounted for by a model according to which a dimer of NADH oxidase forms a 1:1 equilibrium complex with a decamer of peroxiredoxin under the conditions of these experiments. The best-fit equilibrium constants for heteroassociation of the two proteins obtained from the two measurements were found to be identical to well within the uncertainty of estimate of each of the two methods. The relative virtues of each of the methods are discussed.

Role of bacteriophage T4 baseplate in regulating assembly and infection.

Bacteriophage T4 consists of a head for protecting its genome and a sheathed tail for inserting its genome into a host. The tail terminates with a multiprotein baseplate that changes its conformation from a "high-energy" dome-shaped to a "low-energy" star-shaped structure during infection. Although these two structures represent different minima in the total energy landscape of the baseplate assembly, as the dome-shaped structure readily changes to the star-shaped structure when the virus infects a host bacterium, the dome-shaped structure must have more energy than the star-shaped structure. Here we describe the electron microscopy structure of a 3.3-MDa in vitro-assembled star-shaped baseplate with a resolution of 3.8 Å. This structure, together with other genetic and structural data, shows why the high-energy baseplate is formed in the presence of the central hub and how the baseplate changes to the low-energy structure, via two steps during infection. Thus, the presence of the central hub is required to initiate the assembly of metastable, high-energy structures. If the high-energy structure is formed and stabilized faster than the low-energy structure, there will be insufficient components to assemble the low-energy structure.

Cooperative binding of the outer arm-docking complex underlies the regular arrangement of outer arm dynein in the axoneme.

Outer arm dynein (OAD) in cilia and flagella is bound to the outer doublet microtubules every 24 nm. Periodic binding of OADs at specific sites is important for efficient cilia/flagella beating; however, the molecular mechanism that specifies OAD arrangement remains elusive. Studies using the green alga Chlamydomonas reinhardtii have shown that the OAD-docking complex (ODA-DC), a heterotrimeric complex present at the OAD base, functions as the OAD docking site on the doublet. We find that the ODA-DC has an ellipsoidal shape ∼24 nm in length. In mutant axonemes that lack OAD but retain the ODA-DC, ODA-DC molecules are aligned in an end-to-end manner along the outer doublets. When flagella of a mutant lacking ODA-DCs are supplied with ODA-DCs upon gamete fusion, ODA-DC molecules first bind to the mutant axonemes in the proximal region, and the occupied region gradually extends toward the tip, followed by binding of OADs. This and other results indicate that a cooperative association of the ODA-DC underlies its function as the OAD-docking site and is the determinant of the 24-nm periodicity.

Unusual Reversible Oligomerization of Unfolded Dengue Envelope Protein Domain 3 at High Temperatures and Its Abolition by a Point Mutation.

We report differential scanning calorimetry (DSC) experiments between 10 and 120 °C of Dengue 4 envelope protein domain 3 (DEN4 ED3), a small 107-residue monomeric globular protein domain. The thermal unfolding of DEN4 ED3 was fully reversible and exhibited two peculiar endothermic peaks. AUC (analytical ultracentrifugation) experiments at 25 °C indicated that DEN4 ED3 was monomeric. Detailed thermodynamic analysis indicated that the two endothermic peaks separated with an increasing protein concentration, and global fitting of the DSC curves strongly suggested the presence of unfolded tetramers at temperatures around 80-90 °C, which dissociated to unfolded monomers at even higher temperatures. To further characterize this rare thermal unfolding process, we designed and constructed a DEN4 ED3 variant that would unfold according to a two-state model, typical of globular proteins. We thus substituted Val 380, the most buried residue at the dimeric interface in the protein crystal, with less hydrophobic amino acids (Ala, Ser, Thr, Asn, and Lys). All variants showed a single heat absorption peak, typical of small globular proteins. In particular, the DSC thermogram of DEN4 V380K indicated a two-state reversible thermal unfolding independent of protein concentration, indicating that the high-temperature oligomeric state was successfully abolished by a single mutation. These observations confirmed the standard view that small monomeric globular proteins undergo a two-state unfolding. However, the reversible formation of unfolded oligomers at high temperatures is a truly new phenomenon, which was fully inhibited by an accurately designed single mutation.

Structure of a dominant-negative helix-loop-helix transcriptional regulator suggests mechanisms of autoinhibition.

Helix-loop-helix (HLH) family transcription factors regulate numerous developmental and homeostatic processes. Dominant-negative HLH (dnHLH) proteins lack DNA-binding ability and capture basic HLH (bHLH) transcription factors to inhibit cellular differentiation and enhance cell proliferation and motility, thus participating in patho-physiological processes. We report the first structure of a free-standing human dnHLH protein, HHM (Human homologue of murine maternal Id-like molecule). HHM adopts a V-shaped conformation, with N-terminal and C-terminal five-helix bundles connected by the HLH region. In striking contrast to the common HLH, the HLH region in HHM is extended, with its hydrophobic dimerization interfaces embedded in the N- and C-terminal helix bundles. Biochemical and physicochemical analyses revealed that HHM exists in slow equilibrium between this V-shaped form and the partially unfolded, relaxed form. The latter form is readily available for interactions with its target bHLH transcription factors. Mutations disrupting the interactions in the V-shaped form compromised the target transcription factor specificity and accelerated myogenic cell differentiation. Therefore, the V-shaped form of HHM may represent an autoinhibited state, and the dynamic conformational equilibrium may control the target specificity.

Protein aggregate turbidity: Simulation of turbidity profiles for mixed-aggregation reactions.

Due to their colloidal nature, all protein aggregates scatter light in the visible wavelength region when formed in aqueous solution. This phenomenon makes solution turbidity, a quantity proportional to the relative loss in forward intensity of scattered light, a convenient method for monitoring protein aggregation in biochemical assays. Although turbidity is often taken to be a linear descriptor of the progress of aggregation reactions, this assumption is usually made without performing the necessary checks to provide it with a firm underlying basis. In this article, we outline utilitarian methods for simulating the turbidity generated by homogeneous and mixed-protein aggregation reactions containing fibrous, amorphous, and crystalline structures. The approach is based on a combination of Rayleigh-Gans-Debye theory and approximate forms of the Mie scattering equations.

Asymmetrically fused polyoxometalate-silver alkynide composite cluster.

We demonstrate that an asymmetric composite cluster, [Ag25{C≡CC(CH3)3}16(CH3CN)4(P2W15Nb3O62)] (1), consisting of directly fused polyoxometalate and silver alkynide moieties can be facilely synthesized by a one-pot reaction between a Nb-substituted Dawson-type polyoxometalate, H4[α-P2W15Nb3O62](5-), and the mixture of (CH3)3CC≡CAg and CF3SO3Ag. Single-crystal X-ray diffraction revealed the structure of 1, where Ag atoms are selectively attached to the Nb-substituted hemisphere of the pedestal Dawson anion. Its structural integrity in the solution was demonstrated by (31)P NMR spectroscopy and analytical ultracentrifugation. The latter method also unveiled the stepwise formation mechanism of 1.

Crystal structure of Cex1p reveals the mechanism of tRNA trafficking between nucleus and cytoplasm.

In all eukaryotes, transcribed precursor tRNAs are maturated by processing and modification processes in nucleus and are transported to the cytoplasm. The cytoplasmic export protein (Cex1p) captures mature tRNAs from the nuclear export receptor (Los1p) on the cytoplasmic side of the nuclear pore complex, and it delivers them to eukaryotic elongation factor 1α. This conserved Cex1p function is essential for the quality control of mature tRNAs to ensure accurate translation. However, the structural basis of how Cex1p recognizes tRNAs and shuttles them to the translational apparatus remains unclear. Here, we solved the 2.2 Å resolution crystal structure of Saccharomyces cerevisiae Cex1p with C-terminal 197 disordered residues truncated. Cex1p adopts an elongated architecture, consisting of N-terminal kinase-like and a C-terminal α-helical HEAT repeat domains. Structure-based biochemical analyses suggested that Cex1p binds tRNAs on its inner side, using the positively charged HEAT repeat surface and the C-terminal disordered region. The N-terminal kinase-like domain acts as a scaffold to interact with the Ran-exportin (Los1p·Gsp1p) machinery. These results provide the structural basis of Los1p·Gsp1p·Cex1p·tRNA complex formation, thus clarifying the dynamic mechanism of tRNA shuttling from exportin to the translational apparatus.

Archaeal ribosomal stalk protein interacts with translation factors in a nucleotide-independent manner via its conserved C terminus.

Protein synthesis on the ribosome requires translational GTPase factors to bind to the ribosome in the GTP-bound form, take individual actions that are coupled with GTP hydrolysis, and dissociate, usually in the GDP-bound form. The multiple copies of the flexible ribosomal stalk protein play an important role in these processes. Using biochemical approaches and the stalk protein from a hyperthermophilic archaeon, Pyrococcus horikoshii, we here provide evidence that the conserved C terminus of the stalk protein aP1 binds directly to domain I of the elongation factor aEF-2, irrespective of whether aEF-2 is bound to GTP or GDP. Site-directed mutagenesis revealed that four hydrophobic amino acids at the C terminus of aP1, Leu-100, 103, 106, and Phe-107, are crucial for the direct binding. P1 was also found to bind to the initiation factor aIF5B, as well as aEF-1α, but not aIF2γ, via its C terminus. Moreover, analytical ultracentrifugation and gel mobility shift analyses showed that a heptameric complex of aP1 and aP0, aP0(aP1)(2)(aP1)(2)(aP1)(2), can bind multiple aEF-2 molecules simultaneously, which suggests that individual copies of the stalk protein are accessible to the factor. The functional significance of the C terminus of the stalk protein was also shown using the eukaryotic proteins P1/P2 and P0. It is likely that the conserved C terminus of the stalk proteins of archaea and eukaryotes can bind to translation factors both before and after GTP hydrolysis. This consistent binding ability of the stalk protein may contribute to maintaining high concentrations of translation factors around the ribosome, thus promoting translational efficiency.

Structure of the 3.3MDa, in vitro assembled, hubless bacteriophage T4 baseplate.

The bacteriophage T4 baseplate is the control center of the virus, where the recognition of an Escherichiacoli host by the long tail fibers is translated into a signal to initiate infection. The short tail fibers unfold from the baseplate for firm attachment to the host, followed by shrinkage of the tail sheath that causes the tail tube to enter and cross the periplasmic space ending with injection of the genome into the host. During this process, the 6.5MDa baseplate changes its structure from a "dome" shape to a "star" shape. An in vitro assembled hubless baseplate has been crystallized. It consists of six copies of the recombinantly expressed trimeric gene product (gp) 10, monomeric gp7, dimeric gp8, dimeric gp6 and monomeric gp53. The diffraction pattern extends, at most, to 4.0Å resolution. The known partial structures of gp10, gp8, and gp6 and their relative position in the baseplate derived from earlier electron microscopy studies were used for molecular replacement. An electron density map has been calculated based on molecular replacement, single isomorphous replacement with anomalous dispersion data and 2-fold non-crystallographic symmetry averaging between two baseplate wedges in the crystallographic asymmetric unit. The current electron density map indicates that there are structural changes in the gp6, gp8, and gp10 oligomers compared to their structures when separately crystallized. Additional density is also visible corresponding to gp7, gp53 and the unknown parts of gp10 and gp6.

Role of positions e and g in the fibrous assembly formation of an amphipathic α-helix-forming polypeptide.

We previously characterized α3, a polypeptide that has a three times repeated sequence of seven amino acids (abcdefg: LETLAKA) and forms fibrous assemblies composed of amphipathic α-helices. Upon comparison of the amino acid sequences of α3 with other α-helix forming polypeptides, we proposed that the fibrous assemblies were formed due to the alanine (Ala) residues at positions e and g. Here, we characterized seven α3 analog polypeptides with serine (Ser), glycine (Gly), or charged residues substituted for Ala at positions e and g. The α-helix forming abilities of the substituted polypeptides were less than that of α3. The polypeptides with amino acid substitutions at position g and the polypeptide KEα3, in which Ala was substituted with charged amino acids, formed few fibrous assemblies. In contrast, polypeptides with Ala replaced by Ser at position e formed ß-sheets under several conditions. These results show that Ala residues at position e and particularly at position g are involved in the formation of fibrous assemblies.

Crystallographic analysis reveals octamerization of viroplasm matrix protein P9-1 of Rice black streaked dwarf virus.

The P9-1 protein of Rice black streaked dwarf virus accumulates in viroplasm inclusions, which are structures that appear to play an important role in viral morphogenesis and are commonly found in viruses in the family Reoviridae. Crystallographic analysis of P9-1 revealed structural features that allow the protein to form dimers via hydrophobic interactions. Each dimer has carboxy-terminal regions, resembling arms, that extend to neighboring dimers, thereby uniting sets of four dimers via lateral hydrophobic interactions, to yield cylindrical octamers. The importance of these regions for the formation of viroplasm-like inclusions was confirmed by the absence of such inclusions when P9-1 was expressed without its carboxy-terminal arm. The octamers are vertically elongated cylinders resembling the structures formed by NSP2 of rotavirus, even though there are no significant similarities between the respective primary and secondary structures of the two proteins. Our results suggest that an octameric structure with an internal pore might be important for the functioning of the respective proteins in the events that occur in the viroplasm, which might include viral morphogenesis.

Accumulation of ß-conglycinin in soybean cotyledon through the formation of disulfide bonds between α'- and α-subunits.

ß-Conglycinin, one of the major soybean (Glycine max) seed storage proteins, is folded and assembled into trimers in the endoplasmic reticulum and accumulated into protein storage vacuoles. Prior experiments have used soybean ß-conglycinin extracted using a reducing buffer containing a sulfhydryl reductant such as 2-mercaptoethanol, which reduces both intermolecular and intramolecular disulfide bonds within the proteins. In this study, soybean proteins were extracted from the cotyledons of immature seeds or dry beans under nonreducing conditions to prevent the oxidation of thiol groups and the reduction or exchange of disulfide bonds. We found that approximately half of the α'- and α-subunits of ß-conglycinin were disulfide linked, together or with P34, prior to amino-terminal propeptide processing. Sedimentation velocity experiments, size-exclusion chromatography, and two-dimensional polyacrylamide gel electrophoresis (PAGE) analysis, with blue native PAGE followed by sodium dodecyl sulfate-PAGE, indicated that the ß-conglycinin complexes containing the disulfide-linked α'/α-subunits were complexes of more than 720 kD. The α'- and α-subunits, when disulfide linked with P34, were mostly present in approximately 480-kD complexes (hexamers) at low ionic strength. Our results suggest that disulfide bonds are formed between α'/α-subunits residing in different ß-conglycinin hexamers, but the binding of P34 to α'- and α-subunits reduces the linkage between ß-conglycinin hexamers. Finally, a subset of glycinin was shown to exist as noncovalently associated complexes larger than hexamers when ß-conglycinin was expressed under nonreducing conditions.

Assembly of pyrene-modified DNA/RNA duplexes incorporating a G-rich single strand region.

The structural properties of a DNA/RNA duplex having a pyrene residue at the 5' end of DNA and a G-rich single strand region at the 3' end of RNA were studied in detail. Fluorescence and ultracentrifugation analyses indicated the formation of a complex containing four DNA/RNA duplexes, which required a pyrene residue, G-rich sequence, RNA-type backbone, and high salt concentration.

Dimer-tetramer association equilibria of human adult hemoglobin and its mutants as observed by analytical ultracentrifugation.

Dimer-tetramer equilibrium of human adult hemoglobin in CO form (COHb A) and its mutants were measured by sedimentation velocity and sedimentation equilibrium. In sedimentation velocity, the association constants were estimated by measuring the concentration dependence of the weight average sedimentation coefficients at pH 6 and 7 and fitting the data to the theoretical binding isotherms with association constants as a parameter. Association constants of wild type Hb A and three mutant Hbs, Hb Hirose(ßW37S), recombinant (r)Hb(ßW37H) and rHb(αY42S), in which an amino acid was replaced at the α(1)ß(2) interface, were measured in the presence and absence of inositol hexaphosphate (IHP). All the three mutations lowered the value of association constants, but the presence of IHP shifted the equilibrium toward tetramer. Although the association constant between dimer and tetramer of rHb(ßW37H) and rHb(αY42S) were similar, sedimentation coefficient distribution function, c(s), analysis indicated that the association and dissociation rate constants of the former is higher than the latter.

Screw motion regulates multiple functions of T4 phage protein gene product 5 during cell puncturing.

Bacteriophage T4 penetrates the outer membrane of Escherichia coli using a multifunctional device composed of a gene product 5 (gp5) protein trimer. We report that gp5 sequentially exerts distinct functions along the course of penetration stages induced by screw motion. A triple-stranded ß-helix of gp5 acts as a cell-puncturing drill bit to make a hole on the membrane and then send the lipids upward efficiently by strong charge interactions. The gp5 lysozyme domains, which degrade the peptidoglycan layer later, are shown to play novel roles to enlarge the hole and control the release of the ß-helix. The lysozyme active site is protected from lipid binding during the penetration and is exposed after the ß-helix release. Intrinsic multiple functions of gp5 are shown to be served in turn regulated by gradual change of interdomain interactions, which enables the initial infection process with single protein trimer by continuous screw motion. The results of lysozyme domain should be understood as the case where a single-function protein acquired multiple chemical functions through interplay with other domains in a multidomain protein.

Biochemical analysis and kinetic modeling of the thermal inactivation of MBP-fused heparinase I: implications for a comprehensive thermostabilization strategy.

Enzymatic degradation of heparin by heparin lyases has not only largely facilitated heparin structural analysis and contamination detection, but also showed great potential to be a green and cost-effective way to produce low molecular weight heparin (LMWH). However, the commercial use of heparinase I (HepI), one of the most studied heparin lyases, has been largely hampered by its low productivity and extremely poor thermostability. Here we report the thermal inactivation mechanism and strategic thermal stabilization of maltose-binding protein (MBP)-HepI, a fusion HepI produced in E. coli with high yield, solubility and activity. Biochemical studies demonstrated that the thermal inactivation of MBP-HepI involves an unfolding step that is temperature-dependently reversible, followed by an irreversible dimerization step induced by intermolecular disulfide bonds. A good consistency between the kinetic modeling and experimental data of the inactivation was obtained within a wide range of temperature and enzyme concentration, confirming the adequacy of the proposed inactivation model. Based on the inactivation mechanism, a comprehensive strategy was proposed for the thermal stabilization of MBP-HepI, in which Ca(2+) and Tween 80 were used to inhibit unfolding while site mutation at Cys297 and DTT were employed to suppress dimerization. The engineered enzyme exhibits remarkably improved storage and operational thermostability, for example, 16-fold increase in half-life at its optimum temperature of 30 °C and 8-fold increase in remaining activity of 95% after 1-week storage at 4 °C, and therefore shows great potential as a commercial biocatalyst for heparin degradation in the pharmaceutical industry.