doi:10.1016/j.jmb.2012.04.005 J. Mol. Biol. (2012) 420,87–98 Contents lists available at www.sciencedirect.com Journal of Molecular Biology journal homepage: http://ees.elsevier.com.jmb Impacts of Dystrophin and Utrophin Domains on Actin Structural Dynamics: Implications for Therapeutic Design Ava Yun Lin, Ewa Prochniewicz, Davin M. Henderson, Bin Li, James M. Ervasti and David D. Thomas⁎ Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 6-155 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455, USA Received 27 January 2012; We have used time-resolved phosphorescence anisotropy (TPA) of actin received in revised form to evaluate domains of dystrophin and utrophin, with implications for 26 March 2012; gene therapy in muscular dystrophy. Dystrophin and its homolog accepted 2 April 2012 utrophin bind to cytoskeletal actin to form mechanical linkages that Available online prevent muscular damage. Because these proteins are too large for most 11 April 2012 gene therapy vectors, much effort is currently devoted to smaller constructs. We previously used TPA to show that both dystrophin and Edited by R. Craig utrophin have a paradoxical effect on actin rotational dynamics— restricting amplitude while increasing rate, thus increasing resilience, Keywords: with utrophin more effective than dystrophin. Here, we have evaluated time-resolved individual domains of these proteins. We found that a “mini-dystrophin,” phosphorescence anisotropy; lacking one of the two actin-binding domains, is less effective than TPA; dystrophin in regulating actin dynamics, correlating with its moderate muscular dystrophy; effectiveness in rescuing the dystrophic phenotype in mice. In contrast, gene therapy we found that a “micro-utrophin,” with more extensive internal deletions, is as effective as full-length dystrophin in the regulation of actin dynamics. Each of utrophin's actin-binding domains promotes resilience in actin, while dystrophin constructs require the presence of both actin- binding domains and the C-terminal domain for full function. This work supports the use of a utrophin template for gene or protein therapy designs. Resilience of the actin–protein complex, measured by TPA, correlates remarkably well with previous reports of functional rescue by dystrophin and utrophin constructs in mdx mice. We propose the use of TPA as an in vitro method to aid in the design and testing of emerging gene therapy constructs. © 2012 Elsevier Ltd. All rights reserved. Introduction Duchenne and Becker muscular dystrophies are caused by mutations in dystrophin, a 427-kDa protein localized to the cytoskeletal lattice of the 1 *Corresponding author. E-mail address: [email protected]. sarcolemma. Dystrophin's N-terminus binds to Abbreviations used: TPA, time-resolved cytoskeletal actin, and the C-terminus (CT) binds phosphorescence anisotropy; STR, spectrin-type repeat; to the dystroglycoprotein complex at the sarcolem- wt, wild type; ErIA, erythrosine iodoacetamide; NIH, ma membrane (Fig. 1). Lack of functional dystro- National Institutes of Health. phin in skeletal muscles results in disarray of the 0022-2836/$ - see front matter © 2012 Elsevier Ltd. All rights reserved. 88 Impacts of Dystrophin and Utrophin Domains Fig. 1. Diagram of actin filament rotational motions in complexes with dystrophin and utrophin. The homologous structures of the two proteins are indicated schematically. Each has an N-terminal actin- binding domain (ABD1, contain- ing tandem CH domains) and a C-terminal domain (CT) that binds to the sarcolemma, with intervening STRs (ovals) and hinges (H). On the TPA timescale (1–1000 ms), the detected motions are dominated by twisting,2,3 but the double-helical structure of the actin filament strongly couples its twisting and bending flexibility.4 The ratio of rotational rate to amplitude, detected by TPA, defines the filament's resilience (see Methods), which is increased by both dystrophin and utrophin.5 cytoskeletal organization at key structural regions in the amplitude (increases the order) of actin's – striated muscles,6 8 disabling proper transmission rotational flexibility while also increasing the rate, or diffusion of lateral force9,10 and rendering the both in a highly cooperative manner, thus creating a muscle susceptible to eccentric contraction more resilient complex.5 We found that utrophin is damage.11 Atomic force microscopy studies showed much more effective than dystrophin in increasing a decrease in myocyte stiffness due to lack of this resilience. The goal of the present study is to dystrophin.12 A similar loss in stiffness was ob- understand more clearly how the individual do- served when cytochalasin D was applied to disrupt mains of dystrophin and utrophin influence these the actin cytoskeleton, indicating that the dystro- effects on the resilience of actin. phin–actin interaction plays a central role in main- This goal is important for the rational design of taining mechanical stability in the muscle gene therapy for muscular dystrophy, which is cytoskeleton.12 Our goal is to elucidate the molec- currently being developed using recombinant ular mechanism by which the dystrophin–actin adeno-associated virus vectors. These vectors are interaction contributes to the molecular mechanics size limited; thus, mini- and micro-versions of of force transmission and to use this understanding dystrophin and utrophin have been developed to improve therapeutic strategies. with large deletions of the central STR domains, – Utrophin is a 395-kDa dystrophin homolog that including all or part of ABD2.25 28 Since these is present at the subsarcolemmal region, with TRIT (therapeutically relevant internally truncated) functions similar to dystrophin in fetal or regen- constructs rely on less than a third of the full- erating muscle (Fig. 1).8 As muscle fibers mature, length protein, there is a need to evaluate how – utrophin is replaced by dystrophin,13 15 but individual regions in dystrophin and utrophin utrophin has been proposed as a viable therapeutic affect their interaction with actin. The present replacement in dystrophin-deficient mice.16,17 Both study applies TPA to further evaluate the effects of proteins contain a highly homologous N-terminal isolated regions in dystrophin and utrophin actin-binding domain (ABD1) consisting of tandem through deletion constructs (Fig. 2).29,30 Two CH (calponin homology) motifs, followed by a TRIT constructs, referred to as mini-dystrophin central domain containing a series of triple-helical (Mini-Dys in Fig. 2a) and micro-utrophin (Micro- spectrin-type repeats (STRs), and a C-terminal (CT) Utr in Fig. 2b), were selected due to the availability region.18,19 However, dystrophin and utrophin of consistent physiological studies in dystrophic have distinctly different lateral interactions with (mdx) mice.28,31,32 Additional deletion constructs, actin.20 Dystrophin's second actin-binding domain with either N- or C-terminal truncations, were (ABD2) is separated from ABD1 by 10 STRs,21,22 engineered to test specific domains within dystro- while utrophin's ABD2 is adjacent to ABD123,24 phin and utrophin (Fig. 2).29,30 Our goal is to use (Fig. 1). TPA to (a) create a biophysical blueprint that We previously used time-resolved phosphores- identifies key regions in dystrophin and utrophin cence anisotropy (TPA) of actin to determine how required to regulate actin microsecond structural dystrophin and utrophin affect the structural dy- dynamics, (b) investigate why the most promising namics of the actin filament (Fig. 1).5 We showed therapeutic constructs have limited effects on that the binding of dystrophin or utrophin decreases rescuing the dystrophic phenotype in mice, and Impacts of Dystrophin and Utrophin Domains 89 Fig. 2. Constructs evaluated by TPA in this study. (a) Dystrophin. (b) Utrophin. CT, non-actin-binding C-terminal domain. Intervening cir- cles are STRs. Actin-binding do- mains are depicted in black. This color scheme is used consistently in subsequent figures. (c) establish TPA as a rapid in vitro method for up to 46% of the central STR region is missing but selecting potential therapeutic dystrophin and the patients can remain ambulatory past 60 years of – utrophin constructs according to their ability to age.35 37 Despite the extensive internal deletions, mimic the effects of full-length dystrophin on actin leaving only eight STRs, this construct has shown dynamics. high efficacy in rescuing the dystrophic phenotype in mdx mice.26,33,38 To compare the TPA results Results independently of the different actin-binding affini- ties of the constructs used in this study, we report results as a function of the fractional saturation of ν 2,5 Microsecond dynamics of actin in the presence binding sites on actin [ =y/Bmax; Eq. (7)]. Direct of therapeutically promising mini-dystrophin comparison of the TPA decays when actin binding is 50% saturated (ν=0.5) shows that Mini-Dys is quite To provide motivational context for this study, we similar to full-length dystrophin (Dys) in its ability begin with the mini-dystrophin construct (“Mini- to regulate actin structural dynamics (Fig. 3b). Dys” in Figs. 2aand3a).26,29,30,33 This TRIT Indeed, Mini-Dys decreases the amplitude of rota- (designated DysΔH2-R19 because it lacks the tional dynamics while decreasing the rate (Fig. 3c portion from hinge H2 to STR19) is modeled after and d), corresponding to increased actin resilience. cases of mild Becker muscular dystrophy, in which This supports our hypothesis that increased actin Fig. 3. TPA results comparing dystrophin (Dys) with mini-dystrophin (Mini-Dys). (a) Schematics of Dys and Mini-Dys, – with actin-binding domains in black.20,26,29 33 (b) TPA of actin alone (black) or bound to Dys (green) or Mini-Dys (purple) at 50% saturation of actin binding (n=0.5). Amplitude (c) and Rate (d) of actin rotational motion from TPA decays, as a function of ν [Eq. (7)]. Curves show fits according to Eq. (8), yielding the degree of cooperativity (n).3,34 90 Impacts of Dystrophin and Utrophin Domains resilience is a key to the function of dystrophin or its we used several other deletion constructs (Fig.