Abstract 42: Differential Contribution Of The N- And C-terminal Domains To The Folding And Function Of Tandem Calponin-homology Domains

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Krishna Mallela ◽  
Swati Bandi ◽  
Surinder Singh ◽  
Geoffrey Armstrong
Keyword(s):  
Full Length ◽  
Actin Binding ◽  
Main Function ◽  
Calponin Homology ◽  
Terminal Domain ◽  
Binding Domains ◽  
Binding Function ◽  
Ch2 Domain ◽  
The Stability ◽  
And Function

Tandem calponin-homology (CH) domains constitute a major class of actin-binding domains that include dystrophin and utrophin, the two key proteins involved in muscular dystrophy. Despite their importance, how their structure controls their function is not understood. Here, we study the contribution of individual CH domains to the actin-binding function and thermodynamic stability of utrophin’s tandem CH domain. Traditional actin co-sedimentation assays indicate that the isolated C-terminal CH2 domain binds weakly to F-actin when compared with the full-length tandem CH domain. In contrast, isolated CH1 binds to F-actin with a similar efficiency as that of the full-length tandem CH domain. Thus, the obvious question that arises is why tandem CH domains require CH2, when their actin-binding efficiency is originating primarily from CH1. To answer, we probed the thermodynamic stabilities of individual CH domains. Isolated CH1 domain is unstable and is prone to serious aggregation. Isolated CH2 is very stable, even appears to be more stable than the full-length tandem CH domain. In addition, the CH2 domain, which is more stable, is less functional. These results indicate that the main function of CH2 is to stabilize CH1. Consistently, the proposed structure of utrophin’s tandem CH domain based on earlier X-ray studies indicates a close proximity between the C-terminal helix of CH2 and the N-terminal helix of CH1, and this helix in CH2 is more dynamic in the full-length protein when compared with that in the absence of CH1, suggesting the mechanism by which CH2 stabilizes CH1. These observations indicate that the two CH domains contribute differentially to the folding and function of tandem CH domains, although both domains essentially have the same native structure in the tandem CH domain. The N-terminal domain determines the function, whereas the C-terminal domain determines the stability. This work was funded by the AHA Grant 11SDG4880046.

Abstract 286: Structural Determinants Of Thermodynamic Stability And Actin-binding Function Of Dystrophin And Utrophin

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Krishna Mallela ◽  
Swati Bandi ◽  
Surinder Singh
Keyword(s):  
Three Dimensional ◽  
Molecular Structures ◽  
Actin Binding ◽  
Protein Drug ◽  
Structural Determinants ◽  
Folding Rate ◽  
Incurable Disease ◽  
Binding Function ◽  
The Stability ◽  
And Function

Muscular dystrophy (MD) is an incurable disease, and affects all types of muscles. The decreased function of heart muscles causes heart problems such as cardiomyopathy and congestive heart failure. A deficiency in functional dystrophin protein in muscle cells triggers the onset of Duchenne MD and Becker MD. Utrophin is the closest homologue of dystrophin and is being tested as a protein drug to replace the loss of functional dystrophin in human patients. However, utrophin is less stable and binds to actin through a different mode of contact when compared with dystrophin. To optimize utrophin as the protein drug, we first need to understand how its molecular structure controls its stability and function. Utrophin and dystrophin bind to actin using tandem calponin-homology (CH) domains at their N-terminus. Individual CH domains of utrophin and dystrophin are very similar in amino acid sequence and their three dimensional structures. The major difference is in their relative orientation around the linker region that connects the two CH domains. To determine the role of the linker, we designed constructs with linkers switched between utrophin and dystrophin. Our results indicate that the tandem CH domain of utrophin with dystrophin linker (UDL) is more stable than that of utrophin but less stable than that of dystrophin. Similarly, tandem CH domain of dystrophin with utrophin linker (DUL) is less stable than that of dystrophin but more stable than that of utrophin. Kinetic folding and unfolding studies suggest that the linker predominantly affects the folding rate rather than the unfolding rate. In addition to stability and folding, the linker also controls protein aggregation. UDL is more prone to aggregation when compared with that of utrophin, whereas DUL is less prone to aggregation when compared with that of dystrophin. These results indicate that the linker plays a major role in controlling the stability and function of tandem CH domains, and further suggests that it is possible to engineer utrophin to behave precisely like dystrophin based on their molecular structures.

scholarly journals Overexpression of human fibroblast caldesmon fragment containing actin-, Ca++/calmodulin-, and tropomyosin-binding domains stabilizes endogenous tropomyosin and microfilaments.

1994 ◽  
Vol 125 (2) ◽  
pp. 359-368 ◽  
Author(s):  
K S Warren ◽  
J L Lin ◽  
D D Wamboldt ◽  
J J Lin
Keyword(s):  
Cho Cells ◽  
Actin Filaments ◽  
Actin Binding ◽  
Expression Vectors ◽  
Binding Domains ◽  
Microfilament Bundles ◽  
The Stability ◽  
Triton X

Fibroblast caldesmon is a protein postulated to participate in the modulation of the actin cytoskeleton and the regulation of actin-based motility. The cDNAs encoding the NH2-terminal (aa.1-243, CaD40) and COOH-terminal (aa.244-538, CaD39) fragments of human caldesmon were subcloned into expression vectors and we previously reported that bacterially produced CaD39 protein retains its actin-binding properties as well as its ability to enhance low M(r) tropomyosin (TM) binding to actin and to inhibit TM-actin-activated HMM ATPase activity in vitro (Novy, R. E., J. R. Sellers, L.-F. Liu, and J. J.-C. Lin. 1993. Cell Motil. Cytoskeleton. 26:248-261). Bacterially produced CaD40 does not bind actin. To study the in vivo effects of CaD39 expression on the stability of actin filaments in CHO cells, we isolated and characterized stable CHO transfectants which express varying amounts of CaD39. We found that expression of CaD39 in CHO cells stabilized microfilament bundles as well as endogenous TM. CaD39-expressing clones displayed an increased resistance to cytochalasin B and Triton X-100 treatments and yielded increased amounts of TM-containing actin filaments in microfilament isolation procedures. In addition, analysis of these clones with immunoblotting and indirect immunofluorescence microscopy with anti-TM antibody revealed that stabilized endogenous TM and enhanced TM-containing microfilament bundles parallel increased amounts of CaD39 expression. The increased TM observed corresponded to a decrease in TM turnover rate and did not appear to be due to increased synthesis of endogenous TM. Additionally, the phenomenon of stabilized TM did not occur in stable CHO clones expressing CaD40. Therefore, it is likely that CaD39 can enhance TM's binding to F-actin in vivo, thus reducing TM's rate of turnover and stabilizing actin microfilament bundles.

scholarly journals Structural characterization suggests models for monomeric and dimeric forms of full-length ezrin

2016 ◽  
Vol 473 (18) ◽  
pp. 2763-2782 ◽  
Author(s):  
Juanita M. Phang ◽  
Stephen J. Harrop ◽  
Anthony P. Duff ◽  
Anna V. Sokolova ◽  
Ben Crossett ◽  
...  
Keyword(s):  
Active Form ◽  
Coiled Coil ◽  
Full Length ◽  
Heptad Repeat ◽  
Actin Binding ◽  
Stable Complex ◽  
Globular Domain ◽  
Erm Proteins ◽  
Metazoan Evolution ◽  
Terminal Domain

Ezrin is a member of the ERM (ezrin–radixin–moesin) family of proteins that have been conserved through metazoan evolution. These proteins have dormant and active forms, where the latter links the actin cytoskeleton to membranes. ERM proteins have three domains: an N-terminal FERM [band Four-point-one (4.1) ERM] domain comprising three subdomains (F1, F2, and F3); a helical domain; and a C-terminal actin-binding domain. In the dormant form, FERM and C-terminal domains form a stable complex. We have determined crystal structures of the active FERM domain and the dormant FERM:C-terminal domain complex of human ezrin. We observe a bistable array of phenylalanine residues in the core of subdomain F3 that is mobile in the active form and locked in the dormant form. As subdomain F3 is pivotal in binding membrane proteins and phospholipids, these transitions may facilitate activation and signaling. Full-length ezrin forms stable monomers and dimers. We used small-angle X-ray scattering to determine the solution structures of these species. As expected, the monomer shows a globular domain with a protruding helical coiled coil. The dimer shows an elongated dumbbell structure that is twice as long as the monomer. By aligning ERM sequences spanning metazoan evolution, we show that the central helical region is conserved, preserving the heptad repeat. Using this, we have built a dimer model where each monomer forms half of an elongated antiparallel coiled coil with domain-swapped FERM:C-terminal domain complexes at each end. The model suggests that ERM dimers may bind to actin in a parallel fashion.

scholarly journals Single calponin homology domains are not actin-binding domains

1998 ◽  
Vol 8 (19) ◽  
pp. R673-R675 ◽  
Author(s):  
Mario Gimona ◽  
Steven J. Winder
Keyword(s):  
Actin Binding ◽  
Calponin Homology ◽  
Binding Domains ◽  
Homology Domains

scholarly journals The Nesprin-1/-2 ortholog ANC-1 regulates organelle positioning in C. elegans without its KASH or actin-binding domains

2020 ◽  
Author(s):  
Hongyan Hao ◽  
Shilpi Kalra ◽  
Laura E. Jameson ◽  
Leslie A. Guerrero ◽  
Natalie E. Cain ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Nuclear Membrane ◽  
Actin Binding ◽  
Nuclear Positioning ◽  
Outer Nuclear Membrane ◽  
Calponin Homology ◽  
C Elegans ◽  
Binding Domains ◽  
Null Phenotype ◽  
Er Membrane

AbstractKASH proteins in the outer nuclear membrane comprise the cytoplasmic half of LINC complexes that connect nuclei to the cytoskeleton. Caenorhabditis elegans ANC-1, an ortholog of Nesprin-1/2, contains actin-binding and KASH domains at opposite ends of a long spectrin-like region. Deletion of either the KASH or calponin homology (CH) domains does not completely disrupt nuclear positioning, suggesting neither KASH nor CH domains are essential. Deletions in the spectrin-like region of ANC-1 led to significant defects, but only recapitulated the null phenotype in combination with mutations in the trans-membrane span. In anc-1 mutants, the ER was unanchored, moving throughout the cytoplasm, and often fragmented. The data presented here support a cytoplasmic integrity model where ANC-1 localizes to the ER membrane and extends into the cytoplasm to position nuclei, ER, mitochondria, and likely other organelles in place.

scholarly journals Signature of N-terminal domain (NTD) structural re-orientation in NPC1 for proper alignment of cholesterol transport: Molecular dynamics study with mutation

2020 ◽  
Author(s):  
Hye-Jin Yoon ◽  
Hyunah Jeong ◽  
Hyung Ho Lee ◽  
Soonmin Jang
Keyword(s):  
Molecular Dynamics ◽  
Full Length ◽  
Cholesterol Transport ◽  
Cholesterol Trafficking ◽  
Terminal Domain ◽  
Loop Region ◽  
Niemann Pick ◽  
Dynamics Simulations ◽  
And Function ◽  
Lumenal Domain

AbstractThe lysosomal membrane protein NPC1 (Niemann-Pick type C1) and NPC2 (Niemann-Pick type C2) are main players of cholesterol control in lysosome and it is known that mutation on these proteins leads to cholesterol trafficking related disease, called Niemann-Pick disease type C (NPC) disease. The mutation R518W or R518Q on NPC1 is one of such disease-related mutations, causing reduced cholesterol transport by half, resulting in accumulation of cholesterol and lipids in late endosomal/lysosomal region of the cell. Even though there has been significant progress in understanding cholesterol transport by NPC1 in combination with NPC2, especially after the structural determination of full length NPC1 in 2016, many details such as interaction of full length NPC1 with NPC2, molecular motions responsible for cholesterol transport during and after this interaction, and structure and function relations of many mutations are still not well understood.We report the extensive molecular dynamics simulations to gain insight into the structure and motions of NPC1 lumenal domain for cholesterol transport and disease behind the mutation (R518W). It is found that the mutation induces structural shift of NTD (N-terminal domain), toward the loop region in MLD (middle lumenal domain), which is believed to play central role in interaction with NPC2 protein, such that the interaction with NPC2 protein might be less favorable compare to wild NPC1. Also, the simulation indicates the possible re-orientation of the NTD, aligning to form an internal tunnel, after receiving the cholesterol from NPC2 with wild NPC1 unlike the mutated one, a possible pose for further action in cholesterol trafficking. We believe the current study can provide better understanding on the cholesterol transport by NPC1, especially the role of NTD of NPC1, in combination with NPC2 interaction.Synopsismodeling study of cholesterol binding protein NPC1

scholarly journals Steric Regulation of Tandem Calponin Homology Domain Actin-Binding Affinity

2019 ◽  
Author(s):  
Andrew R Harris ◽  
Brian Belardi ◽  
Pamela Jreij ◽  
Kathy Wei ◽  
Hengameh Shams ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Sequence Similarity ◽  
Point Mutations ◽  
Negative Regulator ◽  
Actin Binding ◽  
Binding Properties ◽  
High Sequence Similarity ◽  
Calponin Homology ◽  
Binding Domains

ABSTRACTTandem calponin homology (CH1-CH2) domains are common actin-binding domains in proteins that interact with and organize the actin cytoskeleton. Despite regions of high sequence similarity, CH1-CH2 domains can have remarkably different actin-binding properties, with disease-associated point mutants known to increase as well as decrease affinity for f-actin. To investigate features that affect CH1-CH2 affinity for f-actin in cells and in vitro, we perturbed the utrophin actin-binding domain by making point mutations at the CH1-CH2 interface, replacing the linker domain, and adding a PEG polymer to CH2. Consistent with a previous model describing CH2 as a steric negative regulator of actin binding, we find that utrophin CH1-CH2 affinity is both increased and decreased by modifications that change the effective ‘openness’ of CH1 and CH2 in solution. We also identified interface mutations that caused a large increase in affinity without changing solution ‘openness’, suggesting additional influences on affinity. Interestingly, we also observe non-uniform sub-cellular localization of utrophin CH1-CH2 that depends on the N-terminal flanking region but not on bulk affinity. These observations provide new insights into how small sequence changes, such as those found in diseases, can affect CH1-CH2 binding properties.

scholarly journals Mutagenesis analysis of human SM22: characterization of actin binding

2000 ◽  
Vol 89 (5) ◽  
pp. 1985-1990 ◽  
Author(s):  
Yiping Fu ◽  
Hong Wei Liu ◽  
Sean M. Forsythe ◽  
Paul Kogut ◽  
John F. McConville ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Full Length ◽  
Serine Phosphorylation ◽  
Site Directed Mutagenesis ◽  
Actin Binding ◽  
Internal Deletion ◽  
Terminal Domain ◽  
Kinase C

SM22 is a 201-amino acid actin-binding protein expressed at high levels in smooth muscle cells. It has structural homology to calponin, but how SM22 binds to actin remains unknown. We performed site-directed mutagenesis to generate a series of NH2-terminal histidine (His)-tagged mutants of human SM22 in Escherichia coli and used these to analyze the functional importance of potential actin binding domains. Purified full-length recombinant SM22 bound to actin in vitro, as demonstrated by cosedimentation assay. Binding did not vary with calcium concentration. The COOH-terminal domain of SM22 is required for actin affinity, because COOH terminally truncated mutants [SM22-(1–186) and SM22-(1–166)] exhibited markedly reduced cosedimentation with actin, and no actin binding of SM22-(1–151) could be detected. Internal deletion of a putative actin binding site (154-KKAQEHKR-161) partially prevented actin binding, as did point mutation to neutralize either or both pairs of positively charged residues at the ends of this region (KK154LL and/or KR160LL). Internal deletion of amino acids 170–180 or 170–186 also partially or almost completely inhibited actin cosedimentation, respectively. Of the three consensus protein kinase C or casein kinase II phosphorylation sites in SM22, only Ser-181 was readily phosphorylated by protein kinase C in vitro, and such phosphorylation greatly decreased actin binding. Substitution of Ser-181 to aspartic acid (to mimic serine phosphorylation) also reduced actin binding. Immunostains of transiently transfected airway myocytes revealed that full-length NH2-terminal FLAG-tagged SM22 colocalizes with actin filaments, whereas FLAG-SM22-(1–151) does not. These data confirm that SM22 binds to actin in vitro and in vivo and, for the first time, demonstrate that multiple regions within the COOH-terminal domain are required for full actin affinity.

scholarly journals Three determinants in ezrin are responsible for cell extension activity.

1997 ◽  
Vol 8 (8) ◽  
pp. 1543-1557 ◽  
Author(s):  
M Martin ◽  
C Roy ◽  
P Montcourrier ◽  
A Sahuquet ◽  
P Mangeat
Keyword(s):  
Amino Acids ◽  
Amino Acid Sequences ◽  
Full Length ◽  
Actin Binding ◽  
Cell Extension ◽  
Erm Proteins ◽  
Terminal Domain ◽  
Extension Activity ◽  
Dependent Cell

The ERM proteins--ezrin, radixin, and moesin--are key players in membrane-cytoskeleton interactions. In insect cells infected with recombinant baculoviruses, amino acids 1-115 of ezrin were shown to inhibit an actin- and tubulin-dependent cell-extension activity located in ezrin C-terminal domain (ezrin310-586), whereas full-length ezrin1-586 did not induce any morphological change. To refine the mapping of functional domains of ezrin, 30 additional constructs were overexpressed in Sf9 cells, and the resulting effect of each was qualitatively and semiquantitatively compared. The removal of amino acids 13-30 was sufficient to release a cell-extension phenotype. This effect was abrogated if the 21 distal-most C-terminal amino acids were subsequently deleted (ezrin31-565), confirming the existence of a head-to-tail regulation in the whole molecule. Surprisingly, the deletion in full-length ezrin of the same 21 amino acids provided strong cell-extension competence to ezrin1-565, and this property was recovered in N-terminal constructs as short as ezrin1-310. Within ezrin1-310, amino acid sequences 13-30 and 281-310 were important determinants and acted in cooperation to induce cytoskeleton mobilization. In addition, these same residues are part of a new actin-binding site characterized in vitro in ezrin N-terminal domain.

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