Troponin Structural Dynamics in the Native Cardiac Thin Filament Revealed by Cryo Electron Microscopy.

Cardiac muscle contraction occurs due to repetitive interactions between myosin thick and actin thin filaments (TF) regulated by Ca2+ levels, active cross-bridges, and cardiac myosin-binding protein C (cMyBP-C). The cardiac TF (cTF) has two nonequivalent strands, each comprised of actin, tropomyosin (Tm), and troponin (Tn). Tn shifts Tm away from myosin-binding sites on actin at elevated Ca2+ levels to allow formation of force-producing actomyosin cross-bridges. The Tn complex is comprised of three distinct polypeptides - Ca2+-binding TnC, inhibitory TnI, and Tm-binding TnT. The molecular mechanism of their collective action is unresolved due to lack of comprehensive structural information on Tn region of cTF. C1 domain of cMyBP-C activates cTF in the absence of Ca2+ to the same extent as rigor myosin. Here we used cryo-EM of native cTFs to show that cTF Tn core adopts multiple structural conformations at high and low Ca2+ levels and that the two strands are structurally distinct. At high Ca2+ levels, cTF is not entirely activated by Ca2+ but exists in either partially or fully activated state. Complete dissociation of TnI C-terminus is required for full activation. In presence of cMyBP-C C1 domain, Tn core adopts a fully activated conformation, even in absence of Ca2+. Our data provide a structural description for the requirement of myosin to fully activate cTFs and explain increased affinity of TnC to Ca2+ in presence of active cross-bridges. We suggest that allosteric coupling between Tn subunits and Tm is required to control actomyosin interactions.

Cardiac troponin T N-domain variant destabilizes the actin interface resulting in disturbed myofilament function.

Missense variant Ile79Asn in human cardiac troponin T (cTnT-I79N) has been associated with hypertrophic cardiomyopathy and sudden cardiac arrest in juveniles. cTnT-I79N is located in the cTnT N-terminal (TnT1) loop region and is known for its pathological and prognostic relevance. A recent structural study revealed that I79 is part of a hydrophobic interface between the TnT1 loop and actin, which stabilizes the relaxed (OFF) state of the cardiac thin filament. Given the importance of understanding the role of TnT1 loop region in Ca2+ regulation of the cardiac thin filament along with the underlying mechanisms of cTnT-I79N-linked pathogenesis, we investigated the effects of cTnT-I79N on cardiac myofilament function. Transgenic I79N (Tg-I79N) muscle bundles displayed increased myofilament Ca2+ sensitivity, smaller myofilament lattice spacing, and slower crossbridge kinetics. These findings can be attributed to destabilization of the cardiac thin filament's relaxed state resulting in an increased number of crossbridges during Ca2+ activation. Additionally, in the low Ca2+-relaxed state (pCa8), we showed that more myosin heads are in the disordered-relaxed state (DRX) that are more likely to interact with actin in cTnT-I79N muscle bundles. Dysregulation of the myosin super-relaxed state (SRX) and the SRX/DRX equilibrium in cTnT-I79N muscle bundles likely result in increased mobility of myosin heads at pCa8, enhanced actomyosin interactions as evidenced by increased active force at low Ca2+, and increased sinusoidal stiffness. These findings point to a mechanism whereby cTnT-I79N weakens the interaction of the TnT1 loop with the actin filament, which in turn destabilizes the relaxed state of the cardiac thin filament.

Actomyosin Complex.

Formation of cross-bridges between actin and myosin occurs ubiquitously in eukaryotic cells and mediates muscle contraction, intracellular cargo transport, and cytoskeletal remodeling. Myosin motors repeatedly bind to and dissociate from actin filaments in a cycle that transduces the chemical energy from ATP hydrolysis into mechanical force generation. While the general layout of surface elements within the actin-binding interface is conserved among myosin classes, sequence divergence within these motifs alters the specific contacts involved in the actomyosin interaction as well as the kinetics of mechanochemical cycle phases. Additionally, diverse lever arm structures influence the motility and force production of myosin molecules during their actin interactions. The structural differences generated by myosin's molecular evolution have fine-tuned the kinetics of its isoforms and adapted them for their individual cellular roles. In this chapter, we will characterize the structural and biochemical basis of the actin-myosin interaction and explain its relationship with myosin's cellular roles, with emphasis on the structural variation among myosin isoforms that enables their functional specialization. We will also discuss the impact of accessory proteins, such as the troponin-tropomyosin complex and myosin-binding protein C, on the formation and regulation of actomyosin cross-bridges.

Optimization of small extracellular vesicle isolation from expressed prostatic secretions in urine for in-depth proteomic analysis.

The isolation and subsequent molecular analysis of extracellular vesicles (EVs) derived from patient samples is a widely used strategy to understand vesicle biology and to facilitate biomarker discovery. Expressed prostatic secretions in urine are a tumor proximal fluid that has received significant attention as a source of potential prostate cancer (PCa) biomarkers for use in liquid biopsy protocols. Standard EV isolation methods like differential ultracentrifugation (dUC) co-isolate protein contaminants that mask lower-abundance proteins in typical mass spectrometry (MS) protocols. Further complicating the analysis of expressed prostatic secretions, uromodulin, also known as Tamm-Horsfall protein (THP), is present at high concentrations in urine. THP can form polymers that entrap EVs during purification, reducing yield. Disruption of THP polymer networks with dithiothreitol (DTT) can release trapped EVs, but smaller THP fibres co-isolate with EVs during subsequent ultracentrifugation. To resolve these challenges, we describe here a dUC method that incorporates THP polymer reduction and alkaline washing to improve EV isolation and deplete both THP and other common protein contaminants. When applied to human expressed prostatic secretions in urine, we achieved relative enrichment of known prostate and prostate cancer-associated EV-resident proteins. Our approach provides a promising strategy for global proteomic analyses of urinary EVs.

The structure of the native cardiac thin filament at systolic Ca2+ levels.

Every heartbeat relies on cyclical interactions between myosin thick and actin thin filaments orchestrated by rising and falling Ca2+ levels. Thin filaments are comprised of two actin strands, each harboring equally separated troponin complexes, which bind Ca2+ to move tropomyosin cables away from the myosin binding sites and, thus, activate systolic contraction. Recently, structures of thin filaments obtained at low (pCa ∼9) or high (pCa ∼3) Ca2+ levels revealed the transition between the Ca2+-free and Ca2+-bound states. However, in working cardiac muscle, Ca2+ levels fluctuate at intermediate values between pCa ∼6 and pCa ∼7. The structure of the thin filament at physiological Ca2+ levels is unknown. We used cryoelectron microscopy and statistical analysis to reveal the structure of the cardiac thin filament at systolic pCa = 5.8. We show that the two strands of the thin filament consist of a mixture of regulatory units, which are composed of Ca2+-free, Ca2+-bound, or mixed (e.g., Ca2+ free on one side and Ca2+ bound on the other side) troponin complexes. We traced troponin complex conformations along and across individual thin filaments to directly determine the structural composition of the cardiac native thin filament at systolic Ca2+ levels. We demonstrate that the two thin filament strands are activated stochastically with short-range cooperativity evident only on one of the two strands. Our findings suggest a mechanism by which cardiac muscle is regulated by narrow range Ca2+ fluctuations.

High-Resolution Cryo-EM Structure of the Cardiac Actomyosin Complex.

Heart contraction depends on a complicated array of interactions between sarcomeric proteins required to convert chemical energy into mechanical force. Cyclic interactions between actin and myosin molecules, controlled by troponin and tropomyosin, generate the sliding force between the actin-based thin and myosin-based thick filaments. Alterations in this sophisticated system due to missense mutations can lead to cardiovascular diseases. Numerous structural studies proposed pathological mechanisms of missense mutations at the myosin-myosin, actin-tropomyosin, and tropomyosin-troponin interfaces. However, despite the central role of actomyosin interactions a detailed structural description of the cardiac actomyosin interface remained unknown. Here, we report a cryo-EM structure of a cardiac actomyosin complex at 3.8 Å resolution. The structure reveals the molecular basis of cardiac diseases caused by missense mutations in myosin and actin proteins.

Effects of cardiomyopathy-linked mutations K15N and R21H in tropomyosin on thin-filament regulation and pointed-end dynamics.

Missense mutations K15N and R21H in striated muscle tropomyosin are linked to dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM), respectively. Tropomyosin, together with the troponin complex, regulates muscle contraction and, along with tropomodulin and leiomodin, controls the uniform thin-filament lengths crucial for normal sarcomere structure and function. We used Förster resonance energy transfer to study effects of the tropomyosin mutations on the structure and kinetics of the cardiac troponin core domain associated with the Ca2+-dependent regulation of cardiac thin filaments. We found that the K15N mutation desensitizes thin filaments to Ca2+ and slows the kinetics of structural changes in troponin induced by Ca2+ dissociation from troponin, while the R21H mutation has almost no effect on these parameters. Expression of the K15N mutant in cardiomyocytes decreases leiomodin's thin-filament pointed-end assembly but does not affect tropomodulin's assembly at the pointed end. Our in vitro assays show that the R21H mutation causes a twofold decrease in tropomyosin's affinity for F-actin and affects leiomodin's function. We suggest that the K15N mutation causes DCM by altering Ca2+-dependent thin-filament regulation and that one of the possible HCM-causing mechanisms by the R21H mutation is through alteration of leiomodin's function.

Multimodal and Polymorphic Interactions between Anillin and Actin: Their Implications for Cytokinesis.

Cytokinesis of animal cells requires the assembly of a contractile ring, which promotes daughter cell splitting. Anillin is a conserved scaffold protein involved in organizing the structural components of the contractile ring including filamentous actin (F-actin), myosin, and septins and in forming the subsequent midbody ring. Like other metazoan homologs, Drosophila anillin contains a conserved domain that can bind and bundle F-actin, but the importance and molecular details of its interaction with F-actin remain unclear. Here, we show that in a depletion-and-rescue assay in Drosophila S2 cells, anillin lacking the entire actin-binding domain (ActBD) exhibits defective cortical localization during mitosis and a greatly diminished ability to support cytokinesis. Using in vitro binding assays and electron microscopy on recombinant fragments, we determine that the anillin ActBD harbors three distinct actin-binding sites (ABS 1-3). We show that each ABS binds to a distinct place on F-actin. Importantly, ABS1 and ABS3 partially overlap on the surface of actin and, therefore, interact with F-actin in a mutually exclusive fashion. Although ABS2 and ABS3 are sufficient for bundling, ABS1 contributes to the overall F-actin bundling activity of anillin and enables anillin to switch between two actin-bundling morphologies and promote the formation of three-dimensional F-actin bundles. Finally, we show that in live S2 cells, ABS2 and ABS3 are each required and together sufficient for the robust cortical localization of the ActBD during cytokinesis. Collectively, our structural, biochemical, and cell biological data suggest that multiple anillin-actin interaction modes promote the faithful progression of cytokinesis.

C0 and C1 N-terminal Ig domains of myosin binding protein C exert different effects on thin filament activation.

Mutations in genes encoding myosin, the molecular motor that powers cardiac muscle contraction, and its accessory protein, cardiac myosin binding protein C (cMyBP-C), are the two most common causes of hypertrophic cardiomyopathy (HCM). Recent studies established that the N-terminal domains (NTDs) of cMyBP-C (e.g., C0, C1, M, and C2) can bind to and activate or inhibit the thin filament (TF). However, the molecular mechanism(s) by which NTDs modulate interaction of myosin with the TF remains unknown and the contribution of each individual NTD to TF activation/inhibition is unclear. Here we used an integrated structure-function approach using cryoelectron microscopy, biochemical kinetics, and force measurements to reveal how the first two Ig-like domains of cMyPB-C (C0 and C1) interact with the TF. Results demonstrate that despite being structural homologs, C0 and C1 exhibit different patterns of binding on the surface of F-actin. Importantly, C1 but not C0 binds in a position to activate the TF by shifting tropomyosin (Tm) to the "open" structural state. We further show that C1 directly interacts with Tm and traps Tm in the open position on the surface of F-actin. Both C0 and C1 compete with myosin subfragment 1 for binding to F-actin and effectively inhibit actomyosin interactions when present at high ratios of NTDs to F-actin. Finally, we show that in contracting sarcomeres, the activating effect of C1 is apparent only once low levels of Ca(2+) have been achieved. We suggest that Ca(2+) modulates the interaction of cMyBP-C with the TF in the sarcomere.

Septin 9 Exhibits Polymorphic Binding to F-Actin and Inhibits Myosin and Cofilin Activity.

Septins are a highly conserved family of proteins in eukaryotes that is recognized as a novel component of the cytoskeleton. Septin 9 (SEPT9) interacts directly with actin filaments and functions as an actin stress fiber cross-linking protein that promotes the maturation of nascent focal adhesions and cell migration. However, the molecular details of how SEPT9 interacts with F-actin remain unknown. Here, we use electron microscopy and image analysis to show that SEPT9 binds to F-actin in a highly polymorphic fashion. We demonstrate that the basic domain (B-domain) of the N-terminal tail of SEPT9 is responsible for actin cross-linking, while the GTP-binding domain (G-domain) does not bundle F-actin. We show that the B-domain of SEPT9 binds to three sites on F-actin, and the two of these sites overlap with the binding regions of myosin and cofilin. SEPT9 inhibits actin-dependent ATPase activity of myosin and competes with the weakly bound state of myosin for binding to F-actin. At the same time, SEPT9 significantly reduces the extent of F-actin depolymerization by cofilin. Taken together, these data suggest that SEPT9 protects actin filaments from depolymerization by cofilin and myosin and indicate a mechanism by which SEPT9 could maintain the integrity of growing and contracting actin filaments.

Septins promote stress fiber-mediated maturation of focal adhesions and renal epithelial motility.

Organogenesis and tumor metastasis involve the transformation of epithelia to highly motile mesenchymal-like cells. Septins are filamentous G proteins, which are overexpressed in metastatic carcinomas, but their functions in epithelial motility are unknown. Here, we show that a novel network of septin filaments underlies the organization of the transverse arc and radial (dorsal) stress fibers at the leading lamella of migrating renal epithelia. Surprisingly, septin depletion resulted in smaller and more transient and peripheral focal adhesions. This phenotype was accompanied by a highly disorganized lamellar actin network and rescued by the actin bundling protein α-actinin-1. We show that preassembled actin filaments are cross-linked directly by Septin 9 (SEPT9), whose expression is increased after induction of renal epithelial motility with the hepatocyte growth factor. Significantly, SEPT9 overexpression enhanced renal cell migration in 2D and 3D matrices, whereas SEPT9 knockdown decreased migration. These results suggest that septins promote epithelial motility by reinforcing the cross-linking of lamellar stress fibers and the stability of nascent focal adhesions.

Reversible paralysis of Schistosoma mansoni by forchlorfenuron, a phenylurea cytokinin that affects septins.

Septins are guanosine-5'-triphosphate-binding proteins involved in wide-ranging cellular processes including cytokinesis, vesicle trafficking, membrane remodelling and scaffolds, and with diverse binding partners. Precise roles for these structural proteins in most processes often remain elusive. Identification of small molecules that inhibit septins could aid in elucidating the functions of septins and has become increasingly important, including the description of roles for septins in pathogenic phenomena such as tumorigenesis. The plant growth regulator forchlorfenuron, a synthetic cytokinin known to inhibit septin dynamics, likely represents an informative probe for septin function. This report deals with septins of the human blood fluke Schistosoma mansoni and their interactions with forchlorfenuron. Recombinant forms of three schistosome septins, SmSEPT5, SmSEPT7.2 and SmSEPT10, interacted with forchlorfenuron, leading to rapid polymerization of filaments. Culturing developmental stages (miracidia, cercariae, adult males) of schistosomes in FCF at 50-500 µM rapidly led to paralysis, which was reversible upon removal of the cytokinin. The reversible paralysis was concentration-, time- and developmental stage-dependent. Effects of forchlorfenuron on the cultured schistosomes were monitored by video and/or by an xCELLigence-based assay of motility, which quantified the effect of forchlorfenuron on fluke motility. The findings implicated a mechanism targeting a molecular system controlling movement in these developmental stages: a direct effect on muscle contraction due to septin stabilization might be responsible for the reversible paralysis, since enrichment of septins has been described within the muscles of schistosomes. This study revealed the reversible effect of forchlorfenuron on both schistosome motility and its striking impact in hastening polymerization of septins. These novel findings suggested routes to elucidate roles for septins in this pathogen, and exploitation of derivatives of forchlorfenuron for anti-schistosomal drugs.

Enhancement of RAD51 recombinase activity by the tumor suppressor PALB2.

Homologous recombination mediated by RAD51 recombinase helps eliminate chromosomal lesions, such as DNA double-strand breaks induced by radiation or arising from injured DNA replication forks. The tumor suppressors BRCA2 and PALB2 act together to deliver RAD51 to chromosomal lesions to initiate repair. Here we document a new function of PALB2: to enhance RAD51's ability to form the D loop. We show that PALB2 binds DNA and physically interacts with RAD51. Notably, although PALB2 alone stimulates D-loop formation, it has a cooperative effect with RAD51AP1, an enhancer of RAD51. This stimulation stems from the ability of PALB2 to function with RAD51 and RAD51AP1 to assemble the synaptic complex. Our results demonstrate the multifaceted role of PALB2 in chromosome damage repair. Because PALB2 mutations can cause cancer or Fanconi anemia, our findings shed light on the mechanism of tumor suppression in humans.

Mapping of drebrin binding site on F-actin.

Drebrin is a filament-binding protein involved in organizing the dendritic pool of actin. Previous in vivo studies identified the actin-binding domain of drebrin (DrABD), which causes the same rearrangements in the cytoskeleton as the full-length protein. Site-directed mutagenesis, electron microscopic reconstruction, and chemical cross-linking combined with mass spectrometry analysis were employed here to map the DrABD binding interface on actin filaments. DrABD could be simultaneously attached to two adjacent actin protomers using the combination of 2-iminothiolane (Traut's reagent) and MTS1 [1,1-methanediyl bis(methanethiosulfonate)]. Site-directed mutagenesis combined with chemical cross-linking revealed that residue 238 of DrABD is located within 5.4 A from C374 of actin protomer 1 and that native cysteine 308 of drebrin is near C374 of actin protomer 2. Mass spectrometry analysis revealed that a zero-length cross-linker, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, can link the N-terminal G-S extension of the recombinant DrABD to E99 and/or E100 on actin. Efficient cross-linking of drebrin residues 238, 248, 252, 270, and 271 to actin residue 51 was achieved with reagents of different lengths (5.4-19 A). These results suggest that the "core" DrABD is centered on actin subdomain 2 and may adopt a folded conformation upon binding to F-actin. The results of electron microscopic reconstruction, which are in a good agreement with the cross-linking data, revealed polymorphism in DrABD binding to F-actin and suggested the existence of two binding sites. These results provide new structural insight into the previously observed competition between drebrin and several other F-actin-binding proteins.

ParA2, a Vibrio cholerae chromosome partitioning protein, forms left-handed helical filaments on DNA.

Most bacterial chromosomes contain homologs of plasmid partitioning (par) loci. These loci encode ATPases called ParA that are thought to contribute to the mechanical force required for chromosome and plasmid segregation. In Vibrio cholerae, the chromosome II (chrII) par locus is essential for chrII segregation. Here, we found that purified ParA2 had ATPase activities comparable to other ParA homologs, but, unlike many other ParA homologs, did not form high molecular weight complexes in the presence of ATP alone. Instead, formation of high molecular weight ParA2 polymers required DNA. Electron microscopy and three-dimensional reconstruction revealed that ParA2 formed bipolar helical filaments on double-stranded DNA in a sequence-independent manner. These filaments had a distinct change in pitch when ParA2 was polymerized in the presence of ATP versus in the absence of a nucleotide cofactor. Fitting a crystal structure of a ParA protein into our filament reconstruction showed how a dimer of ParA2 binds the DNA. The filaments formed with ATP are left-handed, but surprisingly these filaments exert no topological changes on the right-handed B-DNA to which they are bound. The stoichiometry of binding is one dimer for every eight base pairs, and this determines the geometry of the ParA2 filaments with 4.4 dimers per 120 A pitch left-handed turn. Our findings will be critical for understanding how ParA proteins function in plasmid and chromosome segregation.

Structural polymorphism of the ParM filament and dynamic instability.

Segregation of the R1 plasmid in bacteria relies on ParM, an actin homolog that segregates plasmids by switching between cycles of polymerization and depolymerization. We find similar polymerization kinetics and stability in the presence of either ATP or GTP and a 10-fold affinity preference for ATP over GTP. We used electron cryo-microscopy to evaluate the heterogeneity within ParM filaments. In addition to variable twist, ParM has variable axial rise, and both parameters are coupled. Subunits in the same ParM filaments can exist in two different structural states, with the nucleotide-binding cleft closed or open, and the bound nucleotide biases the distribution of states. The interface between protomers is different between these states, and in neither state is it similar to F-actin. Our results suggest that the closed state of the cleft is required but not sufficient for ParM polymerization, and provide a structural basis for the dynamic instability of ParM filaments.

Loop 2 in Saccharomyces cerevisiae Rad51 protein regulates filament formation and ATPase activity.

Previous studies showed that the K342E substitution in the Saccharomyces cerevisiae Rad51 protein increases the interaction with Rad54 protein in the two-hybrid system, leads to increased sensitivity to the alkylating agent MMS and hyper-recombination in an oligonucleotide-mediated gene targeting assay. K342 localizes in loop 2, a region of Rad51 whose function is not well understood. Here, we show that Rad51-K342E displays DNA-independent and DNA-dependent ATPase activities, owing to its ability to form filaments in the absence of a DNA lattice. These filaments exhibit a compressed pitch of 81 A, whereas filaments of wild-type Rad51 and Rad51-K342E on DNA form extended filaments with a 97 A pitch. Rad51-K342E shows near normal binding to ssDNA, but displays a defect in dsDNA binding, resulting in less stable protein-dsDNA complexes. The mutant protein is capable of catalyzing the DNA strand exchange reaction and is insensitive to inhibition by the early addition of dsDNA. Wild-type Rad51 protein is inhibited under such conditions, because of its ability to bind dsDNA. No significant changes in the interaction between Rad51-K342E and Rad54 could be identified. These findings suggest that loop 2 contributes to the primary DNA-binding site in Rad51, controlling filament formation and ATPase activity.

Stabilization of RAD-51-DNA filaments via an interaction domain in Caenorhabditis elegans BRCA2.

Mutations in BRCA2 predispose individuals to breast cancer, a consequence of the role of BRCA2 in DNA repair. Human BRCA2 interacts with the recombinase RAD51 via eight BRC repeats. Controversy has existed, however, about whether the BRC interactions are primarily with RAD51 monomers or with the RAD51-DNA helical polymer, and whether there is a single interaction or multiple ones. We show here that the single BRC motif in the Caenorhabditis elegans BRCA2 homolog, CeBRC-2, contains two different RAD-51-binding regions. One of these regions binds only weakly to RAD-51-DNA filaments but strongly to RAD-51 alone and corresponds to the part of human BRC4 crystallized with RAD51. Injection of a peptide corresponding to this region into worms inhibits the normal formation of RAD-51 foci in response to ionizing radiation (IR). Conversely, peptides corresponding to the second region bind strongly to RAD-51-DNA filaments but do not bind to RAD-51 alone. Three-dimensional reconstructions from electron micrographs show that this peptide binds to the RAD-51 N-terminal domain, which has been shown to have a regulatory function. Injection of this peptide into worms before IR leads to a dramatic increase and persistence of IR-induced RAD-51 foci. This peptide also inhibits the RAD-51 ATPase activity, required for filament depolymerization. These results support a model where an interaction with RAD-51 alone is likely involved in filament nucleation, whereas a second independent interaction is involved in stabilization of RAD-51 filaments by BRCA2. The multiple interactions between BRCA2-like molecules and RAD51 provide insights into why mutations in BRCA2 lead to cancer.

Stabilization of RAD51 nucleoprotein filaments by the C-terminal region of BRCA2.

The human breast cancer susceptibility gene BRCA2 is required for the regulation of RAD51-mediated homologous recombinational repair. BRCA2 interacts with RAD51 monomers, as well as nucleoprotein filaments, primarily though the conserved BRC motifs. The unrelated C-terminal region of BRCA2 also interacts with RAD51. Here we show that the BRCA2 C terminus interacts directly with RAD51 filaments, but not monomers, by binding an interface created by two adjacent RAD51 protomers. These interactions stabilize filaments so that they cannot be dissociated by association with BRC repeats. Interaction of the BRCA2 C terminus with the RAD51 filament causes a large movement of the flexible RAD51 N-terminal domain that is important in regulating filament dynamics. We suggest that interactions of the BRCA2 C-terminal region with RAD51 may facilitate efficient nucleation of RAD51 multimers on DNA and thereby stimulate recombination-mediated repair.

Mapping the interaction of cofilin with subdomain 2 on actin.

Cofilin, a member of the actin-depolymerizing factor (ADF)/cofilin family of proteins, is a key regulator of actin dynamics. Cofilin binds to monomer (G-) and filamentous (F-) actin, severs the filaments, and increases their turnover rate. Electron microscopy studies suggested cofilin interactions with subdomains 2 and 1/3 on adjacent actin protomers in F-actin. To probe for the presence of a cryptic cofilin binding site in subdomain 2 in G-actin, we used transglutaminase-mediated cross-linking, which targets Gln41 in subdomain 2. The cross-linking proceeded with up to 85% efficiency with skeletal alpha-actin and WT yeast actin, yielding a single product corresponding to a 1:1 actin-cofilin complex but was strongly inhibited in Q41C yeast actin (in which Q41 was substituted with cysteine). LC-MS/MS analysis of the proteolytic fragments of this complex mapped the cross-linking to Gln41 on actin and Gly1 on recombinant yeast cofilin. The actin-cofilin (AC) heterodimer was purified on FPLC for analytical ultracentrifugation and electron microscopy analysis. Sedimentation equilibrium and velocity runs revealed oligomers of AC in G-actin buffer. In the presence of excess cofilin, the covalent AC heterodimer bound a second cofilin, forming a 2:1 cofilin/actin complex, as revealed by sedimentation results. Under polymerizing conditions the cross-linked AC formed mostly short filaments, which according to image reconstruction were similar to uncross-linked actin-cofilin filaments. Although a majority of the cross-linking occurs at Gln41, a small fraction of the AC cross-linked complex forms in the Q41C yeast actin mutant. This secondary cross-linking site was sequenced by MALDI-MS/MS as linking Gln360 in actin to Lys98 on cofilin. Overall, these results demonstrate that the region around Gln41 (subdomain 2) is involved in a weak binding of cofilin to G-actin.