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Myosin-I by the PNAS PLUS N-Terminal Region

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Myosin-I by the PNAS PLUS N-Terminal Region Mechanochemical tuning of myosin-I by the PNAS PLUS N-terminal region Michael J. Greenberg, Tianming Lin, Henry Shuman, and E. Michael Ostap1 Pennsylvania Muscle Institute and the Department of Physiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104 Edited by James A. Spudich, Stanford University School of Medicine, Stanford, CA, and approved May 13, 2015 (received for review April 3, 2015) Myosins are molecular motors that generate force to power a wide only do these myosins have different responses to force, but also array of motile cellular functions. Myosins have the inherent ability they have different biochemical transitions that are affected by to change their ATPase kinetics and force-generating properties force (i.e., the rate of ADP release for Myo1b and the rate of ATP- when they encounter mechanical loads; however, little is known induced actomyosin detachment for Myo1c). about the structural elements in myosin responsible for force The high-resolution crystal structures of nucleotide-free sensing. Recent structural and biophysical studies have shown that Myo1b (i.e., rigor-like state) (4) and ADP.vanadate-bound Myo1c myosin-I isoforms, Myosin-Ib (Myo1b) and Myosin-Ic (Myo1c), have (i.e., pre–power-stroke state) (8) have recently been determined. similar unloaded kinetics and sequences but substantially different Despite being in different conformational states, these structures responses to forces that resist their working strokes. Myo1b has the show that Myo1b and Myo1c have a high degree of structural properties of a tension-sensing anchor, slowing its actin-detachment homology to each other and to other myosins (4, 16). A prominent kinetics by two orders of magnitude with just 1 pN of resisting feature in the Myo1b structure is the positioning of the N-terminal force, whereas Myo1c has the properties of a slow transporter, region (NTR), which is in a conformation that has not been ob- generating power without slowing under 1-pN loads that would served in other myosin structures (Fig. 1A). In myosin-II, -V, and stall Myo1b. To examine the structural elements that lead to differ- -VI the NTR includes an SH3-like domain that lies to the side of ences in force sensing, we used single-molecule and ensemble ki- the motor domain (17–19). In contrast, the Myo1b NTR sits in a netic techniques to show that the myosin-I N-terminal region (NTR) hydrophobic pocket between the motor and the lever arm helix plays a critical role in tuning myosin-I mechanochemistry. We found (LAH) and interacts with the first calmodulin light chain. As such, that replacing the Myo1c NTR with the Myo1b NTR changes the it is in a position that might enable it to communicate the position identity of the primary force-sensitive transition of Myo1c, resulting of the LAH to the nucleotide-binding site (4). Although the motor in sensitivity to forces of <2 pN. Additionally, we found that the – domain sequences of myosin-I isoforms are highly conserved, in- NTR plays an important role in stabilizing the post power-stroke cluding the residues that create the hydrophobic pocket in which conformation. These results identify the NTR as an important struc- the NTR sits, the NTR sequences are not conserved (Fig. 1B). We tural element in myosin force sensing and suggest a mechanism for hypothesized that the NTR plays a role in establishing kinetic generating diversity of function among myosin isoforms. diversity among myosins (4). To investigate whether the NTR of Myo1c plays a role in mechanochemistry | optical tweezers | mechanosensing | mechanosensing, we expressed Myo1c constructs with the native transient kinetics | single molecule NTR present, with the NTR deleted, and with the Myo1b NTR in place of the native sequence and measured their kinetic and me- yosin motors use the energy from ATP hydrolysis to power chanical properties in the presence and absence of force. We find Ma wide array of cellular processes including muscle con- that the NTR of Myo1c plays important roles in tuning Myo1c’s traction, cell migration, membrane trafficking, cell division, and kinetics in the presence and absence of load and in stabilizing the intracellular transport (for review, see ref. 1). To optimally function in such diverse processes, different myosin isoforms BIOPHYSICS AND Significance have evolved distinct kinetic and mechanical properties to meet COMPUTATIONAL BIOLOGY their physiological demands (for review, see ref. 2), including differing abilities to adapt their ATPase kinetics and power Myosin molecular motors generate forces in the cell and act as outputs in response to mechanical loads (3). Although many mechanosensors, adjusting their power outputs in response to studies have elucidated structural elements important for force mechanical loads. Little is known about the structural elements generation in myosin motors, not much is known about the re- involved in myosin mechanosensing. Our results identify the gions important for tuning a myosin’s ability to modulate power N-terminal region (NTR) of the myosin-I protein as having an output in response to mechanical loads. important role in tuning mechanochemistry. Appending the An opportunity to study these structural elements has emerged NTR from a highly tension-sensitive myosin (Myo1b) onto a less with the structural and mechanochemical characterization of two tension-sensitive motor (Myo1c) changes the identity of the myosin-I family members with similar sequences, Myosin-Ib primary force-sensitive transition of Myo1c, making it sensitive < (Myo1b) (4–7) and Myosin-Ic (Myo1c) (8–10). These motors have to forces 2 pN. Moreover, we show that the NTR stabilizes the – similar unloaded ATPase kinetics, where they are both low duty- post power-stroke conformation. These results identify the ratio motors (i.e., they spend a majority of their biochemical cycles NTR as an important structural element in myosin force sensing detached from actin) with motility rates that are limited by the rate and suggest a mechanism for generating diversity of function among myosin isoforms. of ADP release (7, 10–14). Although these motors have similar sequences and unloaded ATPase kinetics, they have very different Author contributions: M.J.G., H.S., and E.M.O. designed research; M.J.G. and T.L. per- mechanical outputs under load (for review, see ref. 15). Myo1b is formed research; M.J.G., T.L., and H.S. contributed new reagents/analytic tools; M.J.G., extraordinarily sensitive to small loads, where forces of 1 pN slow T.L., H.S., and E.M.O. analyzed data; and M.J.G., H.S., and E.M.O. wrote the paper. its motility rate more than 50-fold. As such, Myo1b has the The authors declare no conflict of interest. expected properties of a tension-sensitive anchor (5, 6). In contrast, This article is a PNAS Direct Submission. 1 pN of force does not appreciably slow the rate of Myo1c motility, 1To whom correspondence should be addressed. Email: [email protected]. enabling the motor to generate power over a range of forces. Thus, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Myo1c has the expected properties of a slow transporter (10). Not 1073/pnas.1506633112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1506633112 PNAS | Published online June 8, 2015 | E3337–E3344 Downloaded by guest on September 25, 2021 Scheme 1), where the rate of ADP release limits the Myo1c A motility rate at saturating [ATP] (10). We used stopped-flow transient kinetic techniques to determine whether these steps were altered by deletion or swapping of the NTR (Table 1; see Materials and Methods for details). The rate constant for ATP- induced dissociation was measured by mixing pyrene-labeled actomyosin with varying ATP concentrations (Fig. 2). Similar to Δ Myo1c (10), fluorescence transients obtained with Myo1c N Δ > and Myo1c N- b are best fitted by the sum of two exponential functions. The fast component has a hyperbolic dependence on the [ATP] (Fig. 2A) and was modeled as shown in Scheme 1 (20). Δ Δ > Both Myo1c N and Myo1c N- b have substantially accelerated L11 −1 maximal rates of ATP binding (k+2′ = 160 ± 4.6 s and 160 ± L10 − Δ Δ > 2.8 s 1 for Myo1c N and Myo1c N- b, respectively) compared with −1 Myo1c (k+2′ = 18 ± 0.99 s ; P < 0.001). The rate of the slow M14 ΔN −1 phase measured for both Myo1c (kslow = 56 ± 3.2 s )and ΔN->b −1 Myo1c (kslow = 54 ± 2.0 s )is>10-foldfasterthanthat −1 found for Myo1c (kslow = 4.0 ± 0.034 s ; P < 0.001) (Fig. 2B). In Myo1c, the slow phase is due to an isomerization of actin-bound myosin from a nucleotide-free state that is not capable of binding ATP to a state than can bind ATP (12, 21); however, it does not B 10 20 30 appear that the slow phase observed for the NTR mutants cor- Myo1b MAKKEVKSSLLDNMIGVGDTVLLEPLN-EETF ID relates with the same transition (see below). Myo1c ---MESALTARD-RVGVQDFVLLENFTSEAAFIE The rate of ADP release was measured by preincubating NTR 10 20 30 pyrene-labeled actomyosin with saturating [ADP] and then ob- C serving the increase in fluorescence upon ATP-induced acto- 110 767 myosin dissociation (Fig. 2D). The rate of ADP release from k ′ = ± −1 MESALTARDRVGV Myo1c Motor IQ IQ IQ AviTag Myo1c ( +5 3.9 0.060 s ) was reported previously (10). For Myo1c ΔN ΔN Myo1c , the fluorescence transient is best described by the Myo1c MVGV Myo1c Motor IQ IQ IQ AviTag sum of two exponential functions where the slower phase (0.93 ± −1 ΔN->b Myo1c Motor IQ IQ IQ AviTag 0.010 s ) makes up 79% of the amplitude and the faster phase Myo1c MAKKEVKSSLLDNMVGV − Δ > (4.2 ± 0.18 s 1) makes up 21% of the amplitude.
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