Calponin Homology Research Articles

The N-Terminal Flanking Region Modulates the Actin Binding Affinity of the Utrophin Tandem Calponin-Homology Domain.

Despite sharing a high degree of sequence similarity, the tandem calponin-homology (CH) domain of utrophin binds to actin 30 times stronger than that of dystrophin. We have previously shown that this difference in actin binding affinity could not be ascribed to the differences in inter-CH-domain linkers [Bandi, S., et al. (2015) Biochemistry 54, 5480-5488]. Here, we examined the role of the N-terminal flanking region. The utrophin tandem CH domain contains a 27-residue flanking region before its CH1 domain. We examined its effect by comparing the structure and function of full-length utrophin tandem CH domain Utr(1-261) and its truncated Utr(28-261) construct. Both full-length and truncated constructs are monomers in solution, with no significant differences in their secondary or tertiary structures. Truncated construct Utr(28-261) binds to actin 30 times weaker than that of the full-length Utr(1-261), similar to that of the dystrophin tandem CH domain with a much shorter flanking region. Deletion of the N-terminal flanking region stabilizes the CH1 domain. The magnitude of the change in binding free energy upon truncation is similar to that of the change in thermodynamic stability. The isolated N-terminal peptide by itself is significantly random coil and does not bind to F-actin in the affinity range of Utr(1-261) and Utr(28-261). These results indicate that the N-terminal flanking region significantly affects the actin binding affinity of tandem CH domains. This observation further stresses that protein regions other than the three actin-binding surfaces identified earlier, irrespective of whether they directly bind to actin, also contribute to the actin binding affinity of tandem CH domains.

IFT54 regulates IFT20 stability but is not essential for tubulin transport during ciliogenesis.

Intraflagellar transport (IFT) is required for ciliogenesis by ferrying ciliary components using IFT complexes as cargo adaptors. IFT54 is a component of the IFT-B complex and is also associated with cytoplasmic microtubules (MTs). Loss of IFT54 impairs cilia assembly as well as cytoplasmic MT dynamics. The N-terminal calponin homology (CH) domain of IFT54 interacts with tubulins/MTs and has been proposed to transport tubulin during ciliogenesis, whereas the C-terminal coiled-coil (CC) domain binds IFT20. However, the precise function of these domains in vivo is not well understood. We showed that in Chlamydomonas, loss of IFT54 completely blocks ciliogenesis but does not affect spindle formation and proper cell cycle progression, even though IFT54 interacts with mitotic MTs. Interestingly, IFT54 lacking the CH domain allows proper flagellar assembly. The CH domain is required for the association of IFT54 with the axoneme but not with mitotic MTs, and also regulates the flagellar import of IFT54 but not IFT81 and IFT46. The C-terminal CC domain is essential for IFT54 to bind IFT20, and for its recruitment to the basal body and incorporation into IFT complexes. Complete loss of IFT54 or the CC domain destabilizes IFT20. ift54 mutant cells expressing the CC domain alone rescue the stability of IFT20 and form stunted flagella with accumulation of both IFT-A component IFT43 and IFT-B component IFT46, indicating that IFT54 also functions in IFT turn-around at the flagellar tip.

Crenomytilus grayanus 40 kDa calponin-like protein: cDNA cloning, sequence analysis, tissue expression, and post-translational modifications

Calponin-like protein (CaP-40), a third major protein after actin and tropomyosin, has recently been identified by us in the Ca2+-regulated thin filaments of mussel Crenomytilus grayanus. It contains calponin homology domain, five calponin family repeats and possesses similar biochemical properties as vertebrate smooth muscle calponin. In this paper, we report a full-length cDNA sequence of CaP-40, study its expression pattern on mRNA and protein levels, evaluate CaP-40 post-translational modifications and perform protein-protein interaction analysis.The full-length sequence of CaP-40 consists of 398 amino acids and has high similarity to calponins among molluscan species. CaP-40 gene is widely expressed in mussel tissues, with the highest expression in adductor and mantle. Comparison of these data with protein content established by mass-spectrometry analysis revealed that the high mRNA content is mirrored by high protein levels for adductor smooth muscles. To provide unbiased insight into the function of CaP-40 and effect of its over-expression in adductor smooth muscle, we built protein-protein interaction network of identified Crenomytilus grayanus proteome. In addition, we showed that CaP-40 is subjected to post-translational N- and C-terminal acetylation at N127, G229 and G349 sites which potentially regulates its function in vivo.

Formin' bridges between microtubules and actin filaments in axonal growth cones.

Formin' bridges between microtubules and actin filaments in axonal growth cones.

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

IQGAP1 is a multidomain scaffold protein involved in many cellular processes. We have determined the crystal structure of an N-terminal fragment spanning residues 1-191 (CHDF hereafter) that contains the entire calponin homology domain. The structure of the CHDF is very similar to those of 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-to-head" or parallel dimer through mostly hydrophobic interactions. Binding experiments indicate that the CHDF binds to both F-actin and Ca2+/calmodulin, but binding is mutually exclusive. On the basis of 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 a reduced level of F-actin binding in sedimentation assays and shows no binding to Ca2+/calmodulin in isothermal titration calorimetry (ITC) experiments, indicating that interface residues are utilized for both binding events. The Calmodulin Target Database predicts that residues 93KK94 are important for CaM binding, and indeed, the 93EE94 double mutation displays a reduced level of binding to Ca2+/calmodulin in ITC experiments. Our results indicate that the CHDF dimer interface is used for both F-actin and Ca2+/calmodulin binding, and the 93KK94 pair, near the interface, is also used for Ca2+/calmodulin binding. These results are also consistent with full-length IQGAP1 forming a parallel homodimer.

The Calponin Family Member CHDP-1 Interacts with Rac/CED-10 to Promote Cell Protrusions.

Eukaryotic cells extend a variety of surface protrusions to direct cell motility. Formation of protrusions is mediated by coordinated actions between the plasma membrane and the underlying actin cytoskeleton. Here, we found that the single calponin homology (CH) domain-containing protein CHDP-1 induces the formation of cell protrusions in C. elegans. CHDP-1 is anchored to the cortex through its amphipathic helix. CHDP-1 associates through its CH domain with the small GTPase Rac1/CED-10, which is a key regulator of the actin cytoskeleton. CHDP-1 preferentially binds to the GTP-bound active form of the CED-10 protein and preserves the membrane localization of GTP-CED-10. Hence, by coupling membrane expansion to Rac1-mediated actin dynamics, CHDP-1 promotes the formation of cellular protrusions in vivo.

Specific localization of nesprin-1-α2, the short isoform of nesprin-1 with a KASH domain, in developing, fetal and regenerating muscle, using a new monoclonal antibody.

BackgroundNesprin-1-giant (1008kD) is a protein of the outer nuclear membrane that links nuclei to the actin cytoskeleton via amino-terminal calponin homology domains. The short nesprin-1 isoform, nesprin-1-α2, is present only in skeletal and cardiac muscle and several pathogenic mutations occur within it, but the functions of this short isoform without calponin homology domains are unclear. The aim of this study was to determine mRNA levels and protein localization of nesprin-1-α2 at different stages of muscle development in order to shed light on its functions.ResultsmRNA levels of all known nesprin-1 isoforms with a KASH domain were determined by quantitative PCR. The mRNA for the 111 kD muscle-specific short isoform, nesprin-1-α2, was not detected in pre-differentiation human myoblasts but was present at significant levels in multinucleate myotubes. We developed a monoclonal antibody against the unique amino-terminal sequence of nesprin-1-α2, enabling specific immunolocalization for the first time. Nesprin-1-α2 protein was undetectable in pre-differentiation myoblasts but appeared at the nuclear rim in post-mitotic, multinucleate myotubes and reached its highest levels in fetal muscle. In muscle from a Duchenne muscular dystrophy biopsy, nesprin-1-α2 protein was detected mainly in regenerating fibres expressing neonatal myosin. Nesprin-1-giant was present at all developmental stages, but was also highest in fetal and regenerating fibres. In fetal muscle, both isoforms were present in the cytoplasm, as well as at the nuclear rim. A pathogenic early stop codon (E7854X) in nesprin-1 caused reduced mRNA levels and loss of protein levels of both nesprin-1-giant and (unexpectedly) nesprin-1-α2, but did not affect myogenesis in vitro.ConclusionsNesprin-1-α2 mRNA and protein expression is switched on during myogenesis, alongside other known markers of muscle differentiation. The results show that nesprin-1-α2 is dynamically controlled and may be involved in some process occurring during early myofibre formation, such as re-positioning of nuclei.

RAB-10 Promotes EHBP-1 Bridging of Filamentous Actin and Tubular Recycling Endosomes.

EHBP-1 (Ehbp1) is a conserved regulator of endocytic recycling, acting as an effector of small GTPases including RAB-10 (Rab10). Here we present evidence that EHBP-1 associates with tubular endosomal phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] enriched membranes through an N-terminal C2-like (NT-C2) domain, and define residues within the NT-C2 domain that mediate membrane interaction. Furthermore, our results indicate that the EHBP-1 central calponin homology (CH) domain binds to actin microfilaments in a reaction that is stimulated by RAB-10(GTP). Loss of any aspect of this RAB-10/EHBP-1 system in the C. elegans intestinal epithelium leads to retention of basolateral recycling cargo in endosomes that have lost their normal tubular endosomal network (TEN) organization. We propose a mechanism whereby RAB-10 promotes the ability of endosome-bound EHBP-1 to also bind to the actin cytoskeleton, thereby promoting endosomal tubulation.

In vivo epidermal migration requires focal adhesion targeting of ACF7.

Turnover of focal adhesions allows cell retraction, which is essential for cell migration. The mammalian spectraplakin protein, ACF7 (Actin-Crosslinking Factor 7), promotes focal adhesion dynamics by targeting of microtubule plus ends towards focal adhesions. However, it remains unclear how the activity of ACF7 is regulated spatiotemporally to achieve focal adhesion-specific guidance of microtubule. To explore the potential mechanisms, we resolve the crystal structure of ACF7’s NT (amino-terminal) domain, which mediates F-actin interactions. Structural analysis leads to identification of a key tyrosine residue at the calponin homology (CH) domain of ACF7, whose phosphorylation by Src/FAK (focal adhesion kinase) complex is essential for F-actin binding of ACF7. Using skin epidermis as a model system, we further demonstrate that the phosphorylation of ACF7 plays an indispensable role in focal adhesion dynamics and epidermal migration in vitro and in vivo. Together, our findings provide critical insights into the molecular mechanisms underlying coordinated cytoskeletal dynamics during cell movement.

624. A Single Neonatal Delivery of an Exon 2 Directed AAV9.U7snRNA Vector Results in Long-Term Dystrophin Expression That Prevents Pathologic Features in the Dup2 Mouse

We recently identified an internal ribosome entry site (IRES) within exon 5 of the DMD gene. Mutations that truncate the reading frame 5’ of this exon can result in use of the IRES for alternate translational initiation beginning within exon 6 that results in expression of an N-truncated isoform. Despite lacking the calponin homology domain 1 (CH1) of the actin binding domain 1 (ABD1), this isoform is highly functional, as demonstrated by the minimal symptoms in patients who express it. Consistent with genotype-phenotype correlations in DMD patients, the IRES is not active in the presence of exon 2 duplication but is active when exon 2 is deleted. We developed an AAV9.U7snRNA vector to that truncates DMD reading frame by skipping of exon 2, and have shown that in a Duchenne muscular dystrophy (DMD) mouse model carrying a duplication of exon 2 (the Dup2 mouse), postnatal intramuscular (IM) or intravascular (IV) treatment results in functional and pathologic improvement in skeletal muscle. Relevant to efforts to identify and treat DMD patients at an earlier age, we sought here to determine whether earlier gene transfer might slow down or even prevent the development of pathology. Dup2 mice were injected via facial vein at postnatal day 1 (P1) with 1E12 total vector genomes of the AAV9.U7snRNA vector and sacrificed at either 1, 3, 6, or 12 months post-injection for evaluation of exon 2 skipping by RT-PCR, quantification of dystrophin expression, and characterization of histopathology. To model the applicability of this approach beyond exon 2 duplication patients, the same vector was used to treat 6 human patient fibroblast-derived transdifferentiated myoblasts (FibroMyoD cells) harboring various mutations within exons 1 to 4. In the Dup2 mouse, efficient skipping and abundant dystrophin expression were present up to one year following the single AAV injection. Dystrophic pathology was absent at all-time points; at one year, less than 1% of fibers showed central nucleation, in comparison to ~70% in untreated Dup2 mice. Two tests on the ex vivo diaphragm preparations: isometric force (providing assessment of strength), and eccentric contractions (evaluating sarcolemma stability) were performed at 3 and 6 months following P1 injection. Both tests demonstrated little to no difference between treated animals and wild type mice. In all FibroMyoD cultures, abundant exon 2 skipping and dystrophin expression were detected in myotubes at 14 days of culture after treatment. These results suggest that this exon-skipping vector offers a therapeutic approach not only to patients with exon 2 duplications but with all mutations within the first four DMD exons (~6% of patients), an area of the gene largely ignored by the current therapeutic approaches. This work strongly supports the idea that early treatment of these patients will have longstanding and significant benefit resulting in a better outcome.

Detection of genetic variation affecting milk coagulation properties in Danish Holstein dairy cattle by analyses of pooled whole-genome sequences from phenotypically extreme samples (pool-seq)1

Rennet-induced milk coagulation is an important trait for cheese production. Recent studies have reported an alarming frequency of cows producing poorly coagulating milk unsuitable for cheese production. Several genetic factors are known to affect milk coagulation, including variation in the major milk proteins; however, recent association studies indicate genetic effects from other genomic regions as well. The aim of this study was to detect genetic variation affecting milk coagulation properties, measured as curd-firming rate (CFR) and milk pH. This was achieved by examining allele frequency differences between pooled whole-genome sequences of phenotypically extreme samples (pool-seq).. Curd-firming rate and raw milk pH were measured for 415 Danish Holstein cows, and each animal was sequenced at low coverage. Pools were created containing whole genome sequence reads from samples with "extreme" values (high or low) for both phenotypic traits. A total of 6,992,186 and 5,295,501 SNP were assessed in relation to CFR and milk pH, respectively. Allele frequency differences were calculated between pools and 32 significantly different SNP were detected, 1 for milk pH and 31 for CFR, of which 19 are located on chromosome 6. A total of 9 significant SNP, which were selected based on the possible function of proximal candidate genes, were genotyped in the entire sample set ( = 415) to test for an association. The most significant SNP was located proximal to , explaining 33% of the phenotypic variance. , coding for κ-casein, is the most studied in relation to milk coagulation due to its position on the surface of the casein micelles and the direct involvement in milk coagulation. Three additional SNP located on chromosome 6 showed significant associations explaining 7, 3.6, and 1.3% of the phenotypic variance of CFR. The significant SNP on chromosome 6 were shown to be in linkage disequilibrium with the SNP peaking proximal to ; however, after accounting for the genotype of the peak SNP within this QTL, significant effects (-value < 0.1) could still be detected for 2 of the SNP accounting for 2 and 1% of the phenotypic variance. These 2 interesting SNP were located within introns or proximal to the candidate genes-solute carrier family 4 (sodium bicarbonate cotransporter), member 4 () and LIM and calponin homology domains 1 (), respectively-making them interesting targets for further analysis.

Phosphorylation of EB2 by Aurora B and CDK1 ensures mitotic progression and genome stability.

Temporal regulation of microtubule dynamics is essential for proper progression of mitosis and control of microtubule plus-end tracking proteins by phosphorylation is an essential component of this regulation. Here we show that Aurora B and CDK1 phosphorylate microtubule end-binding protein 2 (EB2) at multiple sites within the amino terminus and a cluster of serine/threonine residues in the linker connecting the calponin homology and end-binding homology domains. EB2 phosphorylation, which is strictly associated with mitotic entry and progression, reduces the binding affinity of EB2 for microtubules. Expression of non-phosphorylatable EB2 induces stable kinetochore microtubule dynamics and delays formation of bipolar metaphase plates in a microtubule binding-dependent manner, and leads to aneuploidy even in unperturbed mitosis. We propose that Aurora B and CDK1 temporally regulate the binding affinity of EB2 for microtubules, thereby ensuring kinetochore microtubule dynamics, proper mitotic progression and genome stability.

Break of symmetry in regenerating tobacco protoplasts is independent of nuclear positioning.

Nuclear migration and positioning are crucial for the morphogenesis of plant cells. We addressed the potential role of nuclear positioning for polarity induction using an experimental system based on regenerating protoplasts, where the induction of a cell axis de novo can be followed by quantification of specific regeneration stages. Using overexpression of fluorescently tagged extranuclear (perinuclear actin basket, kinesins with a calponin homology domain (KCH)) as well as intranuclear (histone H2B) factors of nuclear positioning and time-lapse series of the early stages of regeneration, we found that nuclear position is no prerequisite for polarity formation. However, polarity formation and nuclear migration were both modulated in the transgenic lines, indicating that both phenomena depend on factors affecting cytoskeletal tensegrity and chromatin structure. We integrated these findings into a model where retrograde signals are required for polarity induction. These signals travel via the cytoskeleton from the nucleus toward targets at the plasma membrane.

Modulation of MICAL Monooxygenase Activity by its Calponin Homology Domain: Structural and Mechanistic Insights

MICALs (Molecule Interacting with CasL) are conserved multidomain enzymes essential for cytoskeletal reorganization in nerve development, endocytosis, and apoptosis. In these enzymes, a type-2 calponin homology (CH) domain always follows an N-terminal monooxygenase (MO) domain. Although the CH domain is required for MICAL-1 cellular localization and actin-associated function, its contribution to the modulation of MICAL activity towards actin remains unclear. Here, we present the structure of a fragment of MICAL-1 containing the MO and the CH domains—determined by X-ray crystallography and small angle scattering—as well as kinetics experiments designed to probe the contribution of the CH domain to the actin-modification activity. Our results suggest that the CH domain, which is loosely connected to the MO domain by a flexible linker and is far away from the catalytic site, couples F-actin to the enhancement of redox activity of MICALMO-CH by a cooperative mechanism involving a trans interaction between adjacently bound molecules. Binding cooperativity is also observed in other proteins regulating actin assembly/disassembly dynamics, such as ADF/Cofilins.

On the Interaction Between Human IQGAP1 and Actin.

IQGAPs are eukaryotic proteins which integrate signals from various sources and pass these on the cytoskeleton. Understanding how they do this requires information on the interfaces between the proteins. Here, it is shown that the calponin homology domain of human IQGAP1 (CHD1) can be crosslinked with α-actin. The stoichiometry of the interaction was 1:1. A molecular model was built of the complex and associated bioinformatics analyses predicted that the interaction is likely to involve an electrostatic interaction between Lys-240 of α-actin and Glu-30 of CHD1. These residues are predicted to be accessible and are not involved in many intra-protein interactions; they are thus available for interaction with binding partners. They are both located in regions of the proteins which are predicted to be flexible and disordered; interactions between signalling molecules often involve flexible, disordered regions. The predicted binding region in CHD1 is well conserved in many eukaryotic IQGAP-like proteins. In some cases (e.g Dictyostelium discoideum and Saccharomyces cerevisiae) protein sequence conservation is weak, but molecular modelling reveals that a region of charged, polar residues in a flexible N-terminus is structurally well conserved. Therefore we conclude that the calponin homology domains of IQGAP1-like proteins interact initially through the electrostatic interaction identified here and that there may be subsequent conformational changes to form the final complex.

Congenital macrothrombocytopenia-linked mutations in the actin-binding domain of α-actinin-1 enhance F-actin association.

Mutations in the actin cross-linking protein actinin-1 were recently linked to dominantly inherited congenital macrothrombocytopenia. Here, we report that several disease-associated mutations that are located within the actinin-1 actin-binding domain cause increased binding of actinin-1 to actin filaments and enhance filament bundling in vitro. These actinin-1 mutants are also more stably associated with the cytoskeleton in cultured cells, as assessed by biochemical fractionation and fluorescence recovery after photobleaching experiments. For two mutations the disruption of contacts between the calponin homology domains within the actinin actin-binding domain may explain increased filament binding--providing mechanistic and structural insights into the basis of actinin-1 dysfunction in congenital macrothrombocytopenia.

Intraflagellar transport proteins 172, 80, 57, 54, 38, and 20 form a stable tubulin-binding IFT-B2 complex.

Intraflagellar transport (IFT) relies on the IFT complex and is required for ciliogenesis. The IFT‐B complex consists of 9–10 stably associated core subunits and six “peripheral” subunits that were shown to dissociate from the core structure at moderate salt concentration. We purified the six “peripheral” IFT‐B subunits of Chlamydomonas reinhardtii as recombinant proteins and show that they form a stable complex independently of the IFT‐B core. We suggest a nomenclature of IFT‐B1 (core) and IFT‐B2 (peripheral) for the two IFT‐B subcomplexes. We demonstrate that IFT88, together with the N‐terminal domain of IFT52, is necessary to bridge the interaction between IFT‐B1 and B2. The crystal structure of IFT52N reveals highly conserved residues critical for IFT‐B1/IFT‐B2 complex formation. Furthermore, we show that of the three IFT‐B2 subunits containing a calponin homology (CH) domain (IFT38, 54, and 57), only IFT54 binds αβ‐tubulin as a potential IFT cargo, whereas the CH domains of IFT38 and IFT57 mediate the interaction with IFT80 and IFT172, respectively. Crystal structures of IFT54 CH domains reveal that tubulin binding is mediated by basic surface‐exposed residues.

Crystallization and X-ray diffraction analysis of the CH domain of the cotton kinesin GhKCH2.

GhKCH2 belongs to a group of plant-specific kinesins (KCHs) containing an actin-binding calponin homology (CH) domain in the N-terminus. Previous studies revealed that the GhKCH2 CH domain (GhKCH2-CH) had a higher affinity for F-actin (Kd = 0.42 ± 0.02 µM) than most other CH-domain-containing proteins. To understand the underlying mechanism, prokaryotically expressed GhKCH2-CH (amino acids 30-166) was purified and crystallized. Crystals were grown by the sitting-drop vapour-diffusion method using 0.1 M Tris-HCl pH 7.0, 20%(w/v) PEG 8000 as a precipitant. The crystals diffracted to a resolution of 2.5 Å and belonged to space group P21, with unit-cell parameters a = 41.57, b = 81.92, c = 83.00 Å, α = 90.00, β = 97.31, γ = 90.00°. Four molecules were found in the asymmetric unit with a Matthews coefficient of 2.22 Å(3) Da(-1), corresponding to a solvent content of 44.8%.

A human β-III-spectrin spinocerebellar ataxia type 5 mutation causes high-affinity F-actin binding.

Spinocerebellar ataxia type 5 (SCA5) is a human neurodegenerative disease that stems from mutations in the SPTBN2 gene encoding the protein β-III-spectrin. Here we investigated the molecular consequence of a SCA5 missense mutation that results in a L253P substitution in the actin-binding domain (ABD) of β-III-spectrin. We report that the L253P substitution in the isolated β-III-spectrin ABD causes strikingly high F-actin binding affinity (Kd = 75.5 nM) compared to the weak F-actin binding affinity of the wild-type ABD (Kd = 75.8 μM). The mutation also causes decreased thermal stability (Tm = 44.6 °C vs 59.5 °C). Structural analyses indicate that leucine 253 is in a loop at the interface of the tandem calponin homology (CH) domains comprising the ABD. Leucine 253 is predicted to form hydrophobic contacts that bridge the CH domains. The decreased stability of the mutant indicates that these bridging interactions are probably disrupted, suggesting that the high F-actin binding affinity of the mutant is due to opening of the CH domain interface. These results support a fundamental role for leucine 253 in regulating opening of the CH domain interface and binding of the ABD to F-actin. This study indicates that high-affinity actin binding of L253P β-III-spectrin is a likely driver of neurodegeneration.

Properties and catalytic activities of MICAL1, the flavoenzyme involved in cytoskeleton dynamics, and modulation by its CH, LIM and C-terminal domains

MICAL1 is a cytoplasmic 119 kDa protein participating in cytoskeleton dynamics through the NADPH-dependent oxidase and F-actin depolymerizing activities of its N-terminal flavoprotein domain, which is followed by calponin homology (CH), LIM domains and a C-terminal region with Pro-, Glu-rich and coiled-coil motifs. MICAL1 and truncated forms lacking the C-terminal, LIM and/or CH regions have been produced and characterized. The CH, LIM and C-terminal regions cause an increase of Km,NADPH exhibited by the NADPH oxidase activity of the flavoprotein domain, paralleling changes in the overall protein charge. The C-terminus also determines a ∼10-fold decrease of kcat, revealing its role in establishing an inactive/active conformational equilibrium, which is at the heart of the regulation of MICAL1 in cells. F-actin lowers Km,NADPH (10–50 μM) and increases kcat (10–25 s−1) to similar values for all MICAL forms. The apparent Km,actin of MICAL1 is ∼10-fold higher than that of the other forms (3–5 μM), reflecting the fact that F-actin binds to the flavoprotein domain in the MICAL's active conformation and stabilizes it. Analyses of the reaction in the presence of F-actin indicate that actin depolymerization is mediated by H2O2 produced by the NADPH oxidase reaction, rather than due to direct hydroxylation of actin methionine residues.