Enrique De La Cruz - Academia.edu (original) (raw)

Papers by Enrique De La Cruz

PloS one, 2016

Neuronal calcium sensor-1 (NCS-1 Var1) is a calcium-binding protein expressed in most tissues. We... more Neuronal calcium sensor-1 (NCS-1 Var1) is a calcium-binding protein expressed in most tissues. We examined a poorly characterized variant of NCS-1 (Var2), identified only in humans where the N-terminal 22 amino acid residues of native NCS-1(MGKSNSKLKPEVVEELTRKTY) were replaced with 4 different residues (MATI). Because alterations in the level of expression of NCS-1 Var1 and the expression of NCS-1 variants have been correlated with several neurological diseases, the relative expression and functional role of NCS-1 Var2 was examined. We found that NCS-1 Var2 mRNA levels are not found in mouse tissues and are expressed at levels ~1000-fold lower than NCS-1 Var1 in three different human cell lines (SHSY5Y, HEK293, MB231). Protein expression of both variants was only identified in cell lines overexpressing exogenous NCS-1 Var2. The calcium binding affinity is ~100 times weaker in purified NCS-1 Var2 than NCS-1 Var1. Because truncation of NCS-1 Var1 has been linked to functional changes ...

Current Biology, 2016

Highlights d We generated actin architectures that span the diversity of contractile structures d... more Highlights d We generated actin architectures that span the diversity of contractile structures d These different actin organizations respond differently to myosin-induced contraction d Actin filament organization and connectivity determine the contractile response d Network contraction is dominated by either sarcomeric-like or buckling mechanisms

Nature Communications, 2015

Diseases of ectopic calcification of the vascular wall range from lethal orphan diseases such as ... more Diseases of ectopic calcification of the vascular wall range from lethal orphan diseases such as generalized arterial calcification of infancy (GACI), to common diseases such as hardening of the arteries associated with aging and calciphylaxis of chronic kidney disease (CKD). GACI is a lethal orphan disease in which infants calcify the internal elastic lamina of their medium and large arteries and expire of cardiac failure as neonates, while calciphylaxis of CKD is a ubiquitous vascular calcification in patients with renal failure. Both disorders are characterized by vascular Mönckeburg’s sclerosis accompanied by decreased concentrations of plasma inorganic pyrophosphate (PPi). Here we demonstrate that subcutaneous administration of an ENPP1-Fc fusion protein prevents the mortality, vascular calcifications and sequela of disease in animal models of GACI, and is accompanied by a complete clinical and biomarker response. Our findings have implications for the treatment of rare and com...

Biophysical Journal, 2015

HFSP Journal, 2009

Molecular motors are cellular nanomachines that convert the energy from nucleotide binding, hydro... more Molecular motors are cellular nanomachines that convert the energy from nucleotide binding, hydrolysis, and product release into mechanical work. Because molecular motors contribute to fundamental processes in all living organisms, including genome replication, gene transcription, protein synthesis, organelle transport, and cell division, understanding how the chemical "ATP utilization… and mechanical "motility… cycles are linked is of fundamental importance. A recent study reports the direct visualization of simultaneous nucleotide binding and mechanical displacement of a single myosin 5a molecule, a processive molecular motor protein that takes successive È36-nm steps along actin filaments of the cytoskeleton. This new work demonstrates an exciting advance in single-molecule enzymology and advances our understanding of the link between chemical catalysis and mechanical work in molecular motors, particularly those that operate under internal and external loads.

The EMBO journal, Jan 9, 2008

c-myc is essential for cell homeostasis and growth but lethal if improperly regulated. Transcript... more c-myc is essential for cell homeostasis and growth but lethal if improperly regulated. Transcription of this oncogene is governed by the counterbalancing forces of two proteins on TFIIH--the FUSE binding protein (FBP) and the FBP-interacting repressor (FIR). FBP and FIR recognize single-stranded DNA upstream of the P1 promoter, known as FUSE, and influence transcription by oppositely regulating TFIIH at the promoter site. Size exclusion chromatography coupled with light scattering reveals that an FIR dimer binds one molecule of single-stranded DNA. The crystal structure confirms that FIR binds FUSE as a dimer, and only the N-terminal RRM domain participates in nucleic acid recognition. Site-directed mutations of conserved residues in the first RRM domain reduce FIR's affinity for FUSE, while analogous mutations in the second RRM domain either destabilize the protein or have no effect on DNA binding. Oppositely oriented DNA on parallel binding sites of the FIR dimer results in sp...

Scientific Reports, 2013

Enzyme inhibition due to the reversible binding of reaction products is common and underlies the ... more Enzyme inhibition due to the reversible binding of reaction products is common and underlies the origins of negative feedback inhibition in many metabolic and signaling pathways. Product inhibition generates non-linearity in steady-state time courses of enzyme activity, which limits the utility of well-established enzymology approaches developed under the assumption of irreversible product release. For more than a century, numerous attempts to find a mathematical solution for analysis of kinetic time courses with product inhibition have been put forth. However, no practical general method capable of extracting common enzymatic parameters from such non-linear time courses has been successfully developed. Here we present a simple and practical method of analysis capable of efficiently extracting steady-state enzyme kinetic parameters and product binding constants from non-linear kinetic time courses with product inhibition and/or substrate depletion. The method is general and applicable to all enzyme systems, independent of reaction schemes and pathways.

Science, 2012

Actin Up Actomyosin interactions lie at the heart of fundamental cellular processes—including mor... more Actin Up Actomyosin interactions lie at the heart of fundamental cellular processes—including morphogenesis, establishment of polarity, and overall motility—but the general principles driving the spatiotempotal orchestration of these interactions have remained elusive. Working in vitro, using micropatterned substrates, Reymann et al. (p. 1310 ) demonstrate that myosins can use a “selection orientation” mechanism to pull selectively on actin filaments, contract the actin network and disassemble it, or walk on the filaments, align them, allow their growth, and control filament orientation.

Proceedings of the National Academy of Sciences, 2010

DEAD-box RNA helicase proteins use the energy of ATP hydrolysis to drive the unwinding of duplex ... more 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, 2008

Dimeric myosins V and VI travel long distances in opposite directions along actin filaments in ce... more Dimeric myosins V and VI travel long distances in opposite directions along actin filaments in cells, taking multiple steps in a “hand-over-hand” fashion. The catalytic cycles of both myosins are limited by ADP dissociation, which is considered a key step in the walking mechanism of these motors. Here, we demonstrate that external loads applied to individual actomyosin V or VI bonds asymmetrically affect ADP affinity, such that ADP binds weaker under loads assisting motility. Model-based analysis reveals that forward and backward loads modulate the kinetics of ADP binding to both myosins, although the effect is less pronounced for myosin VI. ADP dissociation is modestly accelerated by forward loads and inhibited by backward loads. Loads applied in either direction slow ADP binding to myosin V but accelerate binding to myosin VI. We calculate that the intramolecular load generated during processive stepping is ≈2 pN for both myosin V and myosin VI. The distinct load dependence of ADP...

Nucleic Acids Research, 2007

We have used 2-aminopurine (2AP) as a fluorescent probe in the template strand of a 13/20mer prim... more We have used 2-aminopurine (2AP) as a fluorescent probe in the template strand of a 13/20mer primer/ template (D) to detect deoxynucleoside triphosphates (N)-dependent conformational changes exhibited by RB69 DNA polymerase (ED) complexes. The rates and amplitudes of fluorescence quenching depend hyperbolically on the [dTTP] when a dideoxy-primer/template (ddP/T) with 2AP as the templating base (n position) is used. No detectable fluorescence changes occur when a ddP/T with 2AP positioned 5' to the templating base (n + 1 position) is used. With a deoxy-primer/template (dP/T) with 2AP in the n position, a rapid fluorescence quenching occurs within 2 ms, followed by a second, slower fluorescence quenching with a rate constant similar to base incorporation as determined by chemical quench. With a dP/T having 2AP in the n + 1 position, there is a [dNTP]-dependent fluorescence enhancement that occurs at a rate comparable to dNMP incorporation. Collectively, the results favor a minimal kinetic scheme in which population of two distinct biochemical states of the ternary EDN complex precedes the nucleotidyl transfer reaction. Observed differences between dP/T and ddP/T ternary complexes indicate that the 3' hydroxyl group of the primer plays a critical role in determining the rate constants of transitions that lead to strong deoxynucleoside triphosphate binding prior to chemistry.

Nature Structural & Molecular Biology, 2004

Myosin V belongs to the myosin superfamily of actin-based molecular motors and is involved in the... more Myosin V belongs to the myosin superfamily of actin-based molecular motors and is involved in the intracellular transport of organelles 1-4. Myosin V consists of two identical heavy chains, each composed of an N-terminal motor domain ('head'), a domain comprising six IQ motifs that bind light chains ('neck'), a coiled coil dimerization domain and a globular cargo-binding tail domain 1,3. Myosin V is a processive motor that 'walks' along an actin filament toward the barbed end over a long distance without dissociating from the filament 5,6. Electron microscopy of actomyosin V in the presence of low ATP concentrations shows both motor domains of myosin V bound to the actin filament at sites spaced 36 nm apart, which corresponds to the half pitch of the filament long-pitch helix 7. Experiments using optical tweezers identified processive 36-nm steps of a bead, on which single myosin V molecules were adsorbed 6,8. Moreover, it was shown that myosin V walks as a left-handed spiral motor along an actin filament, because the average step size is slightly shorter than the half pitch of the long-pitch actin helix 9. The handover hand walking model has received strong support from two recent experiments that (i) observed the orientation of the neck domain of myosin V by monitoring the polarization of a single fluorophore covalently attached to a light chain 10 and (ii) measured the stepwise displacement of a single fluorophore labeled at one of six light chains of myosin V 11. Solution kinetic studies demonstrate that ADP release occurs at ∼15 s-1 and limits the myosin V ATPase cycle 12. Microscopic analysis of myosin V stepping under various nucleotide conditions is consistent with rate-limiting ADP release 13. The next key target is to determine how the mechanical and biochemical cycles are coupled to each other at the single-molecule level. Here, we focused on mechanical events and detected substeps that occur within each regular 36-nm step with high temporal resolution. Each regular 36-nm step is composed of two consecutive substeps, one generating a 12-nm substep and the other a 24-nm substep. To investigate how these substeps and the states attained after the steps are coupled to the ATPase cycle of myosin V, we examined the effects of ATP and ADP concentrations, and 2,3-butanedione 2-monoxime (BDM) 14. We also examined the force dependence of the occurrence frequency of each step and substep, and the dwell time of each state. RESULTS Movement of myosin V along an actin filament A single myosin V-coated bead was trapped with optical tweezers and brought into contact with a fluorescently labeled biotinylated actin filament, which was immobilized on an avidin-coated glass surface through biotinylated BSA (Fig. 1a). A focused red light (685 nm) laser was used to diagonally illuminate the bead, and its dark-field image was projected onto a quadrant photodiode. The bead displacement was determined by measuring the differential output of the quadrant photodiode with nanometer accuracy and a 10-kHz sampling rate 15. The use of a 200-nm-diameter bead here instead of a 1-µm bead was essential to obtain a high spatiotemporal resolution. An example of the time course of bead displacement along an actin filament (Fig. 1b) shows three consecutive runs of a single myosin V molecule along an actin filament at a saturating ATP concentration (1 mM). As the bead began to deviate from the trap center, a positive external load was applied to the myosin V-actin complex (toward the pointed end of an actin filament). Myosin V detached from actin at a stall force of ∼3 pN. After detachment, the bead quickly returned to the

Nature Chemical Biology, 2010

The dimeric motor myosin V transports organelles along actin filament tracks over long distances ... more The dimeric motor myosin V transports organelles along actin filament tracks over long distances in cells. Myosin V is a smart 'walker' that is able to swiftly adjust to variable 'road conditions' to continue its processive movement across dense cellular environments. Coordination between the two heads via intramolecular load modulates biochemical kinetics and ensures highly efficient unidirectional motion. However, little is known about how load-induced regulation of the processive stepping occurs in vivo, where myosin V experiences significant off-axis loads applied in various directions. To reveal how myosin V remains processive in cells, we measured the effect of the off-axis loads, applied to individual actomyosin V bonds in a range of angles, on the coordination between the two heads and myosin V processive stepping. We found that myosin V remains highly processive under diagonal loads owing to asymmetrical ADP affinities and that the native 6IQ lever optimizes the subunit coordination, which indicates that myosin V is designed to be an intracellular transporter. Myosin V transports cargos toward the barbed end of actin filaments in cells 1,2 , taking multiple ~36-nm steps by alternately swinging forward two lever arms in a 'hand-over-hand' fashion 3-7. Single-molecule experiments reveal that one ATP molecule is consumed for each step, which confirms the tight coupling between mechanical and enzymatic events 8. Combined with the unidirectionality of the processive movement, this mechanism implies that the ATPase cycles in two catalytic domains are precisely coordinated. Directional loads modulate ATPase kinetics in myosin V 9-11 , which suggests that two subunits communicate via the intramolecular load, which is generated during binding to actin with both heads, to achieve the coordinated mechanical performance.

Journal of Molecular Biology, 2006

We have evaluated the thermodynamic parameters associated with cooperative cofilin binding to act... more We have evaluated the thermodynamic parameters associated with cooperative cofilin binding to actin filaments, accounting for contributions of ionlinked equilibria, and determined the kinetic basis of cooperative cofilin binding. Ions weaken non-contiguous (isolated, non-cooperative) cofilin binding to an actin filament without affecting cooperative filament interactions. Non-contiguous cofilin binding is coupled to the dissociation of ∼1.7 thermodynamically bound counterions. Counterion dissociation contributes ∼40% of the total cofilin binding free energy (in the presence of 50 mM KCl). The non-contiguous and cooperative binding free energies are driven entirely by large, positive entropy changes, consistent with a cofilin-mediated increase in actin filament structural dynamics. The rate constant for cofilin binding to an isolated site on an actin filament is slow and likely to be limited by filament breathing. Cooperative cofilin binding arises from an approximately tenfold more rapid association rate constant and an approximately twofold slower dissociation rate constant. The more rapid association rate constant is presumably a consequence of cofilin-dependent changes in the average orientation of subdomain 2, subunit angular disorder and filament twist, which increase the accessibility of a neighboring cofilin-binding site on an actin filament. Cooperative association is more rapid than binding to an isolated site, but still slow for a second-order reaction, suggesting that cooperative binding is limited also by binding site accessibility. We suggest that the dissociation of actin-associated ions weakens intersubunit interactions in the actin filament lattice that enhance cofilin-binding site accessibility, favor cooperative binding and promote filament severing.

Journal of Molecular Biology, 2011

Mss116 is a Saccharomyces cerevisiae mitochondrial DEAD-box RNA helicase protein essential for ef... more Mss116 is a Saccharomyces cerevisiae mitochondrial DEAD-box RNA helicase protein essential for efficient in vivo splicing of all group I and II introns and 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 nucleotidelinked RNA binding. RNA accelerates the Mss116 steady-state ATPase ~7-fold by promoting rate-limiting ATP hydrolysis, such that 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 with bound ADP or in the absence of nucleotide. The predominant biochemical intermediate populated during in vivo steadystate cycling is the strong RNA binding, Mss116-ADP-P i state. Strong RNA binding allows Mss116 to fulfill its biological role in 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 RNA-Mss116 complexes, regenerating free Mss116.

Journal of Molecular Biology, 2011

The contractile and enzymatic activities of myosin VI are regulated by calcium binding to associa... more The contractile and enzymatic activities of myosin VI are regulated by calcium binding to associated calmodulin light chains. We have used transient phosphorescence anisotropy (TPA) to monitor the microsecond rotational dynamics of erythrosin iodoacetamide-labeled actin with strongly-bound myosin VI (MVI) and to evaluate the effect of MVI-bound calmodulin (CaM) light chain on actin filament dynamics. MVI binding lowers the amplitude but accelerates actin filament microsecond dynamics in a Ca 2+-and CaM-dependent manner, as indicated from an increase in the final anisotropy and a decrease in the correlation time of TPA decays. MVI with bound apo-CaM or Ca 2+-CaM weakly affects actin filament microsecond dynamics, relative to other myosins (e.g. muscle myosin II and myosin Va). CaM dissociation from bound MVI damps filament rotational dynamics (i.e. increases the torsional rigidity), such that the perturbation is comparable to that induced by other characterized myosins. Analysis of individual actin filament shape fluctuations imaged by fluorescence microscopy reveals a correlated effect on filament bending mechanics. These data support a model in which Ca 2+-dependent CaM binding to the IQ domain of MVI is linked to an allosteric reorganization of the actin-binding site(s), which alters the structural dynamics and the mechanical rigidity of actin filaments. Such modulation of filament dynamics may contribute to the Ca 2+-and CaM-dependent regulation of myosin VI motility and ATP utilization.

Journal of Molecular Biology, 2010

We have used transient phosphorescence anisotropy (TPA) to detect the microsecond rotational dyna... more We have used transient phosphorescence anisotropy (TPA) to detect the microsecond rotational dynamics of erythrosin iodoacetamide (ErIA)-labeled actin strongly bound to single-headed fragments of muscle myosin (muscle S1) and non-muscle myosin V (MV). The conformational dynamics of actin filaments in solution are markedly influenced by the isoform of bound myosin. Both myosins increase the final anisotropy of actin at sub-stoichiometric binding densities, indicating long-range, non-nearest neighbor cooperative restriction of filament rotational dynamics amplitude, but the cooperative unit is larger with MV than muscle S1. Both myosin isoforms also cooperatively affect the actin filament rotational correlation time, but with opposite effects; muscle S1 decreases rates of intrafilament torsional motion, while binding of MV increases the rates of motion. The cooperative effects on the rates of intrafilament motions correlate with the kinetics of myosin binding to actin filaments such that MV binds more rapidly, and muscle myosin more slowly, to partially decorated filaments than to bare filaments. The two isoforms also differ in their effects on the phosphorescence lifetime of the actin-bound ErIA; while muscle S1 increases the lifetime, suggesting decreased aqueous exposure of the probe, MV does not induce a significant change. We conclude that the dynamics and structure of actin in the strongly bound actomyosin complex is determined by the isoform of the bound myosin, in a manner likely to accommodate the diverse functional roles of actomyosin in muscle and non-muscle cells.

Journal of Molecular Biology, 2008

Actin polymerization is a fundamental cellular process involved in cell structure maintenance, fo... more Actin polymerization is a fundamental cellular process involved in cell structure maintenance, force generation, and motility. Phosphate release from filament subunits following ATP hydrolysis destabilizes the filament lattice and increases the critical concentration (C c) for assembly. The structural differences between ATP-and ADP-actin are still debated, as well as the energetic factors that underlie nucleotide-dependent filament stability, particularly under crowded intracellular conditions. Here, we investigate the effect of crowding agents on ATP-and ADP-actin polymerization, and find that ATP-actin polymerization is largely unaffected by solution crowding, while crowding agents lower the C c of ADP-actin in a concentration-dependent manner. The stabilities of ATP-and ADP-actin filaments are comparable in the presence of physiological amounts (~30% w/v) and types (sorbitol) of low molecular weight crowding agents. Crowding agents act to stabilize ADP-F-actin by slowing subunit dissociation. These observations suggest that nucleotide hydrolysis and phosphate release per se do not introduce intrinsic differences in the in vivo filament stability. Rather, the preferential disassembly of ADP-actin filaments in cells is driven through interactions with regulatory proteins. Interpretation of the experimental data according to osmotic stress theory implicates water as an allosteric regulator of actin activity and hydration as the molecular basis for nucleotide-dependent filament stability.

Journal of Biological Chemistry, 2004

We have examined the kinetics of nucleotide binding to actomyosin VI by monitoring the fluorescen... more We have examined the kinetics of nucleotide binding to actomyosin VI by monitoring the fluorescence of pyrene-labeled actin filaments. ATP binds singleheaded myosin VI following a two-step reaction mechanism with formation of a low affinity collision complex (1/K 1 ‫؍‬ 5.6 mM) followed by isomerization (k ؉2 ‫؍‬ 176 s ؊1) to a state with weak actin affinity. The rates and affinity for ADP binding were measured by kinetic competition with ATP. This approach allows a broader range of ADP concentrations to be examined than with fluorescent nucleotide analogs, permitting the identification and characterization of transiently populated intermediates in the pathway. ADP binding to actomyosin VI, as with ATP binding, occurs via a two-step mechanism. The association rate constant for ADP binding is ϳfive times greater than for ATP binding because of a higher affinity in the collision complex (1/K 5b ‫؍‬ 2.2 mM) and faster isomerization rate constant (k ؉5a ‫؍‬ 366 s ؊1). By equilibrium titration, both heads of a myosin VI dimer bind actin strongly in rigor and with bound ADP. In the presence of ATP, conditions that favor processive stepping, myosin VI does not dwell with both heads strongly bound to actin, indicating that the second head inhibits strong binding of the lead head to actin. With both heads bound strongly, ATP binding is accelerated 2.5-fold, and ADP binding is accelerated >10-fold without affecting the rate of ADP release. We conclude that the heads of myosin VI communicate allosterically and accelerate nucleotide binding, but not dissociation, when both are bound strongly to actin.

Journal of Biological Chemistry, 2011

Autotaxin (ATX) is a secreted lysophospholipase D that hydrolyzes lysophosphatidylcholine (LPC) i... more Autotaxin (ATX) is a secreted lysophospholipase D that hydrolyzes lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA), initiating signaling cascades leading to cancer metastasis, wound healing, and angiogenesis. Knowledge of the pathway and kinetics of LPA synthesis by ATX is critical for developing quantitative physiological models of LPA signaling. We measured the individual rate constants and pathway of the LPA synthase cycle of ATX using the fluorescent lipid substrates FS-3 and 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl))-LPC. FS-3 binds rapidly (k 1 >500 M ؊1 s ؊1) and is hydrolyzed slowly (k 2 ‫؍‬ 0.024 s ؊1). Release of the first hydrolysis product is random and rapid (>1 s ؊1), whereas release of the second is slow and rate-limiting (0.005-0.007 s ؊1). Substrate binding and hydrolysis are slow and rate-limiting with LPC. Product release is sequential with choline preceding LPA. The catalytic pathway and kinetics depend strongly on the substrate, suggesting that ATX kinetics could vary for the various in vivo substrates. Slow catalysis with LPC reveals the potential for LPA signaling to spread to cells distal to the site of LPC substrate binding by ATX. An ATX mutant in which catalytic threonine at position 210 is replaced with alanine binds substrate weakly, favoring a role for Thr-210 in binding as well as catalysis. FTY720P, the bioactive form of a drug currently used to treat multiple sclerosis, inhibits ATX in an uncompetitive manner and slows the hydrolysis reaction, suggesting that ATX inhibition plays a significant role in lymphocyte immobilization in FTY720P-based therapeutics. Autotaxin (ATX), 4 also known as nucleotide pyrophosphatase/phosphodiesterase 2 (NPP2), was identified as a secreted

PloS one, 2016

Neuronal calcium sensor-1 (NCS-1 Var1) is a calcium-binding protein expressed in most tissues. We... more Neuronal calcium sensor-1 (NCS-1 Var1) is a calcium-binding protein expressed in most tissues. We examined a poorly characterized variant of NCS-1 (Var2), identified only in humans where the N-terminal 22 amino acid residues of native NCS-1(MGKSNSKLKPEVVEELTRKTY) were replaced with 4 different residues (MATI). Because alterations in the level of expression of NCS-1 Var1 and the expression of NCS-1 variants have been correlated with several neurological diseases, the relative expression and functional role of NCS-1 Var2 was examined. We found that NCS-1 Var2 mRNA levels are not found in mouse tissues and are expressed at levels ~1000-fold lower than NCS-1 Var1 in three different human cell lines (SHSY5Y, HEK293, MB231). Protein expression of both variants was only identified in cell lines overexpressing exogenous NCS-1 Var2. The calcium binding affinity is ~100 times weaker in purified NCS-1 Var2 than NCS-1 Var1. Because truncation of NCS-1 Var1 has been linked to functional changes ...

Current Biology, 2016

Highlights d We generated actin architectures that span the diversity of contractile structures d... more Highlights d We generated actin architectures that span the diversity of contractile structures d These different actin organizations respond differently to myosin-induced contraction d Actin filament organization and connectivity determine the contractile response d Network contraction is dominated by either sarcomeric-like or buckling mechanisms

Nature Communications, 2015

Diseases of ectopic calcification of the vascular wall range from lethal orphan diseases such as ... more Diseases of ectopic calcification of the vascular wall range from lethal orphan diseases such as generalized arterial calcification of infancy (GACI), to common diseases such as hardening of the arteries associated with aging and calciphylaxis of chronic kidney disease (CKD). GACI is a lethal orphan disease in which infants calcify the internal elastic lamina of their medium and large arteries and expire of cardiac failure as neonates, while calciphylaxis of CKD is a ubiquitous vascular calcification in patients with renal failure. Both disorders are characterized by vascular Mönckeburg’s sclerosis accompanied by decreased concentrations of plasma inorganic pyrophosphate (PPi). Here we demonstrate that subcutaneous administration of an ENPP1-Fc fusion protein prevents the mortality, vascular calcifications and sequela of disease in animal models of GACI, and is accompanied by a complete clinical and biomarker response. Our findings have implications for the treatment of rare and com...

Biophysical Journal, 2015

HFSP Journal, 2009

Molecular motors are cellular nanomachines that convert the energy from nucleotide binding, hydro... more Molecular motors are cellular nanomachines that convert the energy from nucleotide binding, hydrolysis, and product release into mechanical work. Because molecular motors contribute to fundamental processes in all living organisms, including genome replication, gene transcription, protein synthesis, organelle transport, and cell division, understanding how the chemical "ATP utilization… and mechanical "motility… cycles are linked is of fundamental importance. A recent study reports the direct visualization of simultaneous nucleotide binding and mechanical displacement of a single myosin 5a molecule, a processive molecular motor protein that takes successive È36-nm steps along actin filaments of the cytoskeleton. This new work demonstrates an exciting advance in single-molecule enzymology and advances our understanding of the link between chemical catalysis and mechanical work in molecular motors, particularly those that operate under internal and external loads.

The EMBO journal, Jan 9, 2008

c-myc is essential for cell homeostasis and growth but lethal if improperly regulated. Transcript... more c-myc is essential for cell homeostasis and growth but lethal if improperly regulated. Transcription of this oncogene is governed by the counterbalancing forces of two proteins on TFIIH--the FUSE binding protein (FBP) and the FBP-interacting repressor (FIR). FBP and FIR recognize single-stranded DNA upstream of the P1 promoter, known as FUSE, and influence transcription by oppositely regulating TFIIH at the promoter site. Size exclusion chromatography coupled with light scattering reveals that an FIR dimer binds one molecule of single-stranded DNA. The crystal structure confirms that FIR binds FUSE as a dimer, and only the N-terminal RRM domain participates in nucleic acid recognition. Site-directed mutations of conserved residues in the first RRM domain reduce FIR's affinity for FUSE, while analogous mutations in the second RRM domain either destabilize the protein or have no effect on DNA binding. Oppositely oriented DNA on parallel binding sites of the FIR dimer results in sp...

Scientific Reports, 2013

Enzyme inhibition due to the reversible binding of reaction products is common and underlies the ... more Enzyme inhibition due to the reversible binding of reaction products is common and underlies the origins of negative feedback inhibition in many metabolic and signaling pathways. Product inhibition generates non-linearity in steady-state time courses of enzyme activity, which limits the utility of well-established enzymology approaches developed under the assumption of irreversible product release. For more than a century, numerous attempts to find a mathematical solution for analysis of kinetic time courses with product inhibition have been put forth. However, no practical general method capable of extracting common enzymatic parameters from such non-linear time courses has been successfully developed. Here we present a simple and practical method of analysis capable of efficiently extracting steady-state enzyme kinetic parameters and product binding constants from non-linear kinetic time courses with product inhibition and/or substrate depletion. The method is general and applicable to all enzyme systems, independent of reaction schemes and pathways.

Science, 2012

Actin Up Actomyosin interactions lie at the heart of fundamental cellular processes—including mor... more Actin Up Actomyosin interactions lie at the heart of fundamental cellular processes—including morphogenesis, establishment of polarity, and overall motility—but the general principles driving the spatiotempotal orchestration of these interactions have remained elusive. Working in vitro, using micropatterned substrates, Reymann et al. (p. 1310 ) demonstrate that myosins can use a “selection orientation” mechanism to pull selectively on actin filaments, contract the actin network and disassemble it, or walk on the filaments, align them, allow their growth, and control filament orientation.

Proceedings of the National Academy of Sciences, 2010

DEAD-box RNA helicase proteins use the energy of ATP hydrolysis to drive the unwinding of duplex ... more 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, 2008

Dimeric myosins V and VI travel long distances in opposite directions along actin filaments in ce... more Dimeric myosins V and VI travel long distances in opposite directions along actin filaments in cells, taking multiple steps in a “hand-over-hand” fashion. The catalytic cycles of both myosins are limited by ADP dissociation, which is considered a key step in the walking mechanism of these motors. Here, we demonstrate that external loads applied to individual actomyosin V or VI bonds asymmetrically affect ADP affinity, such that ADP binds weaker under loads assisting motility. Model-based analysis reveals that forward and backward loads modulate the kinetics of ADP binding to both myosins, although the effect is less pronounced for myosin VI. ADP dissociation is modestly accelerated by forward loads and inhibited by backward loads. Loads applied in either direction slow ADP binding to myosin V but accelerate binding to myosin VI. We calculate that the intramolecular load generated during processive stepping is ≈2 pN for both myosin V and myosin VI. The distinct load dependence of ADP...

Nucleic Acids Research, 2007

We have used 2-aminopurine (2AP) as a fluorescent probe in the template strand of a 13/20mer prim... more We have used 2-aminopurine (2AP) as a fluorescent probe in the template strand of a 13/20mer primer/ template (D) to detect deoxynucleoside triphosphates (N)-dependent conformational changes exhibited by RB69 DNA polymerase (ED) complexes. The rates and amplitudes of fluorescence quenching depend hyperbolically on the [dTTP] when a dideoxy-primer/template (ddP/T) with 2AP as the templating base (n position) is used. No detectable fluorescence changes occur when a ddP/T with 2AP positioned 5' to the templating base (n + 1 position) is used. With a deoxy-primer/template (dP/T) with 2AP in the n position, a rapid fluorescence quenching occurs within 2 ms, followed by a second, slower fluorescence quenching with a rate constant similar to base incorporation as determined by chemical quench. With a dP/T having 2AP in the n + 1 position, there is a [dNTP]-dependent fluorescence enhancement that occurs at a rate comparable to dNMP incorporation. Collectively, the results favor a minimal kinetic scheme in which population of two distinct biochemical states of the ternary EDN complex precedes the nucleotidyl transfer reaction. Observed differences between dP/T and ddP/T ternary complexes indicate that the 3' hydroxyl group of the primer plays a critical role in determining the rate constants of transitions that lead to strong deoxynucleoside triphosphate binding prior to chemistry.

Nature Structural & Molecular Biology, 2004

Myosin V belongs to the myosin superfamily of actin-based molecular motors and is involved in the... more Myosin V belongs to the myosin superfamily of actin-based molecular motors and is involved in the intracellular transport of organelles 1-4. Myosin V consists of two identical heavy chains, each composed of an N-terminal motor domain ('head'), a domain comprising six IQ motifs that bind light chains ('neck'), a coiled coil dimerization domain and a globular cargo-binding tail domain 1,3. Myosin V is a processive motor that 'walks' along an actin filament toward the barbed end over a long distance without dissociating from the filament 5,6. Electron microscopy of actomyosin V in the presence of low ATP concentrations shows both motor domains of myosin V bound to the actin filament at sites spaced 36 nm apart, which corresponds to the half pitch of the filament long-pitch helix 7. Experiments using optical tweezers identified processive 36-nm steps of a bead, on which single myosin V molecules were adsorbed 6,8. Moreover, it was shown that myosin V walks as a left-handed spiral motor along an actin filament, because the average step size is slightly shorter than the half pitch of the long-pitch actin helix 9. The handover hand walking model has received strong support from two recent experiments that (i) observed the orientation of the neck domain of myosin V by monitoring the polarization of a single fluorophore covalently attached to a light chain 10 and (ii) measured the stepwise displacement of a single fluorophore labeled at one of six light chains of myosin V 11. Solution kinetic studies demonstrate that ADP release occurs at ∼15 s-1 and limits the myosin V ATPase cycle 12. Microscopic analysis of myosin V stepping under various nucleotide conditions is consistent with rate-limiting ADP release 13. The next key target is to determine how the mechanical and biochemical cycles are coupled to each other at the single-molecule level. Here, we focused on mechanical events and detected substeps that occur within each regular 36-nm step with high temporal resolution. Each regular 36-nm step is composed of two consecutive substeps, one generating a 12-nm substep and the other a 24-nm substep. To investigate how these substeps and the states attained after the steps are coupled to the ATPase cycle of myosin V, we examined the effects of ATP and ADP concentrations, and 2,3-butanedione 2-monoxime (BDM) 14. We also examined the force dependence of the occurrence frequency of each step and substep, and the dwell time of each state. RESULTS Movement of myosin V along an actin filament A single myosin V-coated bead was trapped with optical tweezers and brought into contact with a fluorescently labeled biotinylated actin filament, which was immobilized on an avidin-coated glass surface through biotinylated BSA (Fig. 1a). A focused red light (685 nm) laser was used to diagonally illuminate the bead, and its dark-field image was projected onto a quadrant photodiode. The bead displacement was determined by measuring the differential output of the quadrant photodiode with nanometer accuracy and a 10-kHz sampling rate 15. The use of a 200-nm-diameter bead here instead of a 1-µm bead was essential to obtain a high spatiotemporal resolution. An example of the time course of bead displacement along an actin filament (Fig. 1b) shows three consecutive runs of a single myosin V molecule along an actin filament at a saturating ATP concentration (1 mM). As the bead began to deviate from the trap center, a positive external load was applied to the myosin V-actin complex (toward the pointed end of an actin filament). Myosin V detached from actin at a stall force of ∼3 pN. After detachment, the bead quickly returned to the

Nature Chemical Biology, 2010

The dimeric motor myosin V transports organelles along actin filament tracks over long distances ... more The dimeric motor myosin V transports organelles along actin filament tracks over long distances in cells. Myosin V is a smart 'walker' that is able to swiftly adjust to variable 'road conditions' to continue its processive movement across dense cellular environments. Coordination between the two heads via intramolecular load modulates biochemical kinetics and ensures highly efficient unidirectional motion. However, little is known about how load-induced regulation of the processive stepping occurs in vivo, where myosin V experiences significant off-axis loads applied in various directions. To reveal how myosin V remains processive in cells, we measured the effect of the off-axis loads, applied to individual actomyosin V bonds in a range of angles, on the coordination between the two heads and myosin V processive stepping. We found that myosin V remains highly processive under diagonal loads owing to asymmetrical ADP affinities and that the native 6IQ lever optimizes the subunit coordination, which indicates that myosin V is designed to be an intracellular transporter. Myosin V transports cargos toward the barbed end of actin filaments in cells 1,2 , taking multiple ~36-nm steps by alternately swinging forward two lever arms in a 'hand-over-hand' fashion 3-7. Single-molecule experiments reveal that one ATP molecule is consumed for each step, which confirms the tight coupling between mechanical and enzymatic events 8. Combined with the unidirectionality of the processive movement, this mechanism implies that the ATPase cycles in two catalytic domains are precisely coordinated. Directional loads modulate ATPase kinetics in myosin V 9-11 , which suggests that two subunits communicate via the intramolecular load, which is generated during binding to actin with both heads, to achieve the coordinated mechanical performance.

Journal of Molecular Biology, 2006

We have evaluated the thermodynamic parameters associated with cooperative cofilin binding to act... more We have evaluated the thermodynamic parameters associated with cooperative cofilin binding to actin filaments, accounting for contributions of ionlinked equilibria, and determined the kinetic basis of cooperative cofilin binding. Ions weaken non-contiguous (isolated, non-cooperative) cofilin binding to an actin filament without affecting cooperative filament interactions. Non-contiguous cofilin binding is coupled to the dissociation of ∼1.7 thermodynamically bound counterions. Counterion dissociation contributes ∼40% of the total cofilin binding free energy (in the presence of 50 mM KCl). The non-contiguous and cooperative binding free energies are driven entirely by large, positive entropy changes, consistent with a cofilin-mediated increase in actin filament structural dynamics. The rate constant for cofilin binding to an isolated site on an actin filament is slow and likely to be limited by filament breathing. Cooperative cofilin binding arises from an approximately tenfold more rapid association rate constant and an approximately twofold slower dissociation rate constant. The more rapid association rate constant is presumably a consequence of cofilin-dependent changes in the average orientation of subdomain 2, subunit angular disorder and filament twist, which increase the accessibility of a neighboring cofilin-binding site on an actin filament. Cooperative association is more rapid than binding to an isolated site, but still slow for a second-order reaction, suggesting that cooperative binding is limited also by binding site accessibility. We suggest that the dissociation of actin-associated ions weakens intersubunit interactions in the actin filament lattice that enhance cofilin-binding site accessibility, favor cooperative binding and promote filament severing.

Journal of Molecular Biology, 2011

Mss116 is a Saccharomyces cerevisiae mitochondrial DEAD-box RNA helicase protein essential for ef... more Mss116 is a Saccharomyces cerevisiae mitochondrial DEAD-box RNA helicase protein essential for efficient in vivo splicing of all group I and II introns and 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 nucleotidelinked RNA binding. RNA accelerates the Mss116 steady-state ATPase ~7-fold by promoting rate-limiting ATP hydrolysis, such that 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 with bound ADP or in the absence of nucleotide. The predominant biochemical intermediate populated during in vivo steadystate cycling is the strong RNA binding, Mss116-ADP-P i state. Strong RNA binding allows Mss116 to fulfill its biological role in 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 RNA-Mss116 complexes, regenerating free Mss116.

Journal of Molecular Biology, 2011

The contractile and enzymatic activities of myosin VI are regulated by calcium binding to associa... more The contractile and enzymatic activities of myosin VI are regulated by calcium binding to associated calmodulin light chains. We have used transient phosphorescence anisotropy (TPA) to monitor the microsecond rotational dynamics of erythrosin iodoacetamide-labeled actin with strongly-bound myosin VI (MVI) and to evaluate the effect of MVI-bound calmodulin (CaM) light chain on actin filament dynamics. MVI binding lowers the amplitude but accelerates actin filament microsecond dynamics in a Ca 2+-and CaM-dependent manner, as indicated from an increase in the final anisotropy and a decrease in the correlation time of TPA decays. MVI with bound apo-CaM or Ca 2+-CaM weakly affects actin filament microsecond dynamics, relative to other myosins (e.g. muscle myosin II and myosin Va). CaM dissociation from bound MVI damps filament rotational dynamics (i.e. increases the torsional rigidity), such that the perturbation is comparable to that induced by other characterized myosins. Analysis of individual actin filament shape fluctuations imaged by fluorescence microscopy reveals a correlated effect on filament bending mechanics. These data support a model in which Ca 2+-dependent CaM binding to the IQ domain of MVI is linked to an allosteric reorganization of the actin-binding site(s), which alters the structural dynamics and the mechanical rigidity of actin filaments. Such modulation of filament dynamics may contribute to the Ca 2+-and CaM-dependent regulation of myosin VI motility and ATP utilization.

Journal of Molecular Biology, 2010

We have used transient phosphorescence anisotropy (TPA) to detect the microsecond rotational dyna... more We have used transient phosphorescence anisotropy (TPA) to detect the microsecond rotational dynamics of erythrosin iodoacetamide (ErIA)-labeled actin strongly bound to single-headed fragments of muscle myosin (muscle S1) and non-muscle myosin V (MV). The conformational dynamics of actin filaments in solution are markedly influenced by the isoform of bound myosin. Both myosins increase the final anisotropy of actin at sub-stoichiometric binding densities, indicating long-range, non-nearest neighbor cooperative restriction of filament rotational dynamics amplitude, but the cooperative unit is larger with MV than muscle S1. Both myosin isoforms also cooperatively affect the actin filament rotational correlation time, but with opposite effects; muscle S1 decreases rates of intrafilament torsional motion, while binding of MV increases the rates of motion. The cooperative effects on the rates of intrafilament motions correlate with the kinetics of myosin binding to actin filaments such that MV binds more rapidly, and muscle myosin more slowly, to partially decorated filaments than to bare filaments. The two isoforms also differ in their effects on the phosphorescence lifetime of the actin-bound ErIA; while muscle S1 increases the lifetime, suggesting decreased aqueous exposure of the probe, MV does not induce a significant change. We conclude that the dynamics and structure of actin in the strongly bound actomyosin complex is determined by the isoform of the bound myosin, in a manner likely to accommodate the diverse functional roles of actomyosin in muscle and non-muscle cells.

Journal of Molecular Biology, 2008

Actin polymerization is a fundamental cellular process involved in cell structure maintenance, fo... more Actin polymerization is a fundamental cellular process involved in cell structure maintenance, force generation, and motility. Phosphate release from filament subunits following ATP hydrolysis destabilizes the filament lattice and increases the critical concentration (C c) for assembly. The structural differences between ATP-and ADP-actin are still debated, as well as the energetic factors that underlie nucleotide-dependent filament stability, particularly under crowded intracellular conditions. Here, we investigate the effect of crowding agents on ATP-and ADP-actin polymerization, and find that ATP-actin polymerization is largely unaffected by solution crowding, while crowding agents lower the C c of ADP-actin in a concentration-dependent manner. The stabilities of ATP-and ADP-actin filaments are comparable in the presence of physiological amounts (~30% w/v) and types (sorbitol) of low molecular weight crowding agents. Crowding agents act to stabilize ADP-F-actin by slowing subunit dissociation. These observations suggest that nucleotide hydrolysis and phosphate release per se do not introduce intrinsic differences in the in vivo filament stability. Rather, the preferential disassembly of ADP-actin filaments in cells is driven through interactions with regulatory proteins. Interpretation of the experimental data according to osmotic stress theory implicates water as an allosteric regulator of actin activity and hydration as the molecular basis for nucleotide-dependent filament stability.

Journal of Biological Chemistry, 2004

We have examined the kinetics of nucleotide binding to actomyosin VI by monitoring the fluorescen... more We have examined the kinetics of nucleotide binding to actomyosin VI by monitoring the fluorescence of pyrene-labeled actin filaments. ATP binds singleheaded myosin VI following a two-step reaction mechanism with formation of a low affinity collision complex (1/K 1 ‫؍‬ 5.6 mM) followed by isomerization (k ؉2 ‫؍‬ 176 s ؊1) to a state with weak actin affinity. The rates and affinity for ADP binding were measured by kinetic competition with ATP. This approach allows a broader range of ADP concentrations to be examined than with fluorescent nucleotide analogs, permitting the identification and characterization of transiently populated intermediates in the pathway. ADP binding to actomyosin VI, as with ATP binding, occurs via a two-step mechanism. The association rate constant for ADP binding is ϳfive times greater than for ATP binding because of a higher affinity in the collision complex (1/K 5b ‫؍‬ 2.2 mM) and faster isomerization rate constant (k ؉5a ‫؍‬ 366 s ؊1). By equilibrium titration, both heads of a myosin VI dimer bind actin strongly in rigor and with bound ADP. In the presence of ATP, conditions that favor processive stepping, myosin VI does not dwell with both heads strongly bound to actin, indicating that the second head inhibits strong binding of the lead head to actin. With both heads bound strongly, ATP binding is accelerated 2.5-fold, and ADP binding is accelerated >10-fold without affecting the rate of ADP release. We conclude that the heads of myosin VI communicate allosterically and accelerate nucleotide binding, but not dissociation, when both are bound strongly to actin.

Journal of Biological Chemistry, 2011

Autotaxin (ATX) is a secreted lysophospholipase D that hydrolyzes lysophosphatidylcholine (LPC) i... more Autotaxin (ATX) is a secreted lysophospholipase D that hydrolyzes lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA), initiating signaling cascades leading to cancer metastasis, wound healing, and angiogenesis. Knowledge of the pathway and kinetics of LPA synthesis by ATX is critical for developing quantitative physiological models of LPA signaling. We measured the individual rate constants and pathway of the LPA synthase cycle of ATX using the fluorescent lipid substrates FS-3 and 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl))-LPC. FS-3 binds rapidly (k 1 >500 M ؊1 s ؊1) and is hydrolyzed slowly (k 2 ‫؍‬ 0.024 s ؊1). Release of the first hydrolysis product is random and rapid (>1 s ؊1), whereas release of the second is slow and rate-limiting (0.005-0.007 s ؊1). Substrate binding and hydrolysis are slow and rate-limiting with LPC. Product release is sequential with choline preceding LPA. The catalytic pathway and kinetics depend strongly on the substrate, suggesting that ATX kinetics could vary for the various in vivo substrates. Slow catalysis with LPC reveals the potential for LPA signaling to spread to cells distal to the site of LPC substrate binding by ATX. An ATX mutant in which catalytic threonine at position 210 is replaced with alanine binds substrate weakly, favoring a role for Thr-210 in binding as well as catalysis. FTY720P, the bioactive form of a drug currently used to treat multiple sclerosis, inhibits ATX in an uncompetitive manner and slows the hydrolysis reaction, suggesting that ATX inhibition plays a significant role in lymphocyte immobilization in FTY720P-based therapeutics. Autotaxin (ATX), 4 also known as nucleotide pyrophosphatase/phosphodiesterase 2 (NPP2), was identified as a secreted