Arm Domain Research Articles

The Unstructured Linker Arms of Mlh1–Pms1 Are Important for Interactions with DNA during Mismatch Repair

DNA mismatch repair (MMR) models have proposed that MSH (MutS homolog) proteins identify DNA polymerase errors while interacting with the DNA replication fork. MLH (MutL homolog) proteins (primarily Mlh1-Pms1 in baker's yeast) then survey the genome for lesion-bound MSH proteins. The resulting MSH-MLH complex formed at a DNA lesion initiates downstream steps in repair. MLH proteins act as dimers and contain long (20-30 nm) unstructured arms that connect two terminal globular domains. These arms can vary between 100 and 300 amino acids in length, are highly divergent between organisms, and are resistant to amino acid substitutions. To test the roles of the linker arms in MMR, we engineered a protease cleavage site into the Mlh1 linker arm domain of baker's yeast Mlh1-Pms1. Cleavage of the Mlh1 linker arm in vitro resulted in a defect in Mlh1-Pms1 DNA binding activity, and in vivo proteolytic cleavage resulted in a complete defect in MMR. We then generated a series of truncation mutants bearing Mlh1 and Pms1 linker arms of varying lengths. This work revealed that MMR is greatly compromised when portions of the Mlh1 linker are removed, whereas repair is less sensitive to truncation of the Pms1 linker arm. Purified complexes containing truncations in Mlh1 and Pms1 linker arms were analyzed and found to have differential defects in DNA binding that also correlated with the ability to form a ternary complex with Msh2-Msh6 and mismatch DNA. These observations are consistent with the unstructured linker domains of MLH proteins providing distinct interactions with DNA during MMR.

Conformation of the Troponin Core Complex in the Thin Filaments of Skeletal Muscle during Relaxation and Active Contraction

Contraction of skeletal and cardiac muscles is regulated by Ca2+ binding to troponin in the actin-containing thin filaments, leading to an azimuthal movement of tropomyosin around the filament that uncovers the myosin binding sites on actin. Here, we use polarized fluorescence to determine the orientation of the C-terminal lobe of troponin C (TnC) in skeletal muscle cells as a step toward elucidating the molecular mechanism of troponin-mediated regulation. Assuming, as shown by X-ray crystallography, that this lobe of TnC is part of a well-defined troponin domain called the IT arm, we show that the coiled coil formed by troponin components I and T makes an angle of about 55° with the thin filament axis in relaxed muscle, in contrast with previous models based on electron microscopy in which this angle is close to 0°. The E helix of TnC makes an angle of about 45° with the thin filament axis. Both the IT coiled coil and the TnC E helix tilt by about 10° on muscle activation. By combining in situ measurements of the orientation of the IT arm and regulatory domain of troponin, which together form the troponin core complex, with published intermolecular distances between thin filament components, we derive models of thin filament structure in which the IT arm of troponin holds its regulatory domain close to the actin surface. Although the structure and function of troponin regions outside the core complex remain to be characterized, the present results provide useful constraints for molecular models of the mechanism of muscle regulation.

Acceptability of coitally-associated versus daily use of 1% tenofovir vaginal gel among women in Pune, India

This study reports on the acceptability of 1% tenofovir microbicide gel among participants randomised to the coitally-associated use (n=50) or daily use (n=50) arms of a Phase II clinical trial in Pune, India. In a 6-month follow-up study, information on behavioural domains was collected on a 6-point Likert scale and gel acceptability was measured on a 5-point Likert scale. Random intercept logistic modelling was performed to examine the simultaneous effects of study arm, follow-up time, sociodemographic factors and behavioural domains on gel acceptability. The mean age of female participants was 32.7 years. Women in both study arms had similar sociodemographic profiles. Women liked features such as easy use of the gel and its protective effect against HIV. Messiness was the most disliked feature. Gel acceptability increased during subsequent follow-up visits in both arms, especially in the coitally-associated use arm. Non-acceptability of the gel was almost two and a half times higher in daily users (adjusted odds ratio 2.55, 95% CI 1.18-5.51; p=0.017). Acceptability differed significantly between the two study arms at 2 months (68% vs 40%; p=0.006) and 6 months (64% vs 46%; p=0.07). Acceptability was significantly lower in those participants who reported 'messiness' as the most disliked feature (odds ratio 2.42, 95% CI 1.02-5.72; p=0.045). In conclusion, microbicides were more acceptable in coitally-associated users than in daily users. Leakage was a problem that requires attention. Positioning of the product in a setting such as India where the majority of decision-making is done by men would need extensive and systematic education of men.

Specific Armadillo Repeat Sequences Facilitate β-Catenin Nuclear Transport in Live Cells via Direct Binding to Nucleoporins Nup62, Nup153, and RanBP2/Nup358

β-Catenin transduces the Wnt signal from the membrane to nucleus, and certain gene mutations trigger its nuclear accumulation leading to cell transformation and cancer. β-Catenin shuttles between the nucleus and cytoplasm independent of classical Ran/transport receptor pathways, and this movement was previously hypothesized to involve the central Armadillo (Arm) domain. Fluorescence recovery after photobleaching (FRAP) assays were used to delineate functional transport regions of the Arm domain in living cells. The strongest nuclear import/export activity was mapped to Arm repeats R10-12 using both in vivo FRAP and in vitro export assays. By comparison, Arm repeats R3-8 of β-catenin were highly active for nuclear import but displayed a comparatively weak export activity. We show for the first time using purified components that specific Arm sequences of β-catenin interact directly in vitro with the FG repeats of the nuclear pore complex (NPC) components Nup62, Nup98, and Nup153, indicating an independent ability of β-catenin to traverse the NPC. Moreover, a proteomics screen identified RanBP2/Nup358 as a binding partner of Arm R10-12, and β-catenin was confirmed to interact with endogenous and ectopic forms of Nup358. We further demonstrate that knock-down of endogenous Nup358 and Nup62 impeded the rate of nuclear import/export of β-catenin to a greater extent than that of importin-β. The Arm R10-12 sequence facilitated transport even when β-catenin was bound to the Arm-binding partner LEF-1, and its activity was stimulated by phosphorylation at Tyr-654. These findings provide functional evidence that the Arm domain contributes to regulated β-catenin transport through direct interaction with the NPC.

Solution Conformation of Extracellular Matrix Proteins

The extracellular matrix (ECM) is composed of large, multi-domain proteins with varying degree of shapes and post-translational modifications. Therefore, it is challenging to study such proteins with a single technique to gain reliable information about their size, shape and conformation. We have employed a multidisciplinary approach using a variety of techniques to investigate the solution conformation of laminin gamma-1 short arm, netrin-4 and G3 domain of agrin fused with human IgG-Fc (G3Fc). Briefly, the hydrodynamic properties of entire multi-domain proteins were studied using analytical ultracentrifugation, dynamic light scattering, and small angle X-ray scattering (SAXS). Further, the information from SAXS for entire multi-domain protein was then either used to determine ab initio structure or combined with high-resolution data for individual domains to obtain a detailed solution structure of desired multi-domain protein. Additionally, the SAXS models were verified by comparing the experimentally determined hydrodynamic parameters with the parameters calculated from solution structures. The ab initio structures for laminin gamma-1 short arm revealed an extended and curved assembly. We could also combine small portion of high-resolution structure from X-ray Crystallography data to obtain rigid-body models. Further, we found that the netrin4 has an elongated shape in solution using ab initio methods. Remarkably, we found that the G3Fc chimera is T-shaped and not Y-shaped like an antibody in solution by rigid body modelling. Thus, we conclude that by combining various methods, it is possible to explore large muldi-domain protein and protein-protein assemblies that will help enable us to understand structure-function relationship of these proteins with various forms of diseases.

Crystal Structures of the Armadillo Repeat Domain of Adenomatous Polyposis Coli and Its Complex with the Tyrosine-Rich Domain of Sam68

Adenomatous polyposis coli (APC) is a tumor suppressor protein commonly mutated in colorectal tumors. APC plays important roles in Wnt signaling and other cellular processes. Here, we present the crystal structure of the armadillo repeat (Arm) domain of APC, which facilitates the binding of APC to various proteins. APC-Arm forms a superhelix with a positively charged groove. We also determined the structure of the complex of APC-Arm with the tyrosine-rich (YY) domain of the Src-associated in mitosis, 68kDa protein (Sam68), which regulates TCF-1 alternative splicing. Sam68-YY forms numerous interactions with the residues on the groove and is thereby fixed in a bent conformation. We assessed the effects of mutations and phosphorylation on complex formation between APC-Arm andSam68-YY. Structural comparisons revealed different modes of ligand recognition between the Arm domains of APC and other Arm-containing proteins.

Crystal structure of the armadillo repeat domain of adenomatous polyposis coli which reveals its inherent flexibility

The conserved armadillo repeat (ARM) domain of adenomatous polyposis coli (APC) protein plays an important role in the recognition of its binding partners. In this study, we report the crystal structure of APC-ARM (residues 407-775), which was determined to 2.9Å resolution. Our structure shows that the seven armadillo repeats of APC-ARM fold together into a compact domain, with Arm2 and Arm5 presenting some deviations from canonical armadillo repeats. There is a positively charged groove on the surface of APC-ARM, which might be the recognition site for APC-binding partners. Comparison of this structure with our previously reported structure of APC (407-751), together with normal mode analysis, reveals that the APC-ARM domain possesses a limited intrinsic flexibility. We propose that this intrinsic flexibility might be an inherent property of ARM domains in general.

Structural and Functional Relationships between the Lectin and Arm Domains of Calreticulin

Calreticulin and calnexin are key components in maintaining the quality control of glycoprotein folding within the endoplasmic reticulum. Although their lectin function of binding monoglucosylated sugar moieties of glycoproteins is well documented, their chaperone activity in suppressing protein aggregation is less well understood. Here, we use a series of deletion mutants of calreticulin to demonstrate that its aggregation suppression function resides primarily within its lectin domain. Using hydrophobic peptides as substrate mimetics, we show that aggregation suppression is mediated through a single polypeptide binding site that exhibits a K(d) for peptides of 0.5-1 μM. This site is distinct from the oligosaccharide binding site and differs from previously identified sites of binding to thrombospondin and GABARAP (4-aminobutyrate type A receptor-associated protein). Although the arm domain of calreticulin was incapable of suppressing aggregation or binding hydrophobic peptides on its own, it did contribute to aggregation suppression in the context of the whole molecule. The high resolution x-ray crystal structure of calreticulin with a partially truncated arm domain reveals a marked difference in the relative orientations of the arm and lectin domains when compared with calnexin. Furthermore, a hydrophobic patch was detected on the arm domain that mediates crystal packing and may contribute to calreticulin chaperone function.

Clinician's Commentary on Dang et al.

Clinician's Commentary on Dang et al.

Structure of the Flagellar Motor Protein Complex PomAB: Implications for the Torque-Generating Conformation

The bacterial flagellar motor is driven by an ion flux through a channel called MotAB in Escherichia coli or Salmonella and PomAB in Vibrio alginolyticus. PomAB is composed of two transmembrane (TM) components, PomA and PomB, and converts a sodium ion flux to rotation of the flagellum. Its homolog, MotAB, utilizes protons instead of sodium ions. PomB/MotB has a peptidoglycan (PG)-binding motif in the periplasmic domain, allowing it to function as the stator by being anchored to the PG layer. To generate torque, PomAB/MotAB is thought to undergo a conformational change triggered by the ion flux and to interact directly with FliG, a component of the rotor. Here, we present the first three-dimensional structure of this torque-generating stator unit analyzed by electron microscopy. The structure of PomAB revealed two arm domains, which contain the PG-binding site, connected to a large base made of the TM and cytoplasmic domains. The arms lean downward to the membrane surface, likely representing a "plugged" conformation, which would prevent ions leaking through the channel. We propose a model for how PomAB units are placed around the flagellar basal body to function as torque generators.

Helicobacter pylori NikR Protein Exhibits Distinct Conformations When Bound to Different Promoters

Helicobacter pylori NikR (HpNikR) is a ribbon-helix-helix (RHH) DNA-binding protein that binds to several different promoter regions. The binding site sequences are not absolutely conserved. The ability of HpNikR to discriminate specific DNA sites resides partly in its nine-amino acid N-terminal arm. Previously, indirect evidence indicated that the arm exists in different conformations when HpNikR is bound to the nixA and ureA promoters. Here, we directly examined HpNikR conformation when it was bound to nixA and ureA DNA fragments by tethering (S)-1{[bis(carboxymethyl)amino]methyl}-2-{4-[(2-bromoacetyl)amino]phenylethyl}(carboxymethyl)amino]acetic acid, iron(III) to different positions in the N-terminal arm and RHH DNA binding domain. Different cleavage patterns at each promoter directly demonstrated that both the RHH domain and the arm adopt different conformations on the nixA and ureA promoters. Additionally, the two RHH domain dimers of the HpNikR tetramer are in distinct conformations at ureA. Site-directed mutagenesis identified an interchain salt bridge (Lys(48)-Glu(47')) in the RHH domain remote from the DNA binding interface that is required for high affinity binding to ureA but not nixA. Finally, DNA affinity measurements of wild-type HpNikR and a salt bridge mutant (K48A) to hybrid nixA-ureA promoters demonstrated that inverted repeat half-sites, spacers, and flanking DNA are all required for sequence-specific DNA binding by HpNikR. Notably, the spacer region made the largest contribution to DNA affinity. HpNikR exhibits a substantially expanded regulon compared with other NikR proteins. The results presented here provide a molecular basis for understanding regulatory network expansion by NikR as well as other prokaryotic regulatory proteins.

FHC-Linked Myosin Regulatory Light Chain Mutations A13T and K104E Do Not Generate Major Structural Changes Sufficient to Affect Binding of Myosin to Actin

A sarcomere, the essential unit of cardiac muscle, consists of myosin, actin, troponin and tropomyosin. The interaction among these proteins generates muscle contraction. The myosin Regulatory Light Chain (RLC) attaches to the myosin heavy chain (MHC) IQ motif and structurally supports the lever arm domain, known to propagate minute conformational changes between the actin and myosin filaments. Mutations in the RLC may therefore cause alterations in the lever arm structure, thus leading to cardiac dysfunction. Here, we investigated two Familial Hypertrophic Cardiomyopathy (FHC) mutations in RLC, A13T (alanine to threonine) and K104E (lysine to glutamate) and hypothesized that these RLC mutants may lead to cardiomyopathy by changing the lever arm structure, the interaction with the MHC and ultimately impairment of the binding of myosin to actin. To test our hypothesis, I performed the following experiments: 1) Titration of the RLC-depleted porcine myosin with different concentrations of recombinant human cardiac RLC wild-type (WT) and A13T and K104E mutants to study the RLC-MHC binding properties (affinity, kinetics); 2) Study of the actin-myosin interaction by fluorescence and light scattering measurements. My preliminary data shows that the WT-RLC displays a clearly cooperative binding to the MHC, however, a reduction in cooperativity was observed in both mutants. In parallel, the binding affinity of A13T and K104E was slightly decreased (1.3 fold and 1.4-fold increase in Kd, respectively). Fluorescence and light scattering measurements monitoring the binding of WT or mutant-myosin to pyrene-labeled actin demonstrated a very strong binding with no mutant dependent changes in Kd. These results suggest that any structural changes that may be caused by these two FHC-RLC mutations are not sufficient to affect the myosin-actin binding. Supported by NIH-HL071778 (DSC).

Electron microscopic evidence for the myosin head lever arm mechanism in hydrated myosin filaments using the gas environmental chamber

Muscle contraction results from an attachment–detachment cycle between the myosin heads extending from myosin filaments and the sites on actin filaments. The myosin head first attaches to actin together with the products of ATP hydrolysis, performs a power stroke associated with release of hydrolysis products, and detaches from actin upon binding with new ATP. The detached myosin head then hydrolyses ATP, and performs a recovery stroke to restore its initial position. The strokes have been suggested to result from rotation of the lever arm domain around the converter domain, while the catalytic domain remains rigid. To ascertain the validity of the lever arm hypothesis in muscle, we recorded ATP-induced movement at different regions within individual myosin heads in hydrated myosin filaments, using the gas environmental chamber attached to the electron microscope. The myosin head were position-marked with gold particles using three different site-directed antibodies. The amplitude of ATP-induced movement at the actin binding site in the catalytic domain was similar to that at the boundary between the catalytic and converter domains, but was definitely larger than that at the regulatory light chain in the lever arm domain. These results are consistent with the myosin head lever arm mechanism in muscle contraction if some assumptions are made.

Allosteric Modification, the Primary ATP Activation Mechanism of Atrial Natriuretic Factor Receptor Guanylate Cyclase

ANF-RGC is the prototype receptor membrane guanylate cyclase being both the receptor and the signal transducer of the most hypotensive hormones, ANF and BNP. It is a single transmembrane-spanning protein. After binding these hormones at the extracellular domain it at its intracellular domain signals activation of the C-terminal catalytic module and accelerates the production of its second messenger, cyclic GMP, which controls blood pressure, cardiac vasculature, and fluid secretion. ATP is obligatory for the posttransmembrane dynamic events leading to ANF-RGC activation. It functions through the ATP-regulated module, ARM (KHD) domain, of ANF-RGC. In the current over a decade held model "phosphorylation of the KHD is absolutely required for hormone-dependent activation of NPR-A" [Potter, L. R., and Hunter, T. (1998) Mol. Cell. Biol. 18, 2164-2172]. The presented study challenges this concept. It demonstrates that, instead, ATP allosteric modification of ARM is the primary signaling step of ANF-GC activation. In this two-step new dynamic model, ATP in the first step binds ARM. This triggers in it a chain of transduction events, which cause its allosteric modification. The modification partially activates (about 50%) ANF-RGC and, concomitantly, also prepares the ARM for the second successive step. In this second step, ARM is phosphorylated and ANF-RGC achieves additional (∼50%) full catalytic activation. The study defines a new paradigm of the ANF-RGC signaling mechanism.

The p40/ARPC1 Subunit of Arp2/3 Complex Performs Multiple Essential Roles in WASp-regulated Actin Nucleation

The Arp2/3 complex is a conserved seven-subunit actin-nucleating machine activated by WASp (Wiskott Aldrich syndrome protein). Despite its central importance in a broad range of cellular processes, many critical aspects of the mechanism of the Arp2/3 complex have yet to be resolved. In particular, some of the individual subunits in the complex have not been assigned clear functional roles, including p40/ARPC1. Here, we dissected the structure and function of Saccharomyces cerevisiae p40/ARPC1, which is encoded by the essential ARC40 gene, by analyzing 39 integrated alleles that target its conserved surfaces. We identified three distinct sites on p40/ARPC1 required for function in vivo: one site contacts p19/ARPC4, one contacts p15/ARPC5, and one site resides in an extended structural "arm" of p40/ARPC1. Using a novel strategy, we purified the corresponding lethal mutant Arp2/3 complexes from yeast and compared their actin nucleation activities. Lethal mutations at the contact with p19/ARPC4 specifically impaired WASp-induced nucleation. In contrast, lethal mutations at the contact with p15/ARPC5 led to unregulated ("leaky") nucleation in the absence of WASp. Lethal mutations in the extended arm drastically reduced nucleation, and the same mutations disrupted the ability of the purified p40/ARPC1 arm domain to bind the VCA domain of WASp. Together, these data indicate that p40/ARPC1 performs at least three distinct, essential functions in regulating Arp2/3 complex-mediated actin assembly: 1) suppression of spontaneous nucleation by the Arp2/3 complex, which requires proper contacts with p15/ARPC5; 2) propagation of WASp activation signals via contacts with p19/ARPC2; and 3) direct facilitation of actin nucleation through interactions of the extended arm with the VCA domain of WASp.

Involvement of p120 in LPS-induced NF-κB activation and IL-8 production in human bronchial epithelial cells

P120-catenin (p120), a prototypic member of a subfamily of Armadillo repeat domain (Arm domain) proteins, not only participates in cell–cell adhesion, but also mediates inflammatory responses in the skin. In the present study, we demonstrated the effect of p120 on lipopolysaccharide (LPS)-induced inflammatory responses in human bronchial epithelial cells (BECs). We first confirmed that p120 expression was significantly reduced after LPS stimulation in BECs, the p65 subunit of nuclear factor-κB (NF-κB) nuclear translocation was promoted and NF-κB activity was rapidly induced. Moreover, the expression level of interleukin-8 (IL-8) increased after LPS treatment. Over-expression of p120 attenuated LPS-stimulated NF-κB reporter gene expression and IL-8 mRNA expression and protein synthesis. On the contrary, transfection with p120 small interfering RNA (siRNA) significantly elevated LPS-stimulated NF-κB transcriptional activity, p65 nuclear translocation and IL-8 expression. Collectively, these results indicate an anti-inflammatory effect of p120 in BECs, through its modulation of NF-κB signaling.

Electron Microscopic Evidence for the Cross-Bridge Lever Arm Mechanism in Living Muscle Thick Filaments Obtained using the Gas Environmental Chamber

We have succeeded in recording the ATP-induced cross-bridge recovery stroke in living bipolar muscle thick filaments using the gas environmental chamber (EC) (Sugi et al., PNAS 105:17396, 2008). It is generally believed that the distal part of the cross-bridge (catalytic domain) is rigid, while its proximal part acts as a lever arm moving around the hinge to produce force and motion in muscle. To ascertain the validity of this mechanism by our experimental methods, we prepared three different antibodies, directed to the peptide in the cross-bridge catalytic domain (antibodiy 1), to the reactive lysine residue at interface between the catalytic and lever arm domains (antibody 2), and to the peptide in the cross-bridge lever arm domain (antibody 3), respectively. These antibodies, attached to the cross-bridges on the thick filaments, were position-marked with colloidal gold particles.The peak amplitude of the ATP-induced movement of the cross-bridges with antibody 1 (5∼7.5nm) did not differ significantly from that of the cross-bridges with antibody 2, being consistent with the idea that the cross-bridge catalytic domain remains rigid during the cross-bridge stroke. On the other hand, the amplitude of the ATP-induced movement in the cross-bridges with antibody 3 was found to be extremely small and in most cases just barely detectable (2.5nm or less), indicating that the proximal part of the cross-bridge (close to the lever arm hinge region) does not move appreciably during the ATP-induced cross-bridge stroke. These results may constitute the first direct evidence for the cross-bridge lever arm mechanism in muscle contraction.

Interactions of Plakoglobin and β-Catenin with Desmosomal Cadherins

Plakoglobin and beta-catenin are homologous armadillo repeat proteins found in adherens junctions, where they interact with the cytoplasmic domain of classical cadherins and with alpha-catenin. Plakoglobin, but normally not beta-catenin, is also a structural constituent of desmosomes, where it binds to the cytoplasmic domains of the desmosomal cadherins, desmogleins and desmocollins. Here, we report structural, biophysical, and biochemical studies aimed at understanding the molecular basis of selective exclusion of beta-catenin and alpha-catenin from desmosomes. The crystal structure of the plakoglobin armadillo domain bound to phosphorylated E-cadherin shows virtually identical interactions to those observed between beta-catenin and E-cadherin. Trypsin sensitivity experiments indicate that the plakoglobin arm domain by itself is more flexible than that of beta-catenin. Binding of plakoglobin and beta-catenin to the intracellular regions of E-cadherin, desmoglein1, and desmocollin1 was measured by isothermal titration calorimetry. Plakoglobin and beta-catenin bind strongly and with similar thermodynamic parameters to E-cadherin. In contrast, beta-catenin binds to desmoglein-1 more weakly than does plakoglobin. beta-Catenin and plakoglobin bind with similar weak affinities to desmocollin-1. Full affinity binding of desmoglein-1 requires sequences C-terminal to the region homologous to the catenin-binding domain of classical cadherins. Although pulldown assays suggest that the presence of N- and C-terminal beta-catenin "tails" that flank the armadillo repeat region reduces the affinity for desmosomal cadherins, calorimetric measurements show no significant effects of the tails on binding to the cadherins. Using purified proteins, we show that desmosomal cadherins and alpha-catenin compete directly for binding to plakoglobin, consistent with the absence of alpha-catenin in desmosomes.

Highly Sensitive Detection of Individual HEAT and ARM Repeats with HHpred and COACH

BackgroundHEAT and ARM repeats occur in a large number of eukaryotic proteins. As these repeats are often highly diverged, the prediction of HEAT or ARM domains can be challenging. Except for the most clear-cut cases, identification at the individual repeat level is indispensable, in particular for determining domain boundaries. However, methods using single sequence queries do not have the sensitivity required to deal with more divergent repeats and, when applied to proteins with known structures, in some cases failed to detect a single repeat.Methodology and Principal FindingsTesting algorithms which use multiple sequence alignments as queries, we found two of them, HHpred and COACH, to detect HEAT and ARM repeats with greatly enhanced sensitivity. Calibration against experimentally determined structures suggests the use of three score classes with increasing confidence in the prediction, and prediction thresholds for each method. When we applied a new protocol using both HHpred and COACH to these structures, it detected 82% of HEAT repeats and 90% of ARM repeats, with the minimum for a given protein of 57% for HEAT repeats and 60% for ARM repeats. Application to bona fide HEAT and ARM proteins or domains indicated that similar numbers can be expected for the full complement of HEAT/ARM proteins. A systematic screen of the Protein Data Bank for false positive hits revealed their number to be low, in particular for ARM repeats. Double false positive hits for a given protein were rare for HEAT and not at all observed for ARM repeats. In combination with fold prediction and consistency checking (multiple sequence alignments, secondary structure prediction, and position analysis), repeat prediction with the new HHpred/COACH protocol dramatically improves prediction in the twilight zone of fold prediction methods, as well as the delineation of HEAT/ARM domain boundaries.SignificanceA protocol is presented for the identification of individual HEAT or ARM repeats which is straightforward to implement. It provides high sensitivity at a low false positive rate and will therefore greatly enhance the accuracy of predictions of HEAT and ARM domains.

The On–Off Switch in Regulated Myosins: Different Triggers but Related Mechanisms

In regulated myosin, motor and enzymatic activities are toggled between the on-state and off-state by a switch located on its lever arm domain, here called the regulatory domain (RD). This region consists of a long α-helical “heavy chain” stabilized by a “regulatory” light chain (RLC) and an “essential” light chain (ELC). The on-state is activated by phosphorylation of the RLC of vertebrate smooth muscle RD or by direct binding of Ca 2+ to the ELC of molluscan RD. Crystal structures are available only for the molluscan RD. To understand in more detail the pathway between the on-state and the off-state, we have now also determined the crystal structure of a molluscan (scallop) RD in the absence of Ca 2+. Our results indicate that loss of Ca 2+ abolishes most of the interactions between the light chains and may increase the flexibility of the RD heavy chain. We propose that disruption of critical links with the C-lobe of the RLC is the key event initiating the off-state in both smooth muscle myosins and molluscan myosins.