Dimeric Constructs Research Articles

Structural links to kinesin directionality and movement.

The kinesin motor proteins generate directional movement along microtubules and are involved in many vital processes, including cell division, in eukaryotes. The kinesin superfamily is characterized by a conserved motor domain of approximately 320 residues. Dimeric constructs of N and C class kinesins, with the motor domains at opposite ends of the heavy chain, move towards microtubule plus and minus ends, respectively. Their crystal structures differ mainly in the region linking the motor domain core to the alpha-helical coiled coil dimerization domain. Chimeric kinesins show that regions outside of the motor domain core determine the direction of movement and mutations in the linker region have a strong effect on motility. Recent work on chimeras and mutants is discussed in a structural context giving insights to possible molecular mechanisms of kinesin directionality and motility.

Changes in the Inactivation of Rat Kv1.4 K+ Channels Induced by Varying the Number of Inactivation Particles

N-type inactivation of rat Kv1.4 channels with one, two, or four inactivation balls was investigated using homogeneous populations of channels expressed in Xenopus oocytes. Tandem dimeric and tetrameric constructs of Kv1.4 were made. Channels encoded by tandem cDNAs Kv1. 4-Kv1.4Delta1-145 and Kv1.4-[Kv1.4Delta1-145](3) have two or only one tethered inactivation ball, respectively, whereas Kv1.4 itself encodes channels having four inactivation balls. The time constants for inactivation of macroscopic currents were increased significantly as the number of inactivation balls was decreased, whereas the time constants for recovery from inactivation were not modified. The ratios of the rate constants of inactivation (k(inact)) of Kv1.4-Kv1.4Delta1-145 and Kv1.4-[Kv1.4Delta1-145](3) channels to that of the Kv1.4 channel were 0.65 and 0.4, respectively, whereas the ratios of the rate constant of recovery (k(rec)) of these channels to that of Kv1.4 were almost unity. The rate constants k(inact) for channels having two and four inactivation balls are smaller than those that would be expected if inactivation balls on each channel are independent, suggesting some interaction occurs between inactivation balls. Furthermore, noninactivating current became apparent as the number of inactivation balls on a channel was decreased.

Surface topography of microtubule walls decorated with monomeric and dimeric kinesin constructs.

The surface topography of opened-up microtubule walls (sheets) decorated with monomeric and dimeric kinesin motor domains was investigated by freeze-drying and unidirectional metal shadowing. Electron microscopy of surface-shadowed specimens produces images with a high signal/noise ratio, which enable a direct observation of surface features below 2 nm detail. Here we investigate the inner and outer surface of microtubules and tubulin sheets with and without decoration by kinesin motor domains. Tubulin sheets are flattened walls of microtubules, keeping lateral protofilament contacts intact. Surface shadowing reveals the following features: (i) when the microtubule outside is exposed the surface relief is dominated by the bound motor domains. Monomeric motor constructs generate a strong 8 nm periodicity, corresponding to the binding of one motor domain per alpha-beta-tubulin heterodimer. This surface periodicity largely disappears when dimeric kinesin motor domains are used for decoration, even though it is still visible in negatively stained or frozen hydrated specimens. This could be explained by disorder in the binding of the second (loosely tethered) kinesin head, and/or disorder in the coiled-coil tail. (ii) Both surfaces of undecorated sheets or microtubules, as well as the inner surface of decorated sheets, reveal a strong 4 nm repeat (due to the periodicity of tubulin monomers) and a weak 8 nm repeat (due to slight differences between alpha- and beta-tubulin). The differences between alpha- and beta-tubulin on the inner surface are stronger than expected from cryo-electron microscopy of unstained microtubules, indicating the existence of tubulin subdomain-specific surface properties that reflect the surface corrugation and hence metal deposition during evaporation. The 16 nm periodicity visible in some negatively stained specimens (caused by the pairing of cooperatively bound kinesin dimers) is not detected by surface shadowing.

Soluble TNF-alpha receptors bind and neutralize over-expressed transmembrane TNF-alpha on macrophages, but do not inhibit its processing.

Tumor necrosis factor alpha (TNF-alpha) is initially synthesized as a type II integral membrane protein (transmembrane TNF-alpha) after macrophage activation. In this study we have investigated some aspects of the regulation of expression and biological activity of transmembrane TNF-alpha by both soluble TNF-alpha receptors (sTNF-alphaR) and inhibitors of TNF-alpha processing. We show, using the technique of receptor-mediated ligand precipitation (RMLP), that a dimeric construct of the type I sTNF-alphaR binds to transmembrane TNF-alpha, expressed on the mouse macrophage cell line RAW264.7, under cell culture conditions. This interaction between sTNF-alphaR and transmembrane TNF-alpha does not prevent processing and release of soluble TNF-alpha. A specific hydroxamic acid-based inhibitor of processing, BB1101 (British Biotech), was found to increase the total cellular levels of whole-cell, 26-kDa, precursor TNF-alpha by 2.2-fold. However, the inhibitor increased the levels of precursor TNF-alpha present solely on the cell surface (i.e., transmembrane TNF-alpha) by 5.1- to 7.5-fold. This increase in the levels of transmembrane TNF-alpha on the activated human monocytoid cell line mono mac 6 was associated with a similar (6.7-fold) increase in TNF-alpha-mediated cytotoxicity toward the human adenocarcinoma cell line Colo 205, which is sensitive only to the transmembrane form of TNF-alpha. Mono mac 6 cells, expressing transmembrane TNF-alpha, were found to be killing the Colo 205 target cells through apoptosis. This cytotoxicity could be neutralized by pre-incubating the mono mac 6 cells with either sTNF-alphaR or polyclonal anti-TNF-alpha serum.

Characterization of monomeric and dimeric B domain of Staphylococcal protein A.

Both monomeric and dimeric constructs of the B domain of protein A from Staphylococcus aureus have been characterized by NMR, CD and fluorescence spectroscopy. The monomeric form of the protein was synthesized using a novel method incorporating the use of a recombinant, folded, chimeric protein. A comparison of the recombinant monomeric form with the commercially available dimeric form indicates that, although the dimer retains the integrity of the three-helix bundle structure present in the monomer, there are interdomain contacts in the dimeric form. A single long-lived water molecule in the hydrophobic core of the three-helix bundle of monomeric protein A may represent an important stabilizing factor for the three-helix bundle topology.

Binding characteristics and tumor targeting of a covalently linked divalent CC49 single-chain antibody.

Multivalency is a recognized means of increasing the functional affinity of single-chain Fvs (scFvs) for optimizing tumor uptake. A unique divalent single-chain Fv protein [sc(Fv)2], based on the variable regions of the monoclonal antibody (MAb) CC49, has been generated that differs from other dimeric single-chain constructs in that a linker sequence (L) is encoded between the repeated V(L) and V(H) domains (V(L)-L-V(H)-L-V(L)-L-V(H)). This construct was expressed in soluble form in Escherichia coli and purified by ion-exchange and gel-filtration chromatography. Purity and immunoreactivity were determined by SDS-PAGE, HPLC and competitive RIA. sc(Fv)2 exhibited a relative K(A) (3.34 x 10(7) M(-1)) similar to that of the native IgG (1.14 x 10(8) M(-1)) as determined by BIAcore analysis. Pharmacokinetic studies showed rapid blood clearance for sc(Fv)2, with a T(1/2) less than 40 min. Whole-body clearance analysis also revealed rapid clearance, suggesting no significant retention in the extravascular space or normal tissues. Biodistribution studies of radiolabeled sc(Fv)2 showed tumor uptake greater than 6% ID/g after 30 min, which remained at this level for 6 hr. High tumor uptake and retention of sc(Fv)2 coupled with rapid blood and whole-body clearance makes this dimeric scFv of MAb CC49 a strong candidate for imaging and therapeutic applications.

Binding characteristics and tumor targeting of a covalently linked divalent CC49 single‐chain antibody

Multivalency is a recognized means of increasing the functional affinity of single-chain Fvs (scFvs) for optimizing tumor uptake. A unique divalent single-chain Fv protein [sc(Fv)2], based on the variable regions of the monoclonal antibody (MAb) CC49, has been generated that differs from other dimeric single-chain constructs in that a linker sequence (L) is encoded between the repeated VL and VH domains (VL-L-VH-L-VL-L-VH). This construct was expressed in soluble form in Escherichia coli and purified by ion-exchange and gel-filtration chromatography. Purity and immunoreactivity were determined by SDS-PAGE, HPLC and competitive RIA. sc(Fv)2 exhibited a relative KA (3.34 × 107 M−1) similar to that of the native IgG (1.14 × 108 M−1) as determined by BIAcore analysis. Pharmacokinetic studies showed rapid blood clearance for sc(Fv)2, with a T1/2 less than 40 min. Whole-body clearance analysis also revealed rapid clearance, suggesting no significant retention in the extravascular space or normal tissues. Biodistribution studies of radiolabeled sc(Fv)2 showed tumor uptake greater than 6% ID/g after 30 min, which remained at this level for 6 hr. High tumor uptake and retention of sc(Fv)2 coupled with rapid blood and whole-body clearance makes this dimeric scFv of MAb CC49 a strong candidate for imaging and therapeutic applications. Int. J. Cancer 81:911–917, 1999. © 1999 Wiley-Liss, Inc.

Individual Subunits Contribute Independently to Slow Gating of Bovine EAG Potassium Channels

The bovine ether à go-go gene encodes a delayed rectifier potassium channel. In contrast to other delayed rectifiers, its activation kinetics is largely determined by the holding potential and the concentration of extracellular Mg2+, giving rise to slowly activating currents with a characteristic sigmoidal rising phase. Replacement of a single amino acid in the extracellular linker between transmembrane segments S3 and S4 (L322H) strongly reduced the prepulse dependence and accelerated activation by 1 order of magnitude. In addition, compared with the wild type, the half-activation voltage of this mutant was shifted by more than 30 mV to more negative potentials. We used dimeric and tetrameric constructs of the bovine eag1 gene to analyze channels with defined stoichiometry of mutated and wild-type subunits within the tetrameric channel complexes. With increasing numbers of mutated subunits, the channel activation was progressively accelerated, and the sigmoidicity of the current traces was reduced. Based on a quantitative analysis, we show that the slow gating, typical for EAG channels, is mediated by independent conformational transitions of individual subunits, which gain their voltage dependence from the S4 segment. At a given voltage, external Mg2+ increases the probability of a channel subunit to be in the slowly activating conformation, whereas mutation L322H strongly reduces this probability.

Ellagitannin Chemistry. Syntheses of Tellimagrandin II and a Dehydrodigalloyl Ether-Containing Dimeric Gallotannin Analogue of Coriariin A.

The first chemical synthesis of the naturally occurring ellagitannin tellimagrandin II is reported. Key steps of the synthesis include the atropselective oxidative coupling of suitably protected galloyl rings at the O(4) and O(6) positions of a glucopyranose core, and the stereoselective acylation of the derived anomeric alcohol with a galloyl chloride. In addition, the synthesis of a novel gallotannin-ellagitannin hybrid is described. This dimeric construct relied on a hetero Diels-Alder cycloaddition/reductive rearrangement sequence to deliver the intact skeleton from a monomeric pentagalloylglucose-based orthoquinone.

Equilibrium Binding Studies of Non-claret Disjunctional Protein (Ncd) Reveal Cooperative Interactions between the Motor Domains

Non-claret disjunctional protein (Ncd) is a minus end-directed microtubule motor required for normal spindle assembly and integrity during Drosophila oogenesis. We have pursued equilibrium binding experiments to examine the affinity of Ncd for microtubules in the presence of the ATP nonhydrolyzable analog 5'-adenylyl-beta, gamma-imidodiphosphate (AMP-PNP), ADP, or ADP + Pi using both dimeric (MC1) and monomeric (MC6) Ncd constructs expressed in Escherichia coli. Both MC1 and MC6 sediment with microtubules in the absence of added nucleotide as well as in the presence of either ADP or AMP-PNP. Yet, in the presence of ADP + Pi, there is a decrease in the affinity of both MC1 and MC6 for microtubules. The data for dimeric MC1 show that release of the dimer to the supernatant is sigmoidal with the apparent Kd(Pi) for the two phosphate sites at 23.3 and 1.9 mM, respectively. The results indicate that binding at the first phosphate site enhances binding at the second site, thus cooperatively stimulating release. Stopped-flow kinetics indicate that MgATP promotes dissociation of the Mt.MC1 complex at 14 s-1, yet AMP-PNP has no effect on the Mt.MC1 complex. These results are consistent with a model for the ATPase cycle in which ATP hydrolysis occurs on the microtubule followed by detachment as the Ncd.ADP.Pi intermediate.

Nucleotide-dependent structural changes in dimeric NCD molecules complexed to microtubules

Complexes consisting of motor domains of the kinesin-like protein ncd bound to reassembled brain microtubules were visualised using cryoelectron microscopy and helical image reconstruction. Different nucleotide-associated states of a dimeric construct (NΔ295-700) of ncd were analysed to reveal ADP-containing, AMP.PNP-containing and empty (rigor) conformations. In these three states, each thought to mimic a different stage in ATP turnover, the double-headed motors attach to the microtubules by one head only, with the free head tethered in relatively fixed positions. The three structures differ both in the way the attached heads interact with tubulin and in the position of the tethered heads. In the strongly binding rigor and AMP.PNP (ATP-like) states, the attached head makes close contact with both subunits of a tubulin heterodimer. In the weakly bound ADP state, the contact made by the attached head with the monomer closer to the plus end appears to be more loose. Also, in the ATP-like state, the free head tilts nearer to the plus end than in the other two states. The data argue against model mechanisms in which a conformational change in the bound head guides the free head closer to its next binding site; on the contrary, the transition from ADP-filled via rigor to the AMP.PNP (ATP-like) state of the bound head produces a small motion of the free head in the counter-productive direction. However, the observation that the tethered head points towards the minus end, in all three states, is consistent with the idea that the relative arrangement of the heads in a dimer is a major determinant of directionality.

Image reconstructions of microtubules decorated with monomeric and dimeric kinesins: comparison with x-ray structure and implications for motility.

We have decorated microtubules with monomeric and dimeric kinesin constructs, studied their structure by cryoelectron microscopy and three-dimensional image reconstruction, and compared the results with the x-ray crystal structure of monomeric and dimeric kinesin. A monomeric kinesin construct (rK354, containing only a short neck helix insufficient for coiled-coil formation) decorates microtubules with a stoichiometry of one kinesin head per tubulin subunit (alpha-beta-heterodimer). The orientation of the kinesin head (an anterograde motor) on the microtubule surface is similar to that of ncd (a retrograde motor). A longer kinesin construct (rK379) forms a dimer because of the longer neck helix forming a coiled-coil. Unexpectedly, this construct also decorates the microtubule with a stoichiometry of one head per tubulin subunit, and the orientation is similar to that of the monomeric construct. This means that the interaction with microtubules causes the two heads of a kinesin dimer to separate sufficiently so that they can bind to two different tubulin subunits. This result is in contrast to recent models and can be explained by assuming that the tubulin-kinesin interaction is antagonistic to the coiled-coil interaction within a kinesin dimer.

Role of the kinesin neck region in processive microtubule-based motility.

Kinesin is a dimeric motor protein that can move along a microtubule for several microns without releasing (termed processive movement). The two motor domains of the dimer are thought to move in a coordinated, hand-over-hand manner. A region adjacent to kinesin's motor catalytic domain (the neck) contains a coiled coil that is sufficient for motor dimerization and has been proposed to play an essential role in processive movement. Recent models have suggested that the neck enables head-to-head communication by creating a stiff connection between the two motor domains, but also may unwind during the mechanochemical cycle to allow movement to new tubulin binding sites. To test these ideas, we mutated the neck coiled coil in a 560-amino acid (aa) dimeric kinesin construct fused to green fluorescent protein (GFP), and then assayed processivity using a fluorescence microscope that can visualize single kinesin-GFP molecules moving along a microtubule. Our results show that replacing the kinesin neck coiled coil with a 28-aa residue peptide sequence that forms a highly stable coiled coil does not greatly reduce the processivity of the motor. This result argues against models in which extensive unwinding of the coiled coil is essential for movement. Furthermore, we show that deleting the neck coiled coil decreases processivity 10-fold, but surprisingly does not abolish it. We also demonstrate that processivity is increased by threefold when the neck helix is elongated by seven residues. These results indicate that structural features of the neck coiled coil, although not essential for processivity, can tune the efficiency of single molecule motility.

Octameric stoichiometry of the KATP channel complex.

ATP-sensitive potassium (KATP) channels link cellular metabolism to electrical activity in nerve, muscle, and endocrine tissues. They are formed as a functional complex of two unrelated subunits-a member of the Kir inward rectifier potassium channel family, and a sulfonylurea receptor (SUR), a member of the ATP-binding cassette transporter family, which includes cystic fibrosis transmembrane conductance regulators and multidrug resistance protein, regulators of chloride channel activity. This recent discovery has brought together proteins from two very distinct superfamilies in a novel functional complex. The pancreatic KATP channel is probably formed specifically of Kir6.2 and SUR1 isoforms. The relationship between SUR1 and Kir6.2 must be determined to understand how SUR1 and Kir6.2 interact to form this unique channel. We have used mutant Kir6.2 subunits and dimeric (SUR1-Kir6.2) constructs to examine the functional stoichiometry of the KATP channel. The data indicate that the KATP channel pore is lined by four Kir6.2 subunits, and that each Kir6.2 subunit requires one SUR1 subunit to generate a functional channel in an octameric or tetradimeric structure.

Systemic Infection of Vigna mungo Using the Cloned DNAs of the Blackgram Isolate of Mungbean Yellow Mosaic Geminivirus through Agroinoculation and Transmission of the Progeny Virus by Whiteflies

AbstractAgroinoculation of blackgram (Vigna mungo) by the cloned genomic components (DNA‐A and DNA‐B) of a non‐sap transmissible isolate of mungbean yellow mosaic geminivirus (MYMV) has been demonstrated. It has been shown that MYMV has a bipartite genome. Tandem dimeric constructs of DNA‐A and DNA‐B were infectious when inoculated together and induced systemic yellow mosaic symptoms. Agroinoculation of germinated seeds of blackgram was superior to inoculation of stems of seedlings. Infection of blackgram plants was confirmed by ELISA, dot blot and Southern hybridization. Agroinfected plants contained different forms of DNA characteristic of whitefly transmitted geminiviruses. The virus from agroinfected plants was transmissible by the whitefly Bemisia tabaci.

The Directional Preference of Kinesin Motors Is Specified by an Element outside of the Motor Catalytic Domain

Members of the kinesin superfamily share a similar motor catalytic domain yet move either toward the plus end (e.g., conventional kinesin) or the minus end (e.g., Ncd) of microtubules. The structural features that determine the polarity of movement have remained enigmatic. Here, we show that kinesin's catalytic domain (316 residues) in a dimeric construct (560 residues) can be replaced with the catalytic domain of Ncd and that the resultant motor moves in the kinesin direction. We also demonstrate that this chimera does not move processively over many tubulin subunits, which is similar to Ncd but differs from the highly processive motion of conventional kinesin. These findings reveal that the catalytic domain contributes to motor processivity but does not control the polarity of movement. We propose that a region adjacent to the catalytic domain serves as a mechanical transducer that determines directionality.

Inwardly rectifying potassium channels expressed by gene transfection into the green Monkey kidney cell line COS-1.

1. cDNA encoding a functional inwardly rectifying K+ (IRK1) channel was transfected into COS-1 cells (a Green Monkey kidney cell line) using the liposome method, and voltage clamp experiments were done after 48-72 h. 2. Transfected cells showed inward rectification under whole-cell recording. The unitary current-voltage relationships in the inside-out configuration were almost linear in the absence of internal Mg2+ and polyamines, and the channel conductance averaged 34.1 +/- 2.0 pS (n = 15) at 23-26 degrees C. 3. Internal Mg2+ (2-10 microM) induced sublevels in the outward current with one-third and two-thirds of the unitary amplitude as in native channels. 4. To determine the subunit stoichiometry, we constructed tandem multimeric cDNAs consisting of the coding sequences of the IRK1 gene linked in a head-to-tail fashion. Cells transfected with tandem homomultimers up to octamers showed similar inwardly rectifying K+ channels. 5. A mutation (E138Q) eliminated the ionic conductance of the channel. Channels expressed by dimeric constructs containing a single mutant have a conductance ranging between 5 and 35 pS. 6. The E138Q mutant cotransfected with a wild-type dimeric, trimeric or tetrameric construct did not alter the channel conductance. The results do not support the notion that IRK1 channel proteins consist of four subunits.

Interacting Head Mechanism of Microtubule-Kinesin ATPase

Kinetic and equilibrium properties are compared for a monomeric kinesin construct (K332) and a dimeric construct (K379). MtK379 has a low affinity (5 x 10(4) M(-1)) and a high affinity (5 x 10(6) M(-1)) binding site for mant ADP while MtK332 has a single low affinity site (5 x 10(4) M(-1)). Rate constants of dissociation of mant ADP are <1 s(-1) for the high affinity site and 75-100 s(-1) for the low affinity site for MtK379. For MtK332, the effective rate constant is 200-300 s(-1). It is proposed that the two heads of the dimer are different through the interaction with the microtubule, a strongly bound head with low affinity for 2'-(3')-O-(N-methylanthraniloyl) adenosine 5'-diphosphate (mant ADP), similar to the single strongly bound head of the monomer and a weakly bound or detached head with high affinity for mant ADP. Rate of binding of mant ADP gave an "S"-shaped dependence on concentration for MtK379 and a hyperbolic dependence for MtK332. Binding of K379 x mant ADP dimer to microtubules releases only one mant ADP at a rate of 50 s(-1). The second strongly bound mant ADP is released by binding of nucleotides to the other head. Rates are 100 s(-1) for ATP, 30 s(-1) for AMPPNP or ATPgammaS, and 2 s(-1) for ADP. The rate of binding of mant ATP to MtK379 showed an "S"-shaped concentration dependence and limiting rate at zero concentration is <1 s(-1) while MtK332 gave a hyperbolic dependence and limiting rate of 100 s(-1). The limiting rate is determined by the rate of dissociation of mant ADP in the hydrolysis cycle. The evidence is consistent with an interacting site model in which binding of ATP to one head is required for release of ADP from the other head in the hydrolysis cycle. This model, in which the cycles are maintained partly out of phase, is an extension of the alternating site model of Hackney (Hackney, D. D. (1994) Proc. Nat. Acad. Sci. U.S.A. 91, 6865-6869). It provides a basis for a processive mechanism.

Atomic distance estimates from disulfides and high-affinity metal-binding sites in a K+ channel pore

The pore of potassium channels is lined by four identical, highly conserved hairpin loops, symmetrically arranged around a central permeation pathway. Introduction of cysteines into the external mouth of the drk1 K channel pore resulted in the formation of disulfide bonds that were incompatible with channel function. Breaking these bonds restored function and resulted in a high-affinity Cd(2+)-binding site, indicating coordinated ligation by multiple sulfhydryls. Dimeric constructs showed that these disulfide bonds formed between subunits. These results impose narrow constraints on intersubunit atomic distances in the pore that strongly support a radial pore model. The data also suggest an important functional role for the outer mouth of the pore in gating or permeation.

Structural Studies of Kinesin-Nucleotide Intermediates

We have investigated the structural changes that occur in the molecular motor kinesin during its ATPase cycle, utilizing two bacterially expressed constructs. The structure of both constructs has been examined as a function of the nature of the nucleotide intermediate occupying the active site by means of sedimentation velocity, sedimentation equilibrium, fluorescence solute quenching, fluorescence anisotropy decay, and limited proteolysis. While the molecular weight of monomeric and dimeric human kinesin constructs, as measured by sedimentation velocity and sedimentation equilibrium, and the tryptic cleavage pattern are unaffected by the nucleotide intermediate occupying the active site, significant changes in the rotational correlation time of fluorescently labeled kinesin-nucleotide intermediates can be detected. These results suggest that kinesin contains an internal "hinge" whose flexibility varies through the course of the ATPase cycle. In prehydrolytic, "strong" binding states, this hinge is relatively rigid, while in posthydrolytic, "weak" binding states, it is more flexible. Our results, in conjunction with anisotropy decay studies of myosin, suggest that these two molecular motors may share a common structural feature; viz. weak binding states are characterized by segmental flexibility, which is lost upon assumption of a strong binding conformation.