The Actin Binding Affinity of the Utrophin Tandem Calponin-Homology Domain Is Primarily Determined by Its N-Terminal Domain

Singh, Surinder; Bandi, Swati; Winder, Steve J.; Mallela, Krishna

doi:10.1021/bi500149q

Cited by 18 publications

(66 citation statements)

References 33 publications

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“…We separately expressed the minimal CH1 and CH2 domains from utrophin fused to mCherry ( Fig S1). The isolated CH1 domain of utrophin had a high relative bound fraction (0.55±0.09, p*<0.05) ( Fig 1D), although the isolated domain appeared partially insoluble, aggregating within cells ( Fig S1 A,B), consistent with previous observations about its stability in vitro (Singh et al, 2014). The isolated CH2 domain was soluble but distributed throughout the cytosol (Fig S1 C) with a low relative bound fraction (-0.27±0.06, p****<0.05) ( Fig 1D), implying that it alone has minimal actin-binding activity.…”

Section: Measurement Of Ch1-ch2 Domain Binding In Vitro and In Live Csupporting

confidence: 90%

“…An example showing the actin-binding domain of filamin A compared to utrophin ABD is given in Fig 1C. We first quantified the binding of CH1 and CH2 domains alone in live cells and compared to previous measurements of CH1 and CH2 affinity. Affinity of tandem CH1-CH2 domains for f-actin is known to primarily arise from the CH1 domain, as CH2 alone cannot bind to actin (Singh et al, 2014). We separately expressed the minimal CH1 and CH2 domains from utrophin fused to mCherry ( Fig S1).…”

Section: Measurement Of Ch1-ch2 Domain Binding In Vitro and In Live Cmentioning

confidence: 99%

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Steric Regulation of Tandem Calponin Homology Domain Actin-Binding Affinity

Harris¹,

Belardi²,

Jreij³

et al. 2019

Preprint

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Tandem calponin homology domains are common actin-binding motifs found in proteins such as α-actinin, filamin, utrophin, and dystrophin that organize actin filaments into functional structures. Despite a conserved structural fold and regions of high sequence similarity, tandem calponin homology (CH1-CH2) domains can have remarkably different actin-binding properties, with disease-associated point mutants known to cause increased as well as decreased affinity for f-actin. How small variations in sequence can lead to significant differences in binding affinity and resulting actin organization remains unclear. Here, we show that the affinity of CH1-CH2 domains for f-actin can be both increased and decreased by diverse modifications that change the effective 'openness' of CH1 and CH2 that sterically regulates binding to f-actin. We find that mutations to the interdomain linker, as well as changes to the inter-CH domain interface distinct from a well characterized cationπ interaction, 'open' the domain and increases binding affinity, while a modification that adds molecular size to the CH2 'closes' the domain and decreases binding affinity. Interestingly, we observe non-uniform sub-cellular localization of CH1-CH2 domains to f-actin that depend on the N-terminal flanking region of CH1 but not on the overall affinity to f-actin. This dependence of CH1-CH2 binding properties on sequence and 'openness' contributes to a mechanistic understanding of disease-associated mutations and has implications for engineering actin-binding domains with specific properties.

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Section: Measurement Of Ch1-ch2 Domain Binding In Vitro and In Live Csupporting

confidence: 90%

Section: Measurement Of Ch1-ch2 Domain Binding In Vitro and In Live Cmentioning

confidence: 99%

Steric Regulation of Tandem Calponin Homology Domain Actin-Binding Affinity

Harris¹,

Belardi²,

Jreij³

et al. 2019

Preprint

View full text Add to dashboard Cite

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“…The latter does not show much change in structure (movie SM3). The cross play between the two CH subdomains has recently been highlighted . Our study demonstrates how the switch between the open and the closed forms can be brought about by the interaction of residues across CH1 and CH2 subdomains in utrophin.…”

Section: Discussionmentioning

confidence: 66%

“…The cross play between the two CH subdomains has recently been highlighted. 49 Our study demonstrates how the switch between the open and the closed forms can be brought about by the interaction of residues across CH1 and CH2 subdomains in utrophin. The opening/closing of the dystrophin is caused by change in the helicity of the linker region.…”

Section: Discussionmentioning

confidence: 71%

Flexibility in the N‐terminal actin‐binding domain: Clues from in silico mutations and molecular dynamics

2015

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Dystrophin is a long, rod-shaped cytoskeleton protein implicated in muscular dystrophy (MDys). Utrophin is the closest autosomal homolog of dystrophin. Both proteins have N-terminal actin-binding domain (N-ABD), a central rod domain and C-terminal region. N-ABD, composed of two calponin homology (CH) subdomains joined by a helical linker, harbors a few disease causing missense mutations. Although the two proteins share considerable homology (>72%) in N-ABD, recent structural and biochemical studies have shown that there are significant differences (including stability, mode of actin-binding) and their functions are not completely interchangeable. In this investigation, we have used extensive molecular dynamics simulations to understand the differences and the similarities of these two proteins, along with another actin-binding protein, fimbrin. In silico mutations were performed to identify two key residues that might be responsible for the dynamical difference between the molecules. Simulation points to the inherent flexibility of the linker region, which adapts different conformations in the wild type dystrophin. Mutations T220V and G130D in dystrophin constrain the flexibility of the central helical region, while in the two known disease-causing mutants, K18N and L54R, the helicity of the region is compromised. Phylogenetic tree and sequence analysis revealed that dystrophin and utrophin genes have probably originated from the same ancestor. The investigation would provide insight into the functional diversity of two closely related proteins and fimbrin, and contribute to our understanding of the mechanism of MDys.

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“…But the situation may not be this simple. A recent study has found that the isolated utrophin CH1 domain has a similar binding affinity as the intact utrophin ABD (CH1 and CH2 domains) while the isolated utrophin CH2 domain shows only very weak binding 44 . The utrophin CH1 has both the ABS1 and ABS2 which is consistent with a requirement for the ABS2 for F-actin binding.…”

Section: Discussionmentioning

confidence: 99%

The IQGAP1 N-Terminus Forms Dimers, and the Dimer Interface Is Required for Binding F-Actin and Calcium-Bound Calmodulin

et al. 2016

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IQGAP1 is a multi-domain scaffold protein involved in many cellular processes. We have determined the crystal structure of an N-terminal fragment spanning residues 1-191 (CHDF hereafter) which contains the entire calponin homology domain. The structure of the CHDF is very similar to other Type 3 calponin homology domains like those from calponin, Vav, and the yeast IQGAP1 ortholog Rng2. However, in the crystal two CHDF molecules form a "head-tohead" or parallel dimer through mostly hydrophobic interactions. Binding experiments indicate that the CHDF binds to both F-actin and Ca 2+ /calmodulin, but binding is mutually exclusive. Based on the structure, two dimer interface substitutions were introduced. While CHDFL157D disrupts the dimer in gel filtration experiments, oxidized CHDFK161C stabilizes the dimer. These results imply that the CHDF forms the same dimer in solution that is seen in the crystal structure. The disulfide-stabilized dimer displays reduced F-actin binding in sedimentation assays, and shows no binding to Ca 2+ /calmodulin in isothermal titration calorimetry (ITC) experiments indicating that interface residues are utilized for both binding events. The Calmodulin Target Database predicts that residues 93 KK 94 are important for CaM binding, and indeed the 93 EE 94 double mutation displays reduced binding to Ca 2+ /calmodulin in ITC experiments. Our results indicate that the CHDF dimer interface is used for both F-actin and Ca 2+ /calmodulin binding, and 93 KK 94 , near the interface, are also used for Ca 2+ /calmodulin binding. These results are also consistent with full-length IQGAP1 forming a parallel homodimer. Graphical Abstract HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptHuman IQGAP1 has been shown to bind to a number of proteins including F-actin, calmodulin (CaM), Cdc42, Rac1, β-catenin, E-cadherin, CLIP-170 and ezrin [1][2][3] . Through dynamic interaction with these binding partners, IQGAP1 coordinates cellular signaling, cytoskeletal rearrangements, and cell-cell adhesion to define cell polarization, facilitate cell migration and influence other important processes 4 . Over-expression of IQGAP1 has been observed in various cancer types, and in vitro models indicate that over-expression and abnormal localization of IQGAP1 can destabilize cell-cell contacts and promote cell migration and invasion 5,6 . IQGAP1 binds to and cross-links F-actin to form a gel-like meshwork and recently Pelikan et al. found that IQGAP1 can cap the barbed end of F-actin, thereby regulating actin polymerization [7][8][9] . In vitro studies have shown that IQGAP1 Nterminal fragments (residues 2-210, 1-216 and 1-232) can bind to filamentous actin (Factin) 7,10,11 . In addition, a fragment including residues 1-232 has also been shown to bind to calcium and also calcium-bound calmodulin (Ca 2+ /CaM). Heretofore, little was known about the molecular details of the IQGAP1 N-terminus and its interactions with F-actin and Ca 2+ /CaM.Calponin homology (CH) domains are ...

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The Actin Binding Affinity of the Utrophin Tandem Calponin-Homology Domain Is Primarily Determined by Its N-Terminal Domain

Cited by 18 publications

References 33 publications

Steric Regulation of Tandem Calponin Homology Domain Actin-Binding Affinity

Steric Regulation of Tandem Calponin Homology Domain Actin-Binding Affinity

Flexibility in the N‐terminal actin‐binding domain: Clues from in silico mutations and molecular dynamics

The IQGAP1 N-Terminus Forms Dimers, and the Dimer Interface Is Required for Binding F-Actin and Calcium-Bound Calmodulin

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