Crystal structure of the actin-binding domain of α-actinin 1: Evaluating two competing actin-binding models

Borrego-Diaz, Emma; Kerff, Frédéric; Lee, Sung Haeng; Ferrón, François; Liu, Yu; Domínguez, Roberto

doi:10.1016/j.jsb.2006.01.013

Cited by 57 publications

(76 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…EM studies have generated controversy, with competing closed (compact) and open (extended) models proposed for Utr261 (Fig. 1), α-actinin (17,20,22), and fimbrin (21,23). The present study provides direct high-resolution measurements on the relative disposition of tandem CH domains bound to actin, clearly resolving the controversy for utrophin and establishing powerful methodology that should be applicable to the other important members of the spectrin superfamily of actin-binding proteins.…”

Section: Discussionmentioning

confidence: 69%

“…Based on crystal structures showing a variable linkage between CH domains, it has been proposed that tandem CH domains are highly dynamic in both structure and function (13,17,18). Our study provides high-resolution structural information on one of these tandem CH domains in solution, showing that it is in equilibrium between two distinct conformations.…”

Section: Discussionmentioning

confidence: 71%

“…The crystal structure of Utr261 suggests that the central helical region connecting CH1 and CH2 is highly flexible. Even for α-actinin, which has a closed crystal structure, computational analysis suggests the potential for a high degree of dynamic flexibility that facilitates actin binding (17). A method is needed that allows high-resolution detection of multiple structural states in the presence of flexibility and disorder, both free and bound to actin.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Large-scale opening of utrophin’s tandem calponin homology (CH) domains upon actin binding by an induced-fit mechanism

Lin

Próchniewicz

James

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

We have used site-directed spin labeling and pulsed electron paramagnetic resonance to resolve a controversy concerning the structure of the utrophin-actin complex, with implications for the pathophysiology of muscular dystrophy. Utrophin is a homolog of dystrophin, the defective protein in Duchenne and Becker muscular dystrophies, and therapeutic utrophin derivatives are currently being developed. Both proteins have a pair of N-terminal calponin homology (CH) domains that are important for actin binding. Although there is a crystal structure of the utrophin actin-binding domain, electron microscopy of the actin-bound complexes has produced two very different structural models, in which the CH domains are in open or closed conformations. We engineered a pair of labeling sites in the CH domains of utrophin and used dipolar electron-electron resonance to determine the distribution of interdomain distances with high resolution. We found that the two domains are flexibly connected in solution, indicating a dynamic equilibrium between two distinct open structures. Upon actin binding, the two domains become dramatically separated and ordered, indicating a transition to a single open and extended conformation. There is no trace of this open conformation of utrophin in the absence of actin, providing strong support for an induced-fit model of actin binding.pulsed EPR | spectroscopy | cryo-EM U trophin is a homolog protein of dystrophin that has shown high therapeutic promise for the treatment of muscular dystrophy (1). It is endogenously found in fetal or regenerating muscle but is replaced by dystrophin, the defective protein in Duchenne and Becker muscular dystrophies, as the muscle matures (2). Up-regulation of utrophin in mdx mice, which lack dystrophin, has been shown to rescue its dystrophic phenotype, improving muscle morphology and function (1, 3). The full-length protein is not required to improve dystrophic pathology in mdx mice; i.e., substantial internal truncations in utrophin can be tolerated (4). These internally truncated constructs for muscular dystrophy therapeutics support the importance of actin binding by the N-terminal portions of either dystrophin or utrophin (5). Utrophin (395 kD) and dystrophin (427 kD) both contain highly homologous N-terminal actin-binding domains (ABD1), consisting of a pair of calponin homology (CH) domains. Despite additional actin-binding regions identified in the central spectrin-type repeats (6), microutrophin constructs with high potential for clinical applications rely almost exclusively on the N-terminal CH domains for actin interaction (7,8). Therefore, understanding the structural interaction between utrophin CH domains and actin has become crucial for the rational development of therapeutic constructs.More generally, there is an urgent need for a structural blueprint of CH domain-actin complexes for the entire spectrin superfamily of actin-binding proteins (e.g., fimbrin and α-actinin), of which dystrophin and utrophin are members. The diversity of crystal structur...

show abstract

Section: Discussionmentioning

confidence: 69%

Section: Discussionmentioning

confidence: 71%

mentioning

confidence: 99%

See 1 more Smart Citation

Large-scale opening of utrophin’s tandem calponin homology (CH) domains upon actin binding by an induced-fit mechanism

Lin

Próchniewicz

James

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…N-terminal sequencing of the ϳ80-kDa breakdown product resulting from calpain 2 proteolysis of PtdIns(3,4,5)P 3 -bound ␣-actinin revealed that cleavage occurred after tyrosine 246 (Fig. 8) within the final helix of the CH2 domain (31). Although the signal was significantly lower, evidence for a secondary cleavage site after serine 243 was also observed.…”

Section: Ptdins(345)p 3 Binding Increases the Susceptibility Of ␣-Amentioning

confidence: 98%

Phosphoinositide Binding to the Substrate Regulates Susceptibility to Proteolysis by Calpain

Sprague¹,

Fraley

Jang

et al. 2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

Calpain-mediated proteolysis regulates cytoskeletal dynamics and is altered during aging and the progression of numerous diseases or pathological conditions. Although several cytoskeletal proteins have been identified as substrates, how localized calpain activity is regulated and the mechanisms controlling substrate recognition are not clear. In this study, we report that phosphoinositide binding regulates the susceptibility of the cytoskeletal adhesion protein ␣-actinin to proteolysis by calpains 1 and 2. At first, ␣-actinin did not appear to be a substrate for calpain 2; however, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) binding to ␣-actinin resulted in nearly complete proteolysis of the full-length protein, producing stable breakdown products. Calpain 1 was able to cleave ␣-actinin in the absence of phosphoinositide binding; however, PtdIns(3,4,5)P 3 binding increased the rate of proteolysis, and phosphatidylinositol 4,5-diphosphate (PtdIns(4,5)P 2 ) binding significantly inhibited cleavage. Phosphoinositide binding appeared to regulate calpain proteolysis of ␣-actinin by modulating the exposure of a highly sensitive cleavage site within the calponin homology 2 domain. In U87MG glioblastoma cells, which contain elevated levels of PtdIns(3,4,5)P 3 , ␣-actinin colocalized with calpain within dynamic actin cytoskeletal structures. Furthermore, proteolysis of ␣-actinin producing stable breakdown products was observed in U87MG cells treated with calcium ionophore to activate the calcium-dependent calpains. Additional evidence of PtdIns(3,4,5)P 3 -mediated calpain proteolysis of ␣-actinin was observed in rat embryonic fibroblasts. These results suggest that PtdIns(3,4,5)P 3 binding is a critical determinant for ␣-actinin proteolysis by calpain. In conclusion, phosphoinositide binding to the substrate is a potential mechanism for regulating susceptibility to proteolysis by calpain.Calpains 1 and 2 are ubiquitous calcium-dependent proteases that play an important role in the signaling of various cellular processes and have been implicated in the degeneration observed in numerous pathological conditions (1). The requirement of calcium concentrations above physiological levels, micromolar for calpain 1 and millimolar for calpain 2, has stimulated much investigation for other factors involved in the activation of calpain. Autolysis lowers the concentration of calcium required for half-maximal activity from 7.1 to 0.6 M for calpain 1 and from 1000 to 180 M for calpain 2 (2). However, there is currently no evidence that autolysis is required for calpain modulation in cells. PtdIns(4,5)P 2 3 binding also lowers the concentration of calcium required for the activation of calpain (3-5) and is a potential mechanism for regulating calpain during cell migration (6). More recently, Glading et al. (7-9) published a series of studies showing that phosphorylation on serine 50 by extracellular signal-regulated kinase activates calpain 2, mediating detachment of the rear of the cell during epidermal growth factor-i...

show abstract

“…ABS1 is located in the CH1 domain and remains largely buried (16). Recently, the atomic structure of the ␣-actinin-1 ABD was published (17). ␣-Actinin-1 and -4 share Ͼ90% homology in the crystallized region; thus, the structural findings reported are likely true for ␣-actinin-4 as well.…”

mentioning

confidence: 97%

Disease-associated mutant α-actinin-4 reveals a mechanism for regulating its F-actin-binding affinity

Weins¹,

Schlöndorff²,

Nakamura

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

141

158

View full text Add to dashboard Cite

␣-Actinin-4 is a widely expressed protein that employs an actinbinding site with two calponin homology domains to crosslink actin filaments (F-actin) in a Ca 2؉ -sensitive manner in vitro. An inherited, late-onset form of kidney failure is caused by point mutations in the ␣-actinin-4 actin-binding domain. Here we show that ␣-actinin-4/F-actin aggregates, observed in vivo in podocytes of humans and mice with disease, likely form as a direct result of the increased actin-binding affinity of the protein. We document that exposure of a buried actin-binding site 1 in mutant ␣-actinin-4 causes an increase in its actin-binding affinity, abolishes its Ca 2؉ regulation in vitro, and diverts its normal localization from actin stress fibers and focal adhesions in vivo. Inactivation of this buried actin-binding site returns the affinity of the mutant to that of the WT protein and abolishes aggregate formation in cells. In vitro, actin filaments crosslinked by the mutant ␣-actinin-4 exhibit profound changes of structural and biomechanical properties compared with WT ␣-actinin-4. On a molecular level, our findings elucidate the physiological importance of a dynamic interaction of ␣-actinin with F-actin in podocytes in vivo. We propose that a conformational change with full exposure of actin-binding site 1 could function as a switch mechanism to regulate the actin-binding affinity of ␣-actinin and possibly other calponin homology domain proteins under physiological conditions. cytoskeleton ͉ kidney ͉ podocyte ͉ glomerulosclerosis

show abstract

Crystal structure of the actin-binding domain of α-actinin 1: Evaluating two competing actin-binding models

Cited by 57 publications

References 46 publications

Large-scale opening of utrophin’s tandem calponin homology (CH) domains upon actin binding by an induced-fit mechanism

Large-scale opening of utrophin’s tandem calponin homology (CH) domains upon actin binding by an induced-fit mechanism

Phosphoinositide Binding to the Substrate Regulates Susceptibility to Proteolysis by Calpain

Disease-associated mutant α-actinin-4 reveals a mechanism for regulating its F-actin-binding affinity

Contact Info

Product

Resources

About