Figure 4. Transient kinetic features of NM2C and R788E.

(A) Interaction between NM2C/R788K/R788E with ATP under single-turnover conditions. Binding 0.375 µM d-mantATP to 0.5 µM myosin results in a transient fluorescence increase that is followed by a short plateau (hydrolysis) and a slow decrease in mantADP fluorescence that is associated with its release. All three phases are reduced in R788E (grey) compared to NM2C (green) and R788K (yellow). The very slow decrease of the fluorescence signal in R788E indicates that either the ATP hydrolysis rate or a subsequent release rate of the hydrolysis products are severely decreased when compared to NM2C and R788K. (B) Interaction between 0.25 µM pyrene-actoNM2C/R788K/R788E with 0.15 µM ATP under single-turnover conditions. Color coding is according to (A).

Figure 4—figure supplement 1. Nucleotide binding characteristics of NM2C, R788K, and R788E and disease causing NM2C mutations.

(A) Dependence of the observed rate constants (k_obs) upon d-mantATP binding to 0.25 µM NM2C/R788K/R788E on the nucleotide concentration. Linear fits to the data result in second-order binding rate constants of K₁k₊₂ = 0.48±0.01 µM⁻¹s⁻¹ for NM2C (green), a two-fold reduced ATP binding constant K₁k₊₂ = 0.22±0.01 µM⁻¹s⁻¹ for R788K (yellow), and a five-fold reduced binding rate constant of K₁k₊₂ = 0.09±0.005 µM⁻¹s⁻¹ for R788E (grey) compared to NM2C. Inset, The rate constants (k_obs) obtained from the binding reaction of ATP to NM2C/R788K dependent hyperbolically on the ATP concentration. The rate of ATP hydrolysis (k₊₃+k_-3) and the second-order binding rate constants K₁k₊₂ were determined from a fit to the data (NM2C: k₊₃+k_-3=24.32 ± 0.89 s⁻¹; K₁k₊₂ = 0.39±0.01 µM⁻¹s⁻¹; R788K: k₊₃+k_-3=37.31 ± 0.55 s⁻¹; K₁k₊₂ = 0.18±0.01 µM⁻¹s⁻¹). The respective parameters are not experimentally accessible for R788E. (B) The apparent second-order ATP binding rate constants, determined by the ATP-induced dissociation of the pyrene-labeled actoNM2C (green) or actoR788E (grey) complexes are with K₁k₊₂ = 1.86±0.03 µM⁻¹s⁻¹ and K₁k₊₂ = 2.11±0.05 µM⁻¹s⁻¹ similar. The respective constant is reduced to K₁k₊₂ = 1.03±0.03 µM⁻¹s⁻¹ for actoR788K (yellow). Inset, The maximum isomerization rate constants k₊₂ = 643.06±22.6 s⁻¹ (NM2C), k₊₂ = 767.43±21.27 s⁻¹ (R788K), and k₊₂ = 579.27±12.81 s⁻¹ was determined from a hyperbolic fit to the respective data. (C) The R788K (yellow) and the R788E (grey) mutation decrease the second-order ADP-binding rate constant two- to threefold to k_+D = 0.21±0.02 µM⁻¹s⁻¹ and k_+D = 0.12±0.01 µM⁻¹s⁻¹ when compared to k_+D = 0.39±0.01 µM⁻¹s⁻¹ for NM2C (green). (D) The ADP-binding rate constant k_+AD = 0.03±0.001 µM⁻¹s⁻¹, as calculated from the slope of the k_obs versus [d-mantADP] plot, is 85-fold decreased for R788E (grey) when compared to k_+AD = 2.54±0.18 µM⁻¹s⁻¹ for NM2C (green). (E) Time-dependent change in the intrinsic tryptophan signal upon mixing 0.5 mM ATP with 0.25 µM NM2C (grey) or R778E (grey) in the presence of 5 µM ADP. ATP binding increases the fluorescence signal in NM2C but not R788E under identical conditions in a stopped-flow spectrophotometer. (F) Missense mutations G376C and R726S are associated with autosomal dominant hearing impairment (DFNA4) and their location in NM2C is shown in spheres representation. G376C is in proximity to the JK-loop, R726S is in the SH1-SH2 helix. NM2C subdomains are color coded according to Figure 1C and the nucleotide is shown in spheres representation.