Single-molecule force spectroscopy reveals a stepwise unfolding of Caenorhabditis elegans giant protein kinase domains

Abstract

Myofibril assembly and disassembly are complex processes that regulate overall muscle mass. Titin kinase has been implicated as an initiating catalyst in signaling pathways that ultimately result in myofibril growth. In titin, the kinase domain is in an ideal position to sense mechanical strain that occurs during muscle activity. The enzyme is negatively regulated by intramolecular interactions occurring between the kinase catalytic core and autoinhibitory/regulatory region. Molecular dynamics simulations suggest that human titin kinase acts as a force sensor. However, the precise mechanism(s) resulting in the conformational changes that relieve the kinase of this autoinhibition are unknown. Here we measured the mechanical properties of the kinase domain and flanking Ig/Fn domains of the Caenorhabditis elegans titin-like proteins twitchin and TTN-1 using single-molecule atomic force microscopy. Our results show that these kinase domains have significant mechanical resistance, unfolding at forces similar to those for Ig/Fn beta-sandwich domains (30-150 pN). Further, our atomic force microscopy data is consistent with molecular dynamic simulations, which show that these kinases unfold in a stepwise fashion, first an unwinding of the autoinhibitory region, followed by a two-step unfolding of the catalytic core. These data support the hypothesis that titin kinase may function as an effective force sensor.

FIGURE 1

Expression, purification, and enzyme activity of recombinant kinase domains and tandem Ig domain segments from C. elegans giant proteins. (A and B) Twitchin and TTN-1 recombinant protein constructs for AFM experiments. The kinase constructs have an autoregulated kinase domain flanked by Fn and Ig domains. The Ig constructs consist of five tandem Ig domains that immediately follow the kinase domain. The numbers denote the position of the amino acid in the full-length polypeptide. (C) SDS PAGE (12%) stained with Coomassie brilliant blue shows that the proteins were isolated to >95% purity. In each lane, 2 μg of protein was loaded. The lanes are as follows: (1) molecular weight standard; (2) Fn-TwcKin-Ig; (3) Fn-TTN1Kin-Ig; (4) Twc Ig 26-30; (5) TTN-1 Ig 38-42. (D) The purified TTN-1 and twitchin kinases retain phosphotransferase activity in vitro. Using ³²P-ATP and a model peptide as substrates the specific activity values (SPAC) obtained are similar to those published previously (9,10). Enzyme activities were determined in triplicate on freshly purified protein.

FIGURE 2

3D structures of C. elegans twitchin Ig and kinase domains and homology model for TTN-1 kinase. (A) Twitchin kinase is composed of three subdomains: an α-helical rich large lobe (green), a small lobe of mainly -sheets (dark blue), and the autoregulatory tail (red) (structure taken from (12)). The autoregulatory tail is situated between the two lobes making extensive contact with the active site. To orient the active site a conserved lysine residue helping to neutralize the ATP binding pocket is shown in a ball and stick interpretation. (B) 3D homology model of TTN-1 kinase. The N-terminal β-sheet (strands βC1-C3) predicted to be parallel to the pulling force is colored dark blue. This sheet is thought to stabilize the kinase on activation by force. The C-terminal β-sheet is predicted to be perpendicular to the pulling force and is composed of βC10 (dark green), βR1 (red), and βC11 (dark green) strands. The autoinhibitory domain is colored red and includes the βR1 strand and αR1-R2 helices. Shown in ball and stick representation is a key lysine residue from the N-terminal β-strands interacting directly with the ATP binding pocket of the active site. (C) Twitchin Ig domain 26, which immediately follows the twitchin kinase domain (structure taken from (12)). The two β-sheets, characteristic of an Ig fold are depicted in green and brown. All three structures have their N-terminal α-carbons marked with cyan spheres and their C-terminal α-carbons marked with magenta spheres.

FIGURE 3

Force-extension relationships of twitchin and TTN-1 Ig domains. (A and D) Several examples of force-extension curves obtained after stretching twitchin (A) and TTN-1 (D) Ig domains. The gray lines were generated with the WLC equation using a persistence length of 0.4 nm and contour length increments, ΔL, of 30 nm. (B and E) Unfolding force histograms for twitchin and TTN-1 domains. The mean force peak values are 93 ± 25 pN, (n = 88 peaks) and 85 ± 22 pN (n = 193 peaks), respectively. In (E) the red line corresponds to a Monte Carlo simulation of TTN-1 Ig using and Δx_u = 0.35 nm at a pulling speed of 500 nm/s. (C) Histogram of contour length increments observed on unfolding of twitchin Ig domains shows one main peak centered at ∼30 nm (Gaussian fit: 30.6 ± 3.2 nm). (F) The unfolding forces of TTN-1 Ig domains depend on pulling speed. The experimental data (black symbols) can be well described by Monte Carlo simulations (red line) using Δx_u = 0.35 nm.

FIGURE 4

Mechanical properties of C. elegans twitchin kinase. (A) Two examples of force-extension curves obtained for the Fn-Twc kinase-Ig construct. The two small force peaks correspond to the stepwise unfolding of the Twc kinase domain and the last two peaks to the unfolding of the flanking Ig/Fn domains. (B) Histogram of increases in contour length increments observed on unfolding, ΔL, of Fn-Twc kinase-Ig. There are peaks at ∼30 nm, 65 nm, and 95 nm (Gaussian fits: 31 ± 5 nm, 67 ± 18 nm, and 97 ± 10 nm, n = 142), which correspond to the unfolding of Ig/Fn domains (blue bars), and kinase domain (red and green bars). (C) Unfolding force distributions for Ig/Fn domains (blue bars) and kinase domain (red and green bars); the respective force peaks are at 111 ± 67 pN (n = 57), 83 ± 57 pN (n = 41), and 52 ± 11 pN (n = 43).

FIGURE 5

Mechanical properties of C. elegans TTN-1 kinase. (A) Two examples of force-extension curves obtained for the Fn-TTN1 kinase-Ig construct. (B) Histogram of increases in contour length increments observed on unfolding, ΔL, of the Fn-TTN-1 kinase-Ig construct (16 molecules). There are peaks at ∼30 nm, 65 nm, and 95 nm (n = 35). (Gaussian fits: 29 ± 16 nm, 63 ± 15 nm, and 95 ± 13 nm). (C) Plot of the average unfolding force versus the pulling rate for TTN-1 Ig/Fn domains and the kinase domain. For the kinase, we analyzed the first force peak with a ΔL of ∼95 nm. The continuous lines correspond to the result of Monte Carlo simulations of two-state unfolding at the corresponding pulling rates. The parameters used for the Monte Carlo simulation are: Δx_u = 0.35 nm for the Ig/Fn domains and Δx_u = 0.6 nm for the TTN-1 kinase.

FIGURE 6

Constant velocity steered molecular dynamics simulation of the mechanical unfolding of twitchin kinase. (Left) Force-extension curve obtained from SMD simulations by stretching the twitchin kinase domain (1KOA) between its C terminus and its N terminus at a pulling speed of 0.5 Å/ps. The total simulation time was 2.4 ns using 33,428 total atoms including 18 Na⁺ and 9305 water molecules. The fixed atom was Tyr-5915 and the SMD atom Arg-6261. (Right) Four snapshots of twitchin kinase stretched from its termini taken at no extension (rest), after 65 Å (1), 340 Å (2), and 639 Å (3) of extension. At rest, the kinase domain is in a closed conformation. The active site is occupied by the autoinhibitory region (red), which makes extensive contact with the catalytic site, blocking substrate binding. (1) At low forces the regulatory tail will unravel reversibly and expose the active site to its substrates. (2) At high forces the kinase begins to unfold and the integrity of the active site is disrupted. The small lobe (blue), made mainly of β-sheets, unravels first followed by the unfolding of the α-helical rich large lobe (green).