Cytoplasmic b-actin supports fundamental cellular processes in healthy and diseased cells including cell adhesion, migration, cytokinesis and maintenance of cell polarity. Mutations in ACTB, the gene encoding cytoplasmic b-actin, lead to severe disorders with a broad range of symptoms. The two dominant heterozygous gain-of-function b-actin mutations p.R183W and p.E364K were identified in patients with developmental malformations, deafness and juvenile-onset dystonia (p.R183W) and neutrophil dysfunction (p.E364K). Here, we report the recombinant production and functional characterization of the two mutant proteins. Arg183 is located near the nucleotide-binding pocket of actin. Our results from biochemical studies and molecular dynamics simulations show that replacement by a tryptophan residue at position 183 establishes an unusual stacking interaction with Tyr69 that perturbs nucleotide release from actin monomers and polymerization behavior by inducing a closed state conformation. The replacement of Glu364 by a lysine residue appears to act as an allosteric trigger event leading to the preferred formation of the closed state. Thus, our approach indicates that both mutations affect interdomain mobility and nucleotide interactions as a basis for the formation of disease phenotypes in patients. IntroductionActin is a highly conserved and ubiquitous protein found in nearly all eukaryotic cells. Six actin isoforms can be distinguished in vertebrates: three a-actin isoforms (a-skeletal muscle, a-cardiac muscle and a-aortic smooth muscle, also known as a-vascular), one b-isoform (b-cytoplasmic) and two c-isoforms (c-cytoplasmic and c-smooth muscle also known as centeric). Cytoplasmic b-and c-actin are essential for cell migration, cell shape maintenance, mitosis and intracellular transport processes and are expressed at moderate to high levels in nearly all adult tissues [1,2]. The most abundant isoactin in many nonmuscle cells including myeloid and neuronal cells is b-actin [2]. Cellular studies show that the b-isoform is preferentially recruited into cellular protrusions, stress fibers, circular bundles and at cell-cell contacts [3,4]. The rapid cytoskeletal rearrangements observed for these structures appear to be linked to the highly dynamic turnover of actin filaments made from b-actin [5].All actin isoforms share the same architecture with four different subdomains and a common nucleotidebinding site. In vivo, b-actin is post-translationally modified by cleavage of the first methionine followed by N-terminal acetylation. Actin is an ATPase and the hydrophobic nucleotide-binding site is located in the cleft between subdomains SD1-2 and SD3-4 (Fig. 1A). In the presence of divalent cations, monomers with bound ATP assemble into filaments. The growth of actin filaments depends on the addition of actin-ATP monomers predominantly at the fast-growing barbed ends. After monomer addition, the bound ATP is hydrolyzed to ADP and inorganic phosphate (P i ) followed by P i -release. Because dissociation of actin-ADP occurs pr...
The hypothesis that increased heart muscle contractility leads to HCM and reduced contractility leads to DCM is based mainly on studies on myosin motor proteins. This hypothesis predicts that a change in cardiac actin (ACTC) may impact muscle contractility. To test this prediction, we determined the interactions between myosin and 8 of 16 ACTC mutant proteins that are known to cause hypertrophic or dilated cardiomyopathy. R312H showed a decreased actin-activated S1 ATPase rate (13.150.63 mM/min) compared to WT (15.351.58 mM/min), whereas the rate with E99K was significantly higher (20.151.46mM/min). In vitro motility assays were performed with varying ATP concentrations. E99K exhibited increased K M (0.08650.02 mM) compared to WT (0.06850.02 mM). E99K demonstrated a significantly decreased V max (1.9350.1 mm/sec) compared to WT (3.3150.12 mm/sec). In contrast, M305 had a similar K M (0.08850.01 mm/sec) to E99K, but its V max (3.3950.11mm/sec) was similar to WT. Based on a 5 nm myosin step size, we calculated a duty ratio of about 0.04 for WT and most mutant actins; however, the duty ratio was twice as high for E99K. With thin-filament extracted and reconstituted muscle fibers with E99K, a slight, but significant increase in Ca 2þ sensitivity (DpCa=0.1150.04) was observed; consequently tension was slightly larger than that of WT for pCa ranging 5.6-6.0, without changing maximum tension at pCa 4.66. These experiments suggest that the primary change with the E99K actin mutant is enhanced ATP usage and increased tension at partial activation, which lead to HCM.
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