Figure 1.
Residue interactions of loop L7 of the kinesin central β-sheet.
(A) A loop (yellow) that connects two β-strands (gray arrows) of the central β-sheet could alternately interact with residues that bind nucleotide (ADP, purple oval) or the microtubule (MT, dark green oval) in different nucleotide states, bending or distorting the β-sheet. Mutating a key loop residue could affect both nucleotide and microtubule binding, and alter the stable state of the motor. (B) Two conformations of loop L7 between strands β4 and β5 of the central β-sheet in a stalk-rotated Ncd crystal structure (PDB 3L1C; Movie S1) [12]. In head H1 and both heads of unrotated Ncd dimer structures (PDB 2NCD, 1CZ7), Y485 of loop L7 (space-filled, yellow) touches switch I R552 (space-filled, purple). The adjacent residue, S551 (space-filled, magenta), interacts with the Mg2+ coordinating the ADP. In head H2, N600 (space-filled, dark green) at the N terminus of switch II helix ∝4, which interfaces with the microtubule [1], moves towards Y485, and R552 changes in orientation. Images prepared in PyMol [39].
Figure 2.
ADP release by the NcdY485 mutants.
(A) ADP release without (left) and with microtubules (right). Normalized mean fluorescence versus time after adding ATP (left, arrow) or microtubules followed by ATP (right, arrows) to motor bound to mant-ADP. WT, wild type (magenta). Mutants, YE (gray); YN (dark gray); YK (black). s, seconds. (B) NcdY485K ADP release after adding 0–0.5 mM ATP to 1 µM motor bound to mant-ADP. koff (s−1) per active site vs [ATP]. WT (magenta circles; curve fit, black); NcdYK (black circles; curve fit, magenta). Data points fit to the Michaelis-Menten equation to estimate Vmax and KM,ATP. Error bars, mean±SEM. Assays performed in HEM100.
Table 1.
NcdY485 kinetic parameters.
Figure 3.
Microtubule binding and motility of NcdY485 mutants.
(A) Microtubule binding. Top, supernatants (left) and pellets (right) from assays of motors with added ADP pelleted without or with microtubules. MTs, microtubules; M, Mr markers (kDa); Tub, tubulin. Bottom, fraction of motor pelleted with added ADP and microtubules, after correction for motor that pelleted with added ADP but without microtubules. Assays performed in HEM100. (B) Microtubule gliding velocity. Assays in HEM with varying NaCl concentration to optimize motor-microtubule binding and gliding (WT, 50–280 mM NaCl; YE, 100–180 mM NaCl; YN, 250–300 mM NaCl; YK, 250–400 mM NaCl). Error bars, mean±SEM.
Table 2.
NcdY485 mutant motility.
Figure 4.
ATP hydrolysis by NcdY485 mutants.
(A) Left, basal ATPase activity. Data points adjusted to OD340 = 0.6531 (theoretical starting OD of NADH in reaction mix at t = 0) and fit to a line to determine rate of OD340 decrease due to ATP hydrolysis. Right, microtubule-stimulated ATPase activity with 0.5 µM motor+1 µM microtubules. (B) NcdYK vs wild-type Ncd ATPase activity. kcat (s−1) per active site vs [MT]; 0.5 µM motor+0–25 µM microtubules (n = 3). Data points fit to the Michaelis-Menten equation to estimate Vmax and KM,MT. Mean±SEM.
Figure 5.
ncdY485K-gfp mutant oocyte MI spindles.
(A) Wild-type ncd-gfp or (B, C) ncdY485K-gfp late stage 13 spindles. (D) Wild-type ncd-gfp or (E, F) ncdY485K-gfp stage 14 spindles. (A–F) Arrows, frayed or spurred spindle fibers. Bar, 3 µm. (G) MI spindle length in wild-type (WT Ncd) or mutant (NcdY485K) oocytes (St 13, late stage 13, white bars; St 14, stage 14, gray bars). Error bars, mean±SEM. (H) Wild-type ncd-gfp or (I) mutant ncdY485K-gfp stage 14 oocyte cortical region. NcdY485-GFP-decorated cortical microtubules (white) and asters (black arrows) are prominent in ncdY485K mutant oocytes. (H, I) White arrows, MI spindle. Bars, 20 µm.
Figure 6.
Simulations of NcdY485K mutant effects on MI spindle assembly.
(A) Relative microtubule densities (Y axis) versus relative distance from the spindle equator (X axis) in simulations modeling the 11-fold higher microtubule affinity of the NcdY485K mutant as 11-fold faster crosslinking (red) or as 11-fold slower decrosslinking (green), both with 2-fold increased sliding rate to account for faster motility; results for wild-type parameters [18] are shown for comparison (blue). Densities are shown at an early stage of spindle assembly (t = 75, left) and in fully formed spindles (t = 750, right). Top: rho1, unaligned (uncrosslinked) microtubules; middle: rho2, primarily crosslinked microtubules aligned with the long spindle axis, involved in sliding to elongate the spindle; bottom: rho3, microtubules crosslinking different chromosomes, essential for proper spindle shape. (B) Diagrams from the simulations representing spindles at time t = 75 and t = 750.
Figure 7.
Model of Ncd hydrolysis cycle.
(A) Wild-type Ncd exists in a tight-ADP/weak-microtubule binding state in solution (bold arrow) and undergoes cycles of weak-to-tight microtubule binding coupled to nucleotide changes in the head that interacts with the microtubule (D, ADP; T, ATP). (B) NcdY485K and the other two NcdY485 mutants bind tightly to microtubules and may release ADP even when not bound to microtubules; the stable state is the weak-ADP/tight-microtubule binding state (bold arrow), in contrast to wild type. NcdY485K is depicted in a tight microtubule-binding, stalk-rotated conformation like NcdN600K (PDB 1N6M) throughout the cycle [40]; however, the stalk must rotate back to a pre-stroke conformation at least transiently during the cycle for the motor to produce force and support microtubule gliding in motility assays.