Steady-state ATPase Research Articles

Understanding the coupling between DNA damage detection and UvrA's ATPase using bulk and single molecule kinetics.

Nucleotide excision repair (NER) protects cells against diverse types of DNA damage, principally UV irradiation. In Escherichia coli, damage is recognized by 2 key enzymes: UvrA and UvrB. Despite extensive investigation, the role of UvrA’s 2 ATPase domains in NER remains elusive. Combining single-molecule fluorescence microscopy and classic biochemical methods, we have investigated the role of nucleotide binding in UvrA’s kinetic cycle. Measurement of UvrA’s steady-state ATPase activity shows it is stimulated upon binding DNA (kcat 0.71–1.07/s). Despite UvrA’s ability to discriminate damage, we find UV-damaged DNA does not alter the steady-state ATPase. To understand how damage affects UvrA, we studied its binding to DNA under various nucleotide conditions at the single molecule level. We have found that both UV damage and nucleotide cofactors affect the attached lifetime of UvrA. In the presence of ATP and UV damage, the lifetime is significantly greater compared with undamaged DNA. To reconcile these observations, we suggest that UvrA uses negative cooperativity between its ATPase sites that is gated by damage recognition. Only in the presence of damage is the second site activated, most likely in a sequential manner.—Barnett, J. T., Kad, N. M. Understanding the coupling between DNA damage detection and UvrA’s ATPase using bulk and single molecule kinetics.

Acidosis and Phosphate Directly Reduce Myosin's Force-Generating Capacity Through Distinct Molecular Mechanisms.

Elevated levels of the metabolic by-products, including acidosis (i.e., high [H+]) and phosphate (Pi) are putative agents of muscle fatigue; however, the mechanism through which they affect myosin’s function remain unclear. To elucidate these mechanisms, we directly examined the effect of acidosis (pH 6.5 vs. 7.4), alone and in combination with elevated levels of Pi on the force-generating capacity of a mini-ensemble of myosin using a laser trap assay. Acidosis decreased myosin’s average force-generating capacity by 20% (p < 0.05). The reduction was due to both a decrease in the force generated during each actomyosin interaction, as well as an increase in the number of binding events generating negative forces. Adding Pi to the acidic condition resulted in a quantitatively similar decrease in force but was solely due to an elimination of all high force-generating events (>2 pN), resulting from an acceleration of the myosin’s rate of detachment from actin. Acidosis and Pi also had distinct effects on myosin’s steady state ATPase rate with acidosis slowing it by ∼90% (p > 0.05), while the addition of Pi under acidic conditions caused a significant recovery in the ATPase rate. These data suggest that these two fatigue agents have distinct effects on myosin’s cross-bridge cycle that may underlie the synergistic effect that they have muscle force. Thus these data provide novel molecular insight into the mechanisms underlying the depressive effects of Pi and H+ on muscle contraction during fatigue.

Impact of Dilated Cardiomyopathy Mutation and Small Molecule Regulator on Human Beta-Cardiac Myosin

Missense mutations in human β-cardiac myosin are associated with inherited cardiomyopathies. The mechanisms for how mutations alter myosin's motor performance are still unclear. We expressed and purified human beta-cardiac myosin subfragment 1 (M2β-S1) containing a point mutation in the converter domain (F764L) that is associated with dilated cardiomyopathy (DCM) and compared it to wild-type (WT) M2β-S1. We demonstrate that the F764L mutation slows down maximum actin-activated ATPase activity (20-30%). Direct measurements of ADP release in the presence of actin using mant labeled ADP demonstrate the F764L mutant has a 20% slower ADP release rate constant. The in vitro motility assay demonstrates a 10-15% slower actin sliding velocity for F764L. Monitoring ATP binding and hydrolysis with intrinsic tryptophan fluorescence demonstrates that the F764L mutation causes a 2-fold increase in K0.5 without changing the maximal rate. We also examined the impact of the heart failure drug Omecamtiv Mecarbil (OM) on the F764L mutant. The steady-state actin-activated ATPase results suggest that the F764L mutant shares a similar OM binding affinity with WT. Interestingly, the drug does not reduce the maximum ATPase activity and actin sliding velocity as much as it does with WT. We observed no difference in the ionic strength dependence or density dependence of in vitro motility in WT and F764L in the presence and absence of OM. Our previous results suggested OM dramatically slows down the ADP isomerization step in WT M2β-S1, making it the rate-limiting step. Our results demonstrate OM does not slow the ADP isomerization step as much in F764L, but this step is still rate-limiting. Therefore, we propose that OM will increase the myosin duty ratio in the F764L mutant enough to enhance isomeric force while only moderately slowing shortening velocity.

Cardiac Myosin Strutural Kinetics are Modulated by Myk461

The biochemical and structural kinetics of bovine cardiac myosin are perturbed by the putative heart failure therapeutic Myk461. Myosin's lever arm undergoes structural changes that are coupled to its biochemical state during ATPase cycling in muscle. Structural states of myosin's lever arm can be detected directly using the high-precision, high-throughput technique of transient time-resolved FRET revealing kinetics, thermodynamics, and allosteric coupling. We have previously used this technique to investigate mechanochemical coupling in skeletal myosin and cardiac myosin perturbed by the heart failure drug omecamtiv mecarbil. Here we investigate the MyoKardia compound Myk461, in clinical trials for treating hypertrophic cardiomyopathy. We find that the small molecule therapeutic primarily inhibits actomyosin association, both in the absence and presence of nucleotide. This inhibition is also detected in the coupled kinetic steps following actin activation of myosin: the force-producing structural isomerization known as the powerstroke, and the principal free energy-driven biochemical step of Pi release. Other actin-dependent experiments reveal a potent inhibition by Myk461, steady-state ATPase cycling and single-turnover of fluorescent ATP. We also find that the drug's affinity for myosin varies by two-orders of magnitude, dependent on the structural and biochemical state of myosin: binding strongest to actomyosin-ATP and weakest to apo myosin. This work was supported by NIH AR032961 & AR057220 (DDT), American Heart Association Scientist Development Grant (JMM), and Graduate Excellence Fellowship-University of MN (JAR).

W1038 near D-loop of NBD2 is a focal point for inter-domain communication in multidrug transporter Cdr1 of Candida albicans

Candida drug resistance 1 (Cdr1), a PDR subfamily ABC transporter mediates efflux of xenobiotics in Candida albicans. It is one of the prime factors contributing to multidrug resistance in the fungal pathogen. One hallmark of this transporter is its asymmetric nature, characterized by peculiar alterations in its nucleotide binding domains. As a consequence, there exists only one canonical ATP-binding site while the other is atypical. Here, we report suppressor analysis on the drug-susceptible transmembrane domain mutant V532D that identified the suppressor mutation W1038S, close to the D-loop of the non-catalytic ATP-binding site. Introduction of the W1038S mutation in the background of V532D mutant conferred resistance for most of the substrates to the latter. Such restoration is accompanied by a severe reduction of ATPase activity, of about 85%, while that of the V532D mutant is half-reduced. Conversely, alanine substitution of the highly conserved aspartate D1033A in that D-loop rendered cells selectively hyper-susceptible to miconazole without an impact on steady-state ATPase activity, suggesting altogether that ATP hydrolysis may not hold the key to restoration mechanism. Analysis of the ABCG5/ABCG8-based 3D-model of Cdr1p suggested that the W1038S substitution leads to the loss of hydrophobic interactions and H-bond with residues of the neighbor NBD1, in the non-catalytic ATP-binding site area. The compensatory effect within TMDs accounting for transport restoration in the V532D-W1038S variant may, therefore, be mainly due to an increase in NBDs mobility at the non-catalytic interface.

Conformational memory in the association of the transmembrane protein phospholamban with the sarcoplasmic reticulum calcium pump SERCA

The sarcoplasmic reticulum Ca2+-ATPase SERCA promotes muscle relaxation by pumping calcium ions from the cytoplasm into the sarcoplasmic reticulum. SERCA activity is regulated by a variety of small transmembrane peptides, most notably by phospholamban in cardiac muscle and sarcolipin in skeletal muscle. However, how phospholamban and sarcolipin regulate SERCA is not fully understood. In the present study, we evaluated the effects of phospholamban and sarcolipin on calcium translocation and ATP hydrolysis by SERCA under conditions that mimic environments in sarcoplasmic reticulum membranes. For pre-steady-state current measurements, proteoliposomes containing SERCA and phospholamban or sarcolipin were adsorbed to a solid-supported membrane and activated by substrate concentration jumps. We observed that phospholamban altered ATP-dependent calcium translocation by SERCA within the first transport cycle, whereas sarcolipin did not. Using pre-steady-state charge (calcium) translocation and steady-state ATPase activity under substrate conditions (various calcium and/or ATP concentrations) promoting particular conformational states of SERCA, we found that the effect of phospholamban on SERCA depends on substrate preincubation conditions. Our results also indicated that phospholamban can establish an inhibitory interaction with multiple SERCA conformational states with distinct effects on SERCA's kinetic properties. Moreover, we noted multiple modes of interaction between SERCA and phospholamban and observed that once a particular mode of association is engaged it persists throughout the SERCA transport cycle and multiple turnover events. These observations are consistent with conformational memory in the interaction between SERCA and phospholamban, thus providing insights into the physiological role of phospholamban and its regulatory effect on SERCA transport activity.

Nucleotide Exchange Factors for Hsp70 Chaperones.

The ATPase cycle of Hsp70 chaperones controls their transient association with substrates and thus governs their function in protein folding. Nucleotide exchange factors (NEFs) accelerate ADP release from Hsp70, which results in rebinding of ATP and release of the substrate, thereby regulating the lifetime of the Hsp70-substrate complex. This chapter describes several methods suitable to study NEFs of Hsp70 chaperones. On the one hand, steady-state ATPase assays provide information on how the NEF influences progression of the Hsp70 through the entire ATPase cycle. On the other hand, nucleotide release can be measured directly using labeled nucleotides, which enables identification and further characterization of NEFs.

Bacterial Hsp90 ATPase Assays.

Bacterial Hsp90 is an ATP-dependent molecular chaperone involved in protein remodeling and activation. The E. coli Hsp90, Hsp90Ec, collaborates in protein remodeling with another ATP-dependent chaperone, DnaK, the E. coli Hsp70. Both Hsp90Ec and DnaK hydrolyze ATP and client (substrate) proteins stimulate the hydrolysis. Additionally, ATP hydrolysis by the combination of Hsp90Ec and DnaK is synergistically stimulated in the presence of client (substrate). Here, we describe two steady-state ATPase assays used to monitor ATP hydrolysis by Hsp90Ec and DnaK as well as the synergistic stimulation of ATP hydrolysis by the combination of Hsp90Ec and DnaK in the presence of a client (substrate). The first assay is a spectrophotometric assay based on enzyme-coupled reactions that utilize the ADP formed during ATP hydrolysis to oxidize NADH. The second assay is a more sensitive method that directly quantifies the radioactive inorganic phosphate released following the hydrolysis of [γ-33P] ATP or [γ-32P] ATP.

Kinetic signatures of myosin-5B, the motor involved in microvillus inclusion disease

Myosin-5B is a ubiquitous molecular motor that transports cargo vesicles of the endomembrane system in intracellular recycling pathways. Myosin-5B malfunction causes the congenital enteropathy microvillus inclusion disease, underlining its importance in cellular homeostasis. Here we describe the interaction of myosin-5B with F-actin, nucleotides, and the pyrazolopyrimidine compound myoVin-1. We show that single-headed myosin-5B is an intermediate duty ratio motor with a kinetic ATPase cycle that is rate-limited by the release of phosphate. The presence of a second head generates strain and gating in the myosin-5B dimer that alters the kinetic signature by reducing the actin-activated ADP release rate to become rate-limiting. This kinetic transition into a high-duty ratio motor is a prerequisite for the proposed transport function of myosin-5B in cellular recycling pathways. Moreover, we show that the small molecule compound myoVin-1 inhibits the enzymatic and functional activity of myosin-5B in vitro Partial inhibition of the actin-activated steady-state ATPase activity and sliding velocity suggests that caution should be used when probing the effect of myoVin-1 on myosin-5-dependent transport processes in cells.

A small-molecule modulator of cardiac myosin acts on multiple stages of the myosin chemomechanical cycle

Mavacamten, formerly known as MYK-461 is a recently discovered novel small-molecule modulator of cardiac myosin that targets the underlying sarcomere hypercontractility of hypertrophic cardiomyopathy, one of the most prevalent heritable cardiovascular disorders. Studies on isolated cells and muscle fibers as well as intact animals have shown that mavacamten inhibits sarcomere force production, thereby reducing cardiac contractility. Initial mechanistic studies have suggested that mavacamten primarily reduces the steady-state ATPase activity by inhibiting the rate of phosphate release of β-cardiac myosin-S1, but the molecular mechanism of action of mavacamten has not been described. Here we used steady-state and presteady-state kinetic analyses to investigate the mechanism of action of mavacamten. Transient kinetic analyses revealed that mavacamten modulates multiple steps of the myosin chemomechanical cycle. In addition to decreasing the rate-limiting step of the cycle (phosphate release), mavacamten reduced the number of myosin-S1 heads that can interact with the actin thin filament during transition from the weakly to the strongly bound state without affecting the intrinsic rate. Mavacamten also decreased the rate of myosin binding to actin in the ADP-bound state and the ADP-release rate from myosin-S1 alone. We, therefore, conclude that mavacamten acts on multiple stages of the myosin chemomechanical cycle. Although the primary mechanism of mavacamten-mediated inhibition of cardiac myosin is the decrease of phosphate release from β-cardiac myosin-S1, a secondary mechanism decreases the number of actin-binding heads transitioning from the weakly to the strongly bound state, which occurs before phosphate release and may provide an additional method to modulate myosin function.

Kinetic Characterization of the Mitochondrial Transporter Human Myosin XIX

Human myosin XIX is a novel class of actin-based motor implicated in the transport and localization of mitochondria. We expressed myosin XIX using the baculovirus/SF9 cell expression system. The truncated MYO19-3IQ construct was co-expressed with the calmodulin light chain and chaperones Unc45 and Hsp90. The chaperone proteins significantly increased the level of myosin XIX expression 20-fold. To determine the mechanochemical properties that underlie cellular MYO19 activity we carried out transient kinetic experiments and measured elementary rate constants. ADP is released in two steps (k-AD1 = 4.5 s−1 and k-AD2 = 1.5 s−1) from the actomyosinADP complex. Phosphate dissociation is fast and has a kmax of 47 s−1 . The rate of hydrolysis (M·T = M·D·P) determined by quenched flow is >16 s−1 and is favorable with an equilibrium constant of (KH = 2). The maximal rate of ATP induced dissociation of myosin from the actomyosin (k-TA) is 670s−1 . We conclude that the rate-limiting step of the ATPase cycle is the slower step of the ADP release because it's rate is the same as the actin activated steady-state ATPase (Vmax = 1.6 s−1). The human Myosin XIX has a high duty ratio (0.9) which is consistent with its role in transporting mitochondria.

Noncanonical Microtubule Interaction of Yeast Kinesin-5 CIN8

Kinesin-5's were originally described as slow, plus-end-directed motors that are required for proper establishment and maintenance of the mitotic spindle. They share a bipolar homotetrameric structure with four heavy chains organized such that two motor domains are positioned at each end of a coiled-coil stalk, allowing the motor to cross-link and slide two antiparallel microtubules. Interestingly, recent studies have demonstrated bidirectional motility for yeast kinesin-5 motors, which represents a significant biophysical conundrum given our previous understanding of directed kinesin motility. We aim to understand the structural and biochemical features of yeast kinesin-5 motors that promote directional switching of motility. Saccharomyces cerevisiae Cin8 has been shown to switch directionality based on ionic strength, motor coupling and binding between antiparallel microtubules. Using ionic strength to promote switching of directionality, we have determined the microtubule binding behavior, steady-state kinetics, and cryo-EM structure of the Cin8 motor domain. Cosedimentation assays revealed that Cin8 binds super-stoichiometrically along the microtubule lattice with 3±1 motor domains binding per tubulin dimer. Competition assays with human kinesin-5 (Eg5) indicate that Cin8 binds at the canonical kinesin binding site as well as at a yet unidentified non-canonical site. Interestingly, Cin8 has microtubule-stimulated ATPase activity on both the canonical and non-canonical sites. We have probed the structural explanation for these biochemical observations by obtaining a 7A cryo-EM reconstruction of the microtubule-Cin8 complex in the ADP+AlFx state. Deletion of the large insert in Cin8's loop L8 retains similar steady-state ATPase activity yet abolishes super-stoichiometric microtubule binding, providing a powerful tool to dissect its ATPase mechanism. Cin8's microtubule interaction offers a mechanism for bidirectional movement, such that the motor utilizes a non-canonical microtubule site for directional switching.

Investigating the Intersection between DNA Mismatch Repair and Triplet Nucleotide Repeat Expansion

Mismatch repair (MMR) is a highly effective system dedicated to correcting errors produced during DNA replication. Errors arise when DNA polymerases incorporate incorrect nucleotides, leading to base pair mismatches, or slip from the primer‐template junction, leading to insertion‐deletion loops (IDLs). The proteins of interest in this study are the eukaryotic MutS homologs Msh2–Msh6 and Msh2–Msh3, which recognize single base pair mismatches or small IDLs, and larger IDLs, respectively1. Polymerases slip more easily at repeat sequences, which can lead to the formation of triplet nucleotide repeat (TNR) hairpins during replication. Msh2–Msh3 is known to recognize and promote the expansion of these TNR hairpin structures, which in turn is associated with neurological disorders such as Huntington's disease2. The goal of my research project is to understand how Msh2–Msh6 and Msh2–Msh3 interact with IDLs compared to TNRs. Gel mobility shift assays (GMSA) are being used to determine if there are differences in Msh2–Msh6 or Msh2–Msh3 complexes bound to these DNA structures. Preliminary results indicate that Msh2–Msh6 binds CAG repeat TNR hairpins with high affinity, similar to Msh2–Msh3. This leads to the question of why Msh2–Msh6 binding does not affect the TNR structure nor lead to its repair, while Msh2–Msh3 binding leads to its expansion. The answer may lie in differences in the affinity and dynamics of interaction between the proteins and DNA, or their ATPase activity, which is essential for the response to an error in the DNA3. In previous pre‐steady state ATPase studies of Msh2–Msh6, a rapid burst in hydrolysis was observed in the presence of duplex DNA3. In the presence of a mismatched DNA, however, this burst phase is absent, allowing Msh2–Msh6 to adopt a dual ATP‐bound form capable of sliding away from the mismatch, which is critical for signaling mismatch repair3. Due to the presence of ‘A‐A’ mismatches in the hairpin stem, it is possible that Msh2–Msh6 is recognizing this structure as error containing DNA. This hypothesis can be tested by measuring the ATPase response and related actions of Msh2–Msh6 on the TNR hairpin. Currently, a malachite green phosphate release assay is being used to measure the steady state ATPase activity of Msh2–Msh6, to determine any changes in the absence or presence of the TNR and to find optimal conditions for pre‐steady state experiments. In addition, a fluorescence‐based assay is being developed to measure the kinetics of Msh2–Msh6 interaction with the TNR and determine if the protein can adopt an ATP‐bound sliding clamp form on this DNA substrate. It is anticipated that the results of this kinetic analysis will help us understand in a quantitative manner how and why Msh2–Msh6 may not promote TNR expansion, while Msh2–Msh3 does. This mechanistic knowledge may help us find ways to avoid and possibly mitigate the devastating effects of TNR‐induced neurological disorders.Support or Funding InformationNSF grant MCB 1022203; ASBMB Undergraduate Research Award

2-Deoxy-ATP Enhances Multiple Kinetic Parameters to Improve Cardiac Function

Hypertrophic cardiomyopathy (HCM) is a form of genetic heart disease often caused by point mutations in sarcomeric proteins. As the underlying mechanisms of genetic HCM continue to be unraveled, developing novel ways to modulate the actomyosin contractile apparatus is of growing interest and importance. The nucleotide analog 2-deoxyadenosine triphosphate (dATP) has recently garnered interest as potentially having therapeutic benefit for treatment of systolic and/or diastolic heart failure. dATP has been previously reported to enhance cardiac contractility, increase +dP/dt, and improve diastolic relaxation parameters in transgenic mice with elevated levels of dATP in the heart (Korte, 2011). When compared to ATP, dATP has been reported to increase steady state ATPase activity, and sliding velocities in the in vitro motility assay up to 50% (Regnier et al 2000). To better understand potential therapeutic benefits of dATP on actomyosin, we characterize the mechanism of action for dATP using bovine cardiac myosin subfragments S1 and HMM in a variety of steady-state, transient, and single-molecule experiments. We report a 40% increase in unloaded in vitro motility sliding velocities, as well as increased ATPase activity, ADP release rates, and actin-binding affinities with dATP compared to ATP. The combination of transient kinetic rates and equilibrium constants of the actomyosin ATPase cycle, as well as basal myosin parameters, implicate ADP release as the primary contributor to the differences observed between the two nucleotides. We propose a model by which enhancing both cardiac contraction and relaxation kinetics can improve cardiac function and potentially serve as a therapeutic for genetic heart disease.

Non-Canonical Microtubule Interaction by Yeast Kinesin-5, Cin8

Kinesin-5's were originally described as slow, plus-end-directed motors that are required for proper establishment and maintenance of the mitotic spindle. They share a bipolar homotetrameric structure with four heavy chains organized such that two motor domains are positioned at each end of a coiled-coil stalk, allowing the motor to cross-link and slide two antiparallel microtubules. Interestingly, recent studies have demonstrated bidirectional motility for yeast kinesin-5 motors, which represents a significant biophysical conundrum given our previous understanding of directed kinesin motility. We aim to understand the structural and biochemical features of yeast kinesin-5 motors that promote directional switching of motility. Saccharomyces cerevisiae Cin8 has been shown to switch directionality based on ionic strength, motor coupling and binding between antiparallel microtubules. Using ionic strength to promote switching of directionality, we have determined the microtubule binding behavior, steady-state kinetics, and cryo-EM structure of the Cin8 motor domain. Cosedimentation assays revealed that Cin8 binds super-stoichiometrically along the microtubule lattice with 3±1 motor domains binding per tubulin dimer. Competition assays with human kinesin-5 (Eg5) indicate that Cin8 binds at the canonical kinesin binding site as well as at a yet unidentified non-canonical site. Interestingly, Cin8 has microtubule-stimulated ATPase activity on both the canonical and non-canonical sites. We have probed the structural explanation for these biochemical observations by obtaining a 7Å cryo-EM reconstruction of the microtubule-Cin8 complex in the ADP+AlFx state. Deletion of the large insert in Cin8's loop L8 retains similar steady-state ATPase activity yet abolishes super-stoichiometric microtubule binding, providing a powerful tool to dissect its ATPase mechanism. Cin8's microtubule interaction offers a mechanism for bidirectional movement, such that the motor utilizes a non-canonical microtubule site for directional switching.

Chromokinesins NOD and KID use Alternative Nucleotide Mechanisms and One-Dimensional Diffusion to Target Microtubule Plus Ends

Chromokinesins have been found to associate with chromosomes during cell division and play a variety of functions including chromosome positioning, chromosome condensation, spindle bipolarity, and cytokinesis. These motors are thought to generate the elusive polar ejection force that drives chromatin away from the spindle poles. Kinesin-10's are chromokinesins that include human KID (also known as KIF22) and Drosophila melanogaster NOD, which share an N-terminal kinesin-like catalytic domain, a central stalk domain, and a C-terminal DNA-binding domain. Due to their common in vivo physiologies, we propose that kinesin-10's work together in teams of motors to translocate/target to spindle microtubule plus-ends. Using steady-state kinetics, we have characterized the motor domain constructs of NOD and KID by measuring the basal, unpolymerized tubulin-stimulated, and MT-stimulated steady-state ATPase and GTPase kinetics under both low (∼21 mM) and physiologically relevant (∼111 mM) ionic strength conditions. These results suggest both purine nucleotides may be utilized by kinesin-10's in vivo and set the foundation for comparing the ATPase and GTPase mechanisms of these two unconventional kinesins. Using GFP-tagged kinesin-10's and total internal reflection fluorescence (TIRF) microscopy, we observed the microtubule-bound NOD either remaining stationary or engaging in a one-dimensional random walk across the microtubule lattice. These two modes of microtubule binding are interchangeable and movement along the microtubule is rapid (similar to kinesin-13 and other microtubule end-binding proteins) and without directionality preference. Under these conditions, we observe significantly higher incidence of microtubule end-binding events, which is increased after removal of the negatively-charge C-terminal tails of tubulin using subtilisin. Our continued studies reveal mechanistic and functional details of kinesin-10 force generation and therefore provide new insights into the molecular motion that drives the polar ejection force during cell division.

The Relay/Converter Interface Influences Hydrolysis of ATP by Skeletal Muscle Myosin II

The interface between relay and converter domain of muscle myosin is critical for optimal myosin performance. Using Drosophila melanogaster indirect flight muscle S1, we performed a kinetic analysis of the effect of mutations in the converter and relay domain. Introduction of a mutation (R759E) in the converter domain inhibits the steady-state ATPase of myosin S1, whereas an additional mutation in the relay domain (N509K) is able to restore the ATPase toward wild-type values. The R759E S1 construct showed little effect on most steps of the actomyosin ATPase cycle. The exception was a 25–30% reduction in the rate constant of the hydrolysis step, the step coupled to the cross-bridge recovery stroke that involves a change in conformation at the relay/converter domain interface. Significantly, the double mutant restored the hydrolysis step to values similar to the wild-type myosin. Modeling the relay/converter interface suggests a possible interaction between converter residue 759 and relay residue 509 in the actin-detached conformation, which is lost in R759E but is restored in N509K/R759E. This detailed kinetic analysis of Drosophila myosin carrying the R759E mutation shows that the interface between the relay loop and converter domain is important for fine-tuning myosin kinetics, in particular ATP binding and hydrolysis.

Kinesin-2 KIF3AC and KIF3AB Can Drive Long-Range Transport along Microtubules

Mammalian KIF3AC is classified as a heterotrimeric kinesin-2 that is best known for organelle transport in neurons, yet in vitro studies to characterize its single molecule behavior are lacking. The results presented show that a KIF3AC motor that includes the native helix α7 sequence for coiled-coil formation is highly processive with run lengths of ∼1.23 μm and matching those exhibited by conventional kinesin-1. This result was unexpected because KIF3AC exhibits the canonical kinesin-2 neck-linker sequence that has been reported to be responsible for shorter run lengths observed for another heterotrimeric kinesin-2, KIF3AB. However, KIF3AB with its native neck linker and helix α7 is also highly processive with run lengths of ∼1.62 μm and exceeding those of KIF3AC and kinesin-1. Loop L11, a component of the microtubule-motor interface and implicated in activating ADP release upon microtubule collision, is significantly extended in KIF3C as compared with other kinesins. A KIF3AC encoding a truncation in KIF3C loop L11 (KIF3ACΔL11) exhibited longer run lengths at ∼1.55 μm than wild-type KIF3AC and were more similar to KIF3AB run lengths, suggesting that L11 also contributes to tuning motor processivity. The steady-state ATPase results show that shortening L11 does not alter kcat, consistent with the observation that single molecule velocities are not affected by this truncation. However, shortening loop L11 of KIF3C significantly increases the microtubule affinity of KIF3ACΔL11, revealing another structural and mechanistic property that can modulate processivity. The results presented provide new, to our knowledge, insights to understand structure-function relationships governing processivity and a better understanding of the potential of KIF3AC for long-distance transport in neurons.

Development of a Reagentless Biosensor for Inorganic Phosphate, Applicable over a Wide Concentration Range.

A fluorescent reagentless biosensor for inorganic phosphate (Pi), based on the E. coli PstS phosphate binding protein, was redesigned to allow measurements of higher Pi concentrations and at low, substoichiometric concentrations of biosensor. This was achieved by weakening Pi binding of the previous biosensor, and different approaches are described that could enable this change in properties. The readout, providing response to the Pi concentration, is delivered by tetramethylrhodamine fluorescence. In addition to two cysteine mutations for rhodamine labeling at positions 17 and 197, the final variant had an I76G mutation in the hinge region between the two lobes that make up the protein. Upon Pi binding, the lobes rotate on this hinge and the mutation on the hinge lowers affinity ∼200-fold, with a dissociation constant now in the tens to hundreds micromolar range, depending on solution conditions. The signal change on Pi binding was up to 9-fold, depending on pH. The suitability of the biosensor for steady-state ATPase assays was demonstrated with low biosensor usage and its advantage in ability to cope with Pi contamination.

Kinetic characterization of the sole nonmuscle myosin-2 from the model organism Drosophila melanogaster.

Nonmuscle myosin-2 is the primary enzyme complex powering contractility of the F-actin cytoskeleton in the model organism Drosophila. Despite myosin's essential function in fly development and homeostasis, its kinetic features remain elusive. The purpose of this in vitro study is a detailed steady-state and presteady-state kinetic characterization of the Drosophila nonmuscle myosin-2 motor domain. Kinetic features are a slow steady-state ATPase activity, high affinities for F-actin and ADP, and a low duty ratio. Comparative analysis of the overall enzymatic signatures across the nonmuscle myosin-2 complement from model organisms indicates that the Drosophila protein resembles nonmuscle myosin-2s from metazoa rather than protozoa, though modulatory aspects of myosin motor function are distinct. Drosophila nonmuscle myosin-2 is uniquely insensitive toward blebbistatin, a commonly used myosin-2 inhibitor. An in silico modeling approach together with kinetic studies indicate that the nonconsensus amino acid Met466 in the Drosophila nonmuscle myosin-2 active-site loop switch-2 acts as blebbistatin desensitizer. Introduction of the M466I mutation sensitized the protein for blebbistatin, resulting in a half-maximal inhibitory concentration of 36.3 ± 4.1 µM. Together, these data show that Drosophila nonmuscle myosin-2 is a bona fide molecular motor and establish an important link between switch-2 and blebbistatin sensitivity.