D D Hackney

Carnegie Mellon University, Pittsburgh, PA, United States

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Publications (48)393.04 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Human kinesin Eg5 is a target for drug development in cancer chemotherapy with compounds in phase II clinical trials. These agents bind to a well-characterized allosteric pocket involving the loop L5 region, a structural element in kinesin-5 family members thought to provide inhibitor specificity. Using X-ray crystallography, kinetic and biophysical methods, we have identified and characterized a distinct and novel allosteric pocket in Eg5 able to bind inhibitors with nanomolar Kd. This new pocket is formed by key structural elements thought to be pivotal for force generation in kinesins and may represent a novel site for therapeutic intervention in this increasingly well-validated drug target.
    Journal of the American Chemical Society 01/2013; · 10.68 Impact Factor
  • David D Hackney
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    ABSTRACT: It is often possible to obtain a detailed understanding of the forward steps in ATP hydrolysis because they are thermodynamically favored and usually occur rapidly. However, it is difficult to obtain the reverse rates for ATP resynthesis because they are thermodynamically disfavored and little of their product, ATP, accumulates. Isotopic exchange reactions provide access to these reverse reactions because isotopic changes accumulate over time due to multiple reversals of hydrolysis, even in the absence of net resynthesis of significant amounts of ATP. Knowledge of both the forward and reverse rates allows calculation of the free energy changes at each step and how it changes when coupled to an energy-requiring conformational step such as unwinding of an RNA helix. This chapter describes the principal types of oxygen isotopic exchange reactions that are applicable to ATPases, in general, and helicases, in particular, their application and their interpretation.
    Methods in enzymology 01/2012; 511:65-74. · 1.90 Impact Factor
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    ABSTRACT: When not transporting cargo, kinesin-1 is autoinhibited by binding of a tail region to the motor domains, but the mechanism of inhibition is unclear. We report the crystal structure of a motor domain dimer in complex with its tail domain at 2.2 angstroms and compare it with a structure of the motor domain alone at 2.7 angstroms. These structures indicate that neither an induced conformational change nor steric blocking is the cause of inhibition. Instead, the tail cross-links the motor domains at a second position, in addition to the coiled coil. This "double lockdown," by cross-linking at two positions, prevents the movement of the motor domains that is needed to undock the neck linker and release adenosine diphosphate. This autoinhibition mechanism could extend to some other kinesins.
    Science 08/2011; 333(6044):883-5. · 31.20 Impact Factor
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    ABSTRACT: Mss116 is a Saccharomyces cerevisiae mitochondrial DEAD-box RNA helicase protein that is essential for efficient in vivo splicing of all group I and group II introns and for activation of mRNA translation. Catalysis of intron splicing by Mss116 is coupled to its ATPase activity. Knowledge of the kinetic pathway(s) and biochemical intermediates populated during RNA-stimulated Mss116 ATPase is fundamental for defining how Mss116 ATP utilization is linked to in vivo function. We therefore measured the rate and equilibrium constants underlying Mss116 ATP utilization and nucleotide-linked RNA binding. RNA accelerates the Mss116 steady-state ATPase ∼7-fold by promoting rate-limiting ATP hydrolysis such that inorganic phosphate (P(i)) release becomes (partially) rate-limiting. RNA binding displays strong thermodynamic coupling to the chemical states of the Mss116-bound nucleotide such that Mss116 with bound ADP-P(i) binds RNA more strongly than Mss116 with bound ADP or in the absence of nucleotide. The predominant biochemical intermediate populated during in vivo steady-state cycling is the strong RNA-binding Mss116-ADP-P(i) state. Strong RNA binding allows Mss116 to fulfill its biological role in the stabilization of group II intron folding intermediates. ATPase cycling allows for transient population of the weak RNA-binding ADP state of Mss116 and linked dissociation from RNA, which is required for the final stages of intron folding. In cases where Mss116 functions as a helicase, the data collectively favor a model in which ATP hydrolysis promotes a weak-to-strong RNA binding transition that disrupts stable RNA duplexes. The subsequent strong-to-weak RNA binding transition associated with P(i) release dissociates Mss116-RNA complexes, regenerating free Mss116.
    Journal of Molecular Biology 06/2011; 409(3):399-414. · 3.91 Impact Factor
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    David D Hackney
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    ABSTRACT: A simple ADP biosensor would be of broad usefulness in monitoring the large number of metabolic processes that produce ADP. Several new systems have been recently described including one in the current issue of ACS Chemical Biology that provides a simple readout of the ADP concentration without significant interference by ATP.
    ACS Chemical Biology 04/2010; 5(4):353-4. · 5.44 Impact Factor
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    ABSTRACT: DEAD-box RNA helicase proteins use the energy of ATP hydrolysis to drive the unwinding of duplex RNA. However, the mechanism that couples ATP utilization to duplex RNA unwinding is unknown. We measured ATP utilization and duplex RNA unwinding by DbpA, a non-processive bacterial DEAD-box RNA helicase specifically activated by the peptidyl transferase center (PTC) of 23S rRNA. Consumption of a single ATP molecule is sufficient to unwind and displace an 8 base pair rRNA strand annealed to a 32 base pair PTC-RNA "mother strand" fragment. Strand displacement occurs after ATP binding and hydrolysis but before P(i) product release. P(i) release weakens binding to rRNA, thereby facilitating the release of the unwound rRNA mother strand and the recycling of DbpA for additional rounds of unwinding. This work explains how ATPase activity of DEAD-box helicases is linked to RNA unwinding.
    Proceedings of the National Academy of Sciences 02/2010; 107(9):4046-50. · 9.81 Impact Factor
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    ABSTRACT: Human kinesin Eg5 plays an essential role in mitosis by separating duplicated centrosomes and establishing the bipolar spindle. Eg5 is an interesting drug target for the development of cancer chemotherapy, with seven inhibitors already in clinical trials. In the present paper, we report the crystal structure of the Eg5 motor domain complexed with a potent antimitotic inhibitor STLC (S-trityl-L-cysteine) to 2.0 A (1 A=0.1 nm) resolution. The Eg5-STLC complex crystallizes in space group P3(2) with three molecules per asymmetric unit. Two of the molecules reveal the final inhibitor-bound state of Eg5, whereby loop L5 has swung downwards to close the inhibitor-binding pocket, helix alpha4 has rotated by approx. 15 degrees and the neck-linker has adopted a docked conformation. The third molecule, however, revealed an unprecedented intermediate state, whereby local changes at the inhibitor-binding pocket have not propagated to structural changes at the switch II cluster and neck-linker. This provides structural evidence for the sequence of drug-induced conformational changes.
    Biochemical Journal 09/2009; 425(1):55-60. · 4.65 Impact Factor
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    David D Hackney, Nahyeon Baek, Avin C Snyder
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    ABSTRACT: The two heavy chains of kinesin-1 are dimerized through extensive coiled coil regions and fold into an inactive conformation through interaction of the C-terminal tail domains with the N-terminal motor (head) domains. Although this potentially allows a dimer of tail domains to interact symmetrically with a dimer of head domains, we report here that only one of the two available monomeric tail peptides is sufficient for tight binding and inhibition of a dimer of head domains. With a dimeric tail construct, the other tail peptide does not make tight contact with the head dimer and can bind a second head dimer to form a complex containing one tail dimer and two head dimers. The IAK domain and neighboring positively charged region of the tail is sufficient for tight half-site interaction with a dimer of heads. The interaction of tails with monomeric heads is weak, but a head dimer produced by the dimerization of the neck coil is not required because an artificial dimer of head domains also binds monomeric tail peptides with half-site stoichiometry in the complete absence of the native neck coil. The binding of tail peptides to head dimers is fast and readily reversible as determined by FRET between mant-ADP bound to the head dimer and a tail labeled with GFP. The association and dissociation rates are 81 microM(-1) s(-1) and 32 s(-1), respectively. This half-site interaction suggests that the second tail peptide in a folded kinesin-1 might be available to bind other molecules while kinesin-1 remained folded.
    Biochemistry 04/2009; 48(15):3448-56. · 3.38 Impact Factor
  • David D Hackney, Maryanne F Stock
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    ABSTRACT: Kinesin-1 is a vesicle motor that can fold into a compact inhibited conformation that is produced by interaction of the heavy chain C-terminal tail region with the N-terminal motor domains (heads). Binding of the tail domains to the heads inhibits net microtubule-stimulated ATPase activity by blocking the ability of the heads to bind to microtubules with coupled acceleration of ADP release. We now show that folding of kinesin-1 also directly inhibits ADP release even in the absence of microtubules. With long heavy chain constructs such as DKH960 that exhibit a high degree of regulation by folding, the basal rate of ADP release is inhibited up to 30-fold compared to that of truncated DKH894 which has lost the inhibitory tail domains and does not fold. Inhibition of ADP release is also observed when separate head and tail domain constructs are mixed at low salt concentrations. This inhibition of ADP release by tail domains is formally analogous to the action of nucleotide dissociation inhibitors (NDI or GDI) for regulatory GTPases. In contrast to their inhibition of ADP release, tail domains accelerate the rate of ADP binding to nucleotide-free kinesin-1. Inhibition of release of ADP by tail domains is reversed by Unc-76 (FEZ1) which is a potential regulator of kinesin-1. Tail domains only weakly inhibit the initial slow release of Mg (2+) from the kinesin-MgADP complex but strongly inhibit the fast release of Mg (2+)-free ADP.
    Biochemistry 08/2008; 47(29):7770-8. · 3.38 Impact Factor
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    ABSTRACT: DEAD-box proteins are ATPase enzymes that destabilize and unwind duplex RNA. Quantitative knowledge of the ATPase cycle parameters is critical for developing models of helicase activity. However, limited information regarding the rate and equilibrium constants defining the ATPase cycle of RNA helicases is available, including the distribution of populated biochemical intermediates, the catalytic step(s) that limits the enzymatic reaction cycle, and how ATP utilization and RNA interactions are linked. We present a quantitative kinetic and equilibrium characterization of the ribosomal RNA (rRNA)-activated ATPase cycle mechanism of DbpA, a DEAD-box rRNA helicase implicated in ribosome biogenesis. rRNA activates the ATPase activity of DbpA by promoting a conformational change after ATP binding that is associated with hydrolysis. Chemical cleavage of bound ATP is reversible and occurs via a gamma-phosphate attack mechanism. ADP-P(i) and RNA binding display strong thermodynamic coupling, which causes DbpA-ADP-P(i) to bind rRNA with >10-fold higher affinity than with bound ATP, ADP or in the absence of nucleotide. The rRNA-activated steady-state ATPase cycle of DbpA is limited both by ATP hydrolysis and by P(i) release, which occur with comparable rates. Consequently, the predominantly populated biochemical states during steady-state cycling are the ATP- and ADP-P(i)-bound intermediates. Thermodynamic linkage analysis of the ATPase cycle transitions favors a model in which rRNA duplex destabilization is linked to strong rRNA and nucleotide binding. The presented analysis of the DbpA ATPase cycle reaction mechanism provides a rigorous kinetic and thermodynamic foundation for developing testable hypotheses regarding the functions and molecular mechanisms of DEAD-box helicases.
    Journal of Molecular Biology 03/2008; 377(1):193-205. · 3.91 Impact Factor
  • Maryanne F. Stock, David D. Hackney
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    ABSTRACT: Kinesin can generally be expressed to high levels in Escherichia coli and this provides an excellent means for obtaining significant amounts of purified constructs. For detailed work, it is essential to have a native construct uncompromised by extraneous tags, and this can readily be obtained. See refs. 1–3 for examples of vector construction using the T7 RNA polymerase system.
    02/2008: pages 43-48;
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    David D. Hackney, Wei Jiang
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    ABSTRACT: Methods for assay of ATPase rates can be classified into broad categories depending on whether an ATP-regenerating system or a continuous coupled assay is employed versus quenching at discrete times for subsequent analysis for production of Pi or ADP (or, more rarely, for the loss of ATP).
    02/2008: pages 65-71;
  • David D Hackney
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    ABSTRACT: A mechanism that controls the stepwise movement of a molecular motor along a filamentous microtubule track in the cell does not seem to require the track itself.
    Science 05/2007; 316(5821):58-9. · 31.20 Impact Factor
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    David D Hackney
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    ABSTRACT: When it is not actively transporting cargo, conventional Kinesin-1 is present in the cytoplasm in a folded conformation that cannot interact effectively with microtubules (MTs). Two important and largely unexplored aspects of kinesin regulation are how it is converted to an active species when bound to cargo and the related issue of how kinesin discriminates among its many potential cargo molecules. Blasius et al. (see p. 11 of this issue) report that either binding of the cargo linker c-Jun N-terminal kinase-interacting protein 1 (JIP1) to the light chains (LCs) or binding of fasciculation and elongation protein zeta1 (FEZ1) to the heavy chains (HCs) is insufficient for activation but that activation occurs when both are present simultaneously. A related paper by Cai et al. (see p. 51 of this issue) provides structural insight into the conformation of the folded state in the cell obtained by fluorescence resonance energy transfer analysis.
    The Journal of Cell Biology 02/2007; 176(1):7-9. · 10.82 Impact Factor
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    ABSTRACT: Calcium activates full-length myosin Va steady-state enzymatic activity and favors the transition from a compact, folded "off" state to an extended "on" state. However, little is known of how a head-tail interaction alters the individual actin and nucleotide binding rate and equilibrium constants of the ATPase cycle. We measured the effect of calcium on nucleotide and actin filament binding to full-length myosin Va purified from chick brains. Both heads of nucleotide-free myosin Va bind actin strongly, independent of calcium. In the absence of calcium, bound ADP weakens the affinity of one head for actin filaments at equilibrium and upon initial encounter. The addition of calcium allows both heads of myosin Va.ADP to bind actin strongly. Calcium accelerates ADP binding to actomyosin independent of the tail but minimally affects ATP binding. Although 18O exchange and product release measurements favor a mechanism in which actin-activated Pi release from myosin Va is very rapid, independent of calcium and the tail domain, both heads do not bind actin strongly during steady-state cycling, as assayed by pyrene actin fluorescence. In the absence of calcium, inclusion of ADP favors formation of a long lived myosin Va.ADP state that releases ADP slowly, even after mixing with actin. Our results suggest that calcium activates myosin Va by allowing both heads to interact with actin and exchange bound nucleotide and indicate that regulation of actin binding by the tail is a nucleotide-dependent process favored by linked conformational changes of the motor domain.
    Journal of Biological Chemistry 11/2006; 281(42):31326-36. · 4.65 Impact Factor
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    David D Hackney
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    ABSTRACT: Although the steps for the forward reaction of ATP hydrolysis by the motor protein kinesin have been studied extensively, the rates for the reverse reactions and thus the energy changes at each step are not as well defined. Oxygen isotopic exchange between water and P(i) was used to evaluate the reverse rates. The fraction of the kinesin x ADP x P(i) complex that reverts to ATP before release of P(i) during net hydrolysis was approximately 0 and approximately 2.6% in the absence and presence of microtubules (MTs), respectively. The rate of synthesis of bound ATP from free P(i) and the MT x kinesin x ADP complex was approximately 1.7 M(-1) x s(-1) (K0.5 ADP = 70 microM) with monomeric kinesin in the absence of net hydrolysis. Synthesis of bound ATP from the ADP of the tethered head of a dimer-MT complex was 20-fold faster than for the monomer-MT complex. This MT-activated ATP synthesis at the tethered head is in marked contrast to the lack of MT stimulation of ADP release from the same site. The more rapid ATP synthesis with dimers suggests that the tethered head binds behind the strongly attached head, because this positions the neck linker of the tethered head toward the plus end of the MT and would thus facilitate its docking on synthesis of ATP. The observed rate of ATP synthesis also puts limits on the overall energetics that suggest that a significant fraction of the free energy of ATP hydrolysis is available to drive the docking of the neck linker on binding of ATP.
    Proceedings of the National Academy of Sciences 01/2006; 102(51):18338-43. · 9.81 Impact Factor
  • Heidi Browning, David D Hackney
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    ABSTRACT: Tea2 is a kinesin family member from Schizosaccharomyces pombe that is targeted to microtubule tips and cell ends in a process that depends on Mal3. Constructs of Tea2 containing the motor domain only or the motor domain plus the N-terminal extension are monomeric, whereas a construct including the first predicted coiled coil region is dimeric. These constructs have a low basal rate of ATP hydrolysis of <0.1 s(-1), but microtubules stimulate the rate of ATP hydrolysis to a maximum of approximately 15 s(-1). Hydrodynamic analysis of Mal3 indicates that it is dimeric. Mal3 is known to associate with Tea2, and analysis with the above Tea2 constructs indicates that the principal site of interaction of Mal3 with Tea2 is the N-terminal extension, although a weaker interaction is also observed with the motor domain alone. In parallel to the binding studies, Mal3 strongly stimulates the ATPase of constructs containing the N-terminal extension by decreasing the K0.5(MT) for stimulation by microtubules but only weakly stimulates motor domains without the N-terminal extension. Mal3 reduces the K0.5(MT) values without affecting the k(cat) value at saturating microtubule level. Binding of Mal3 to microtubules induces an increase in the binding of Tea2 and a reciprocal stimulation of Mal3 binding by Tea2 is also observed. Tea2 is a plus end directed motor that drives sliding of axonemes when adsorbed to a glass surface. The sliding rate is initially unaffected by Mal3, but axonemes stop moving on continued exposure to Mal3.
    Journal of Biological Chemistry 05/2005; 280(13):12299-304. · 4.65 Impact Factor
  • Maryanne F Stock, Jessica Chu, David D Hackney
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    ABSTRACT: The kinesin family member BimC has a highly positively charged domain of approximately 70 amino acids at the N terminus of the motor domain. Motor domain constructs of BimC were prepared with and without this extra domain to determine its influence. The level of microtubules needed for half saturation of the ATPase of BimC motor domain constructs is reduced by approximately 7000-fold at low ionic strength upon addition of this extra N-terminal extension. Although the change in microtubule affinity is less at higher salt, addition of the N-terminal domain still produces a 20-fold increase in affinity for microtubules in 200 mm potassium acetate. A fusion protein of the N-terminal domain and thioredoxin binds tightly to MTs at low salt, consistent with the increased affinity of motor domain constructs (which contain the N-terminal domain) being due to the additional binding of the N-terminal domain to the microtubule. Hydrodynamic analysis indicates that the N-terminal extension is in a highly extended conformation, suggesting that it may be intrinsically disordered. Fusion of the N-terminal extension of BimC onto the motor domain of conventional kinesin produces a similar large increase in microtubule affinity without significant reduction in kcat or velocity in an in vitro motility assay, suggesting that the N-terminal extension can act in a modular manner to increase the microtubule affinity of kinesin motor domains without a decrease in velocity.
    Journal of Biological Chemistry 01/2004; 278(52):52315-22. · 4.65 Impact Factor
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    ABSTRACT: A series of modifications of the junction of the neck linker and neck coil of dimeric Drosophila kinesin were constructed to determine the influence of head orientation and spacing on the ATPase kinetics. Ala(345) is the first residue in the coiled-coil of the neck, and its replacement with glycine or proline produces no significant change in the k(cat) or K(0.5(MT)) values for activation of their ATPase by microtubules (MTs) or in their k(bi(ratio)) value for the average number of ATP molecules hydrolyzed during a processive encounter with a MT. Addition or deletion of a single amino acid at the junction produces only modest changes with less than a 2-fold reduction in kinetic processivity. Insertion of a spacer of 6 or 12 additional amino acids at the neck linker junction increases the K(0.5(MT)) value by 3-4-fold with a corresponding decrease in kinetic processivity. The sliding velocities of all the mutant constructs under multimotor conditions are within 30% of the wild-type value. All the constructs with single residue changes exhibit half-site ADP release on binding to MTs. The constructs with long insertion, however, rapidly release both ADP molecules per dimer on binding to a MT, indicating that the steric constraints that prevent release of ADP from the tethered head of wild-type kinesin have been relieved by the long insertions. The constructs with long inserts have decreased kinetic processivity and dissociate from the MT during ATP hydrolysis 3-fold faster than wild-type.
    Biochemistry 11/2003; 42(41):12011-8. · 3.38 Impact Factor
  • Heidi Browning, David D Hackney, Paul Nurse
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    ABSTRACT: Kinesins are microtubule-based motor proteins that transport cargo to specific locations within the cell. However, the mechanisms by which cargoes are directed to specific cellular locations have remained elusive. Here, we investigated the in vivo movement of the Schizosaccharomyces pombe kinesin Tea2 to establish how it is targeted to microtubule tips and cell ends. Tea2 is loaded onto microtubules in the middle of the cell, in close proximity to the nucleus, and then travels using its intrinsic motor activity primarily at the tips of polymerizing microtubules. The microtubule-associated protein Mal3, an EB1 homologue, is required for loading and/or processivity of Tea2 and this function can be substituted by human EB1. In addition, the cell-end marker Tea1 is required to anchor Tea2 to cell ends. Movement of Tea1 and the CLIP170 homologue Tip1 to cell ends is abolished in Tea2 rigor (ATPase) mutants. We propose that microtubule-based transport from the vicinity of the nucleus to cell ends can be precisely regulated, with Mal3 required for loading/processivity, Tea2 for movement and Tea1 for cell-end anchoring.
    Nature Cell Biology 10/2003; 5(9):812-8. · 20.76 Impact Factor

Publication Stats

2k Citations
393.04 Total Impact Points

Institutions

  • 1988–2012
    • Carnegie Mellon University
      • Department of Biological Sciences
      Pittsburgh, PA, United States
  • 2009–2011
    • Beatson Institute for Cancer Research
      Glasgow, Scotland, United Kingdom
  • 2006–2011
    • Yale University
      • Department of Molecular Biophysics and Biochemistry
      New Haven, CT, United States