Disease-associated mutations in the p150(Glued) subunit destabilize the CAP-gly domain

Abstract

Point mutations within the CAP-gly domain of the p150(Glued) subunit of the dynactin complex have been identified in patients with distal spinal bulbar muscular atrophy (dSBMA) and Perry's syndrome. Herein, we show by CD and NMR experiments that each mutated CAP-gly domain is folded but less stable than the wild-type (WT) domain. We also demonstrate that the domains harboring these mutations bind to microtubules but fail to bind to EB1. These data indicate that these disease-associated, point mutations affect the stability of this domain and inhibit their interaction with EB1 but do not inhibit their interaction with microtubules.

Figure 1

Ribbon diagram of the p150^Glued CAP-gly domain (yellow) bound to EB1 (cyan). The point mutations identified in Perry’s syndrome are shown as sticks in magenta. These mutations are located in a long loop that connects β3 and β4 strands and part of the ‘GKNDG’ binding motif. The location of G59 is also shown.

Figure 2

Characterization of the CAP-gly point mutations: A) NMR spectrum of the G71P point mutation indicates the domain is folded, but perturbed globally compared to WT. B) Thermal denaturation of each point mutation and WT CAP-gly. The closed symbols represent the ellipticity of the sample at 223 nm upon heating (e.g., 4 to 80 °C), the open symbols represent cooling (e.g., 80 to 4 °C).

Figure 3

Characterization of CAP-gly binding interactions: A) SDS-PAGE gel of the pelleted fraction of CAP-gly constructs in the presence and absence of MTs (see Supporting Information). B) Quantification of the intensities of precipitated fractions indicate that the mutated domains bind to taxol stabilized MTs better than the WT construct (grey bars; n=3). The amount of protein precipitated in the absence of MTs is shown as white bars. C) SEC analysis of the CAP-gly interaction with EB1. D) SDS-PAGE analysis of fractions 1 (first peak) and 2 (second peak) shown in panel C).