Two different unique cardiac isoforms of protein 4.1R in zebrafish, Danio rerio, and insights into their cardiac functions as related to their unique structures

Protein 4.1R (4.1R) has been identified as the major component of the human erythrocyte membrane skeleton. The members of the protein 4.1 gene family are expressed in a tissue-specific alternative splicing manner that increases their functions in each tissue; however, the exact roles of cardiac 4.1R in the developing myocardium are poorly understood. In zebrafish (ZF), we identified two heart-specific 4.1R isoforms, ZF4.1RH2 and ZF4.1RH3, encoding N-terminal 30 kDa (FERM) domain and spectrin-actin binding domain (SABD) and C-terminal domain (CTD), separately. Applying immunohistochemistry using specific antibodies for 30 kDa domain and CTD separately, the gene product of ZF4.1RH2 and ZF4.1RH3 appeared only in the ventricle and in the atrium, respectively, in mature hearts. During embryogenesis, both gene expressions are expressed starting 24 h post-fertilization (hpf). Following whole-mount in situ hybridization, ZF4.1RH3 gene expression was detected in the atrium of 37 hpf embryos. These results indicate that the gene product of ZF4.1RH3 is essential for normal morphological shape of the developing heart and to support the repetitive cycles of its muscle contraction and relaxation.     

The Shuttling Protein Npl3 Promotes Translation Termination Accuracy in Saccharomyces cerevisiae

Heterogeneous nuclear ribonucleoproteins are multifunctional proteins that bind to newly synthesized mRNAs in the nucleus and participate in many subsequent steps of gene expression. A well-studied Saccharomyces cerevisiae heterogeneous nuclear ribonucleoprotein that has several nuclear functions is Npl3p. Here, we provide evidence that Npl3p also has a cytoplasmic role: it functions in translation termination fidelity. Yeast harboring the npl3-95 mutant allele have an impaired ability to translate lacZ, enhanced sensitivity to cycloheximide and paromomycin, and increased ability to read through translation termination codons. Most of these defects are enhanced in yeast that also lack Upf1p, an RNA surveillance factor crucial for translation termination. We show that the npl3-95 mutant allele encodes a form of Npl3p that is part of high molecular-weight complexes that cofractionate with the poly(A)-binding protein Pab1p. Together, these results lead us to propose a model in which Npl3p engenders translational fidelity by promoting the remodeling of mRNPs during translation termination.     

The crystal structure of the actin binding domain from alpha-actinin in its closed conformation: structural insight into phospholipid regulation of alpha-actinin

Alpha-actinin is the major F-actin crosslinking protein in both muscle and non-muscle cells. We report the crystal structure of the actin binding domain of human muscle alpha-actinin-3, which is formed by two consecutive calponin homology domains arranged in a "closed" conformation. Structural studies and available biochemical data on actin binding domains suggest that two calponin homology domains come in a closed conformation in the native apo-form, and that conformational changes involving the relative orientation of the two calponin homology domains are required for efficient binding to actin filaments. The actin binding activity of muscle isoforms is supposed to be regulated by phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), which binds to the second calponin homology domain. On the basis of structural analysis we propose a distinct binding site for PtdIns(4,5)P2, where the fatty acid moiety would be oriented in a direction that allows it to interact with the linker sequence between the actin binding domain and the first spectrin-like repeat, regulating thereby the binding of the C-terminal calmodulin-like domain to this linker.     

Solution NMR structure of the junction between tropomyosin molecules: implications for actin binding and regulation

Tropomyosin is a coiled-coil protein that binds head-to-tail along the length of actin filaments in eukaryotic cells, stabilizing them and providing protection from severing proteins. Tropomyosin cooperatively regulates actin's interaction with myosin and mediates the Ca2+ -dependent regulation of contraction by troponin in striated muscles. The N-terminal and C-terminal ends are critical functional determinants that form an "overlap complex". Here we report the solution NMR structure of an overlap complex formed of model peptides. In the complex, the chains of the C-terminal coiled coil spread apart to allow insertion of 11 residues of the N-terminal coiled coil into the resulting cleft. The plane of the N-terminal coiled coil is rotated 90 degrees relative to the plane of the C terminus. A consequence of the geometry is that the orientation of postulated periodic actin binding sites on the coiled-coil surface is retained from one molecule to the next along the actin filament when the overlap complex is modeled into the X-ray structure of tropomyosin determined at 7 Angstroms. Nuclear relaxation NMR data reveal flexibility of the junction, which may function to optimize binding along the helical actin filament and to allow mobility of tropomyosin on the filament surface as it switches between regulatory states.     

A Structural Model for the DEAD Box Helicase YxiN in Solution: Localization of the RNA Binding Domain

DEAD box proteins consist of a common helicase core formed by two globular RecA domains that are separated by a cleft. The helicase core acts as a nucleotide-dependent switch that alternates between open and closed conformations during the catalytic cycle of duplex separation, thereby providing basic helicase activity. Flanking domains can direct the helicase core to a specific RNA substrate by mediating high-affinity or high-specificity RNA binding. In addition, they may position RNA for the helicase core or may directly contribute to unwinding. While structures of different helicase cores have been determined previously, little is known about the orientation of flanking domains relative to the helicase core.YxiN is a DEAD box protein that consists of a helicase core and a C-terminal RNA binding domain (RBD) that mediates specific binding to hairpin 92 in 23S rRNA. To provide a framework for understanding the functional cooperation of the YxiN helicase core and the RBD, we mapped the orientation of the RBD in single-molecule fluorescence resonance energy transfer experiments. We present a model for the global conformation of YxiN in which the RBD lies above a slightly concave patch that is formed by flexible loops on the surface of the C-terminal RecA domain. The orientation of the RBD is different from the orientations of flanking domains in the Thermus thermophilus DEAD box protein Hera and in Saccharomyces cerevisiae Mss116p, in line with the different functions of these DEAD box proteins and of their RBDs. Interestingly, the corresponding patch on the C-terminal RecA domain that is covered by the YxiN RBD is also part of the interface between the translation factors eIF4A and eIF4G. Possibly, this region constitutes an adaptable interface that generally allows for the interaction of the helicase core with additional domains or interacting factors.     

The Regulation Mechanism for the Auto-Inhibition of Binding of Human Filamin A to Integrin

The ability of adhesion receptors to transmit biochemical signals and mechanical force across cell membranes depends on interactions with the actin cytoskeleton. Human filamins are large actin cross-linking proteins that connect integrins to the cytoskeleton. Filamin binding to the cytoplasmic tail of β integrins has been shown to prevent integrin activation in cells, which is important for controlling cell adhesion and migration. The molecular-level mechanism for filamin binding to integrin has been unclear, however, as it was recently demonstrated that filamin undergoes intramolecular auto-inhibition of integrin binding. In this study, using steered molecular dynamics simulations, we found that mechanical force applied to filamin can expose cryptic integrin binding sites. The forces required for this are considerably lower than those for filamin immunoglobulin domain unfolding. The mechanical-force-induced unfolding of filamin and exposure of integrin binding sites occur through stable intermediates where integrin binding is possible. Accordingly, our results support filamin's role as a mechanotransducer, since force-induced conformational changes allow binding of integrin and other transmembrane and intracellular proteins. This observed force-induced conformational change can also be one of possible mechanisms involved in the regulation of integrin activation.     

Dynamic Interactions between Clathrin and Locally Structured Elements in a Disordered Protein Mediate Clathrin Lattice Assembly

Assembly of clathrin lattices is mediated by assembly/adaptor proteins that contain domains that bind lipids or membrane-bound cargo proteins and clathrin binding domains (CBDs) that recruit clathrin. Here, we characterize the interaction between clathrin and a large fragment of the CBD of the clathrin assembly protein AP180. Mutational, NMR chemical shift, and analytical ultracentrifugation analyses allowed us to precisely define two clathrin binding sites within this fragment, each of which is found to bind weakly to the N-terminal domain of the clathrin heavy chain (TD). The locations of the two clathrin binding sites are consistent with predictions from sequence alignments of previously identified clathrin binding elements and, by extension, indicate that the complete AP180 CBD contains ∼ 12 degenerate repeats, each containing a single clathrin binding site. Sequence and circular dichroism analyses have indicated that the AP180 CBD is predominantly unstructured and our NMR analyses confirm that this is largely the case for the AP180 fragment characterized here. Unexpectedly, unlike the many proteins that undergo binding-coupled folding upon interaction with their binding partners, the AP180 fragment is similarly unstructured in its bound and free states. Instead, we find that this fragment exhibits localized β-turn-like structures at the two clathrin binding sites both when free and when bound to clathrin. These observations are incorporated into a model in which weak binding by multiple, pre-structured clathrin binding elements regularly dispersed throughout a largely unstructured CBD allows efficient recruitment of clathrin to endocytic sites and dynamic assembly of the clathrin lattice.     

Bcl-2 proteins and mitochondria—Specificity in membrane targeting for death

The localization and control of Bcl-2 proteins on mitochondria is essential for the intrinsic pathway of apoptosis. Anti-apoptotic Bcl-2 proteins reside on the outer mitochondrial membrane (OMM) and prevent apoptosis by inhibiting the activation of the pro-apoptotic family members Bax and Bak. The Bcl-2 subfamily of BH3-only proteins can either inhibit the anti-apoptotic proteins or directly activate Bax or Bak. How these proteins interact with each other, the mitochondrial surface and within the OMM are complex processes we are only beginning to understand. However, these interactions are fundamental for the transduction of apoptotic signals to mitochondria and the subsequent release of caspase activating factors into the cytosol. In this review we will discuss our knowledge of how Bcl-2 proteins are directed to mitochondria in the first place, a crucial but poorly understood aspect of their regulation. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.     

The PMS2 subunit of human MutLalpha contains a metal ion binding domain of the iron-dependent repressor protein family

DNA mismatch repair (MMR) is responsible for correcting replication errors. MutLα, one of the main players in MMR, has been recently shown to harbor an endonuclease/metal-binding activity, which is important for its function in vivo. This endonuclease activity has been confined to the C-terminal domain of the hPMS2 subunit of the MutLα heterodimer. In this work, we identify a striking sequence-structure similarity of hPMS2 to the metal-binding/dimerization domain of the iron-dependent repressor protein family and present a structural model of the metal-binding domain of MutLα. According to our model, this domain of MutLα comprises at least three highly conserved sequence motifs, which are also present in most MutL homologs from bacteria that do not rely on the endonuclease activity of MutH for strand discrimination. Furthermore, based on our structural model, we predict that MutLα is a zinc ion binding protein and confirm this prediction by way of biochemical analysis of zinc ion binding using the full-length and C-terminal domain of MutLα. Finally, we demonstrate that the conserved residues of the metal ion binding domain are crucial for MMR activity of MutLα in vitro.     

Retroviral Capsid Assembly: A Role for the CA Dimer in Initiation

In maturing retroviral virions, CA protein assembles to form a capsid shell that is essential for infectivity. The structure of the two folded domains [N-terminal domain (NTD) and C-terminal domain (CTD)] of CA is highly conserved among various retroviruses, and the capsid assembly pathway, although poorly understood, is thought to be conserved as well. In vitro assembly reactions with purified CA proteins of the Rous sarcoma virus (RSV) were used to define factors that influence the kinetics of capsid assembly and provide insights into underlying mechanisms. CA multimerization was triggered by multivalent anions providing evidence that in vitro assembly is an electrostatically controlled process. In the case of RSV, in vitro assembly was a well-behaved nucleation-driven process that led to the formation of structures with morphologies similar to those found in virions. Isolated RSV dimers, when mixed with monomeric protein, acted as efficient seeds for assembly, eliminating the lag phase characteristic of a monomer-only reaction. This demonstrates for the first time the purification of an intermediate on the assembly pathway. Differences in the intrinsic tryptophan fluorescence of monomeric protein and the assembly-competent dimer fraction suggest the involvement of the NTD in the formation of the functional dimer. Furthermore, in vitro analysis of well-characterized CTD mutants provides evidence for assembly dependence on the second domain and suggests that the establishment of an NTD-CTD interface is a critical step in capsid assembly initiation. Overall, the data provide clear support for a model whereby capsid assembly within the maturing virion is dependent on the formation of a specific nucleating complex that involves a CA dimer and is directed by additional virion constituents.     

Superfamily of ankyrin repeat proteins in tomato

The ankyrin repeat (ANK) protein family plays a crucial role in plant growth and development and in response to biotic and abiotic stresses. However, no detailed information concerning this family is available for tomato (Solanum lycopersicum) due to the limited information on whole genome sequences. In this study, we identified a total of 130 ANK genes in tomato genome (SlANK), and these genes were distributed across all 12 chromosomes at various densities. And chromosomal localizations of SlANK genes indicated 25 SlANK genes were involved in tandem duplications. Based on their domain composition, all of the SlANK proteins were grouped into 13 subgroups. A combined phylogenetic tree was constructed with the aligned SlANK protein sequences. This tree revealed that the SlANK proteins comprise five major groups. An analysis of the expression profiles of SlANK genes in tomato in different tissues and in response to stresses showed that the SlANK proteins play roles in plant growth, development and stress responses. To our knowledge, this is the first report of a genome-wide analysis of the tomato ANK gene family. This study provides valuable information regarding the classification and putative functions of SlANK genes in tomato.     

Crystal structure of the receptor binding domain of the botulinum C-D mosaic neurotoxin reveals potential roles of lysines 1118 and 1136 in membrane interactions

The botulinum neurotoxins (BoNTs) produced by different strains of the bacterium Clostridium botulinum are responsible for the disease botulism and include a group of immunologically distinct serotypes (A, B, E, and F) that are considered to be the most lethal natural proteins known for humans. Two BoNT serotypes, C and D, while rarely associated with human infection, are responsible for deadly botulism outbreaks afflicting animals. Also associated with animal infections is the BoNT C–D mosaic protein (BoNT/CD), a BoNT subtype that is essentially a hybrid of the BoNT/C (∼two-third) and BoNT/D (∼one-third) serotypes. While the amino acid sequence of the heavy chain receptor binding (HCR) domain of BoNT/CD (BoNT/CD-HCR) is very similar to the corresponding amino acid sequence of BoNT/D, BoNT/CD-HCR binds synaptosome membranes better than BoNT/D-HCR. To obtain structural insights for the different membrane binding properties, the crystal structure of BoNT/CD-HCR (S867-E1280) was determined at 1.56 Å resolution and compared to previously reported structures for BoNT/D-HCR. Overall, the BoNT/CD-HCR structure is similar to the two sub-domain organization observed for other BoNT HCRs: an N-terminal jellyroll barrel motif and a C-terminal β-trefoil fold. Comparison of the structure of BoNT/CD-HCR with BoNT/D-HCR indicates that K1118 has a similar structural role as the equivalent residue, E1114, in BoNT/D-HCR, while K1136 has a structurally different role than the equivalent residue, G1132, in BoNT/D-HCR. Lysine-1118 forms a salt bridge with E1247 and may enhance membrane interactions by stabilizing the putative membrane binding loop (K1240-N1248). Lysine-1136 is observed on the surface of the protein. A sulfate ion bound to K1136 may mimic a natural interaction with the negatively changed phospholipid membrane surface. Liposome-binding experiments demonstrate that BoNT/CD-HCR binds phosphatidylethanolamine liposomes more tightly than BoNT/D-HCR.     

The sticholysin family of pore-forming toxins induces the mixing of lipids in membrane domains

Sticholysins (Sts) I and II (StI/II) are pore-forming toxins (PFTs) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin family, a unique class of eukaryotic PFTs exclusively found in sea anemones. The role of lipid phase co-existence in the mechanism of the action of membranolytic proteins and peptides is not clearly understood. As for actinoporins, it has been proposed that phase separation promotes pore forming activity. However little is known about the effect of sticholysins on the phase separation of lipids in membranes. To gain insight into the mechanism of action of sticholysins, we evaluated the effect of these proteins on lipid segregation using differential scanning calorimetry (DSC) and atomic force microscopy (AFM). New evidence was obtained reflecting that these proteins reduce line tension in the membrane by promoting lipid mixing. In terms of the relevance for the mechanism of action of actinoporins, we hypothesize that expanding lipid disordered phases into lipid ordered phases decreases the lipid packing at the borders of the lipid raft, turning it into a more suitable environment for N-terminal insertion and pore formation.     

The rheostat in the membrane: BCL-2 family proteins and apoptosis

Apoptosis, a mechanism for programmed cell death, has key roles in human health and disease. Many signals for cellular life and death are regulated by the BCL-2 family proteins and converge at mitochondria, where cell fate is ultimately decided. The BCL-2 family includes both pro-life (e.g. BCL-XL) and pro-death (e.g. BAX, BAK) proteins. Previously, it was thought that a balance between these opposing proteins, like a simple ‘rheostat'', could control the sensitivity of cells to apoptotic stresses. Later, this rheostat concept had to be extended, when it became clear that BCL-2 family proteins regulate each other through a complex network of bimolecular interactions, some transient and some relatively stable. Now, studies have shown that the apoptotic circuitry is even more sophisticated, in that BCL-2 family interactions are spatially dynamic, even in nonapoptotic cells. For example, BAX and BCL-XL can shuttle between the cytoplasm and the mitochondrial outer membrane (MOM). Upstream signaling pathways can regulate the cytoplasmic–MOM equilibrium of BAX and thereby adjust the sensitivity of cells to apoptotic stimuli. Thus, we can view the MOM as the central locale of a dynamic life–death rheostat. BAX invariably forms extensive homo-oligomers after activation in membranes. However, recent studies, showing that activated BAX monomers determine the kinetics of MOM permeabilization (MOMP), perturb the lipid bilayer and form nanometer size pores, pose questions about the role of the oligomerization. Other lingering questions concern the molecular mechanisms of BAX redistribution between MOM and cytoplasm and the details of BAX/BAK–membrane assemblies. Future studies need to delineate how BCL-2 family proteins regulate MOMP, in concert with auxiliary MOM proteins, in a dynamic membrane environment. Technologies aimed at elucidating the structure and function of the full-length proteins in membranes are needed to illuminate some of these critical issues.     

Ezrin Induces Long-Range Interdomain Allostery in the Scaffolding Protein NHERF1

Scaffolding proteins are molecular switches that control diverse signaling events. The scaffolding protein Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1) assembles macromolecular signaling complexes and regulates the macromolecular assembly, localization, and intracellular trafficking of a number of membrane ion transport proteins, receptors, and adhesion/antiadhesion proteins. NHERF1 begins with two modular protein-protein interaction domains—PDZ1 and PDZ2—and ends with a C-terminal (CT) domain. This CT domain binds to ezrin, which, in turn, interacts with cytosekeletal actin. Remarkably, ezrin binding to NHERF1 increases the binding capabilities of both PDZ domains. Here, we use deuterium labeling and contrast variation neutron-scattering experiments to determine the conformational changes in NHERF1 when it forms a complex with ezrin. Upon binding to ezrin, NHERF1 undergoes significant conformational changes in the region linking PDZ2 and its CT ezrin-binding domain, as well as in the region linking PDZ1 and PDZ2, involving very long range interactions over 120 Å. The results provide a structural explanation, at mesoscopic scales, of the allosteric control of NHERF1 by ezrin as it assembles protein complexes. Because of the essential roles of NHERF1 and ezrin in intracellular trafficking in epithelial cells, we hypothesize that this long-range allosteric regulation of NHERF1 by ezrin enables the membrane-cytoskeleton to assemble protein complexes that control cross-talk and regulate the strength and duration of signaling.     

The heat shock protein 70 family: Highly homologous proteins with overlapping and distinct functions

The human heat shock protein 70 (Hsp70) family contains at least eight homologous chaperone proteins. Endoplasmatic reticulum and mitochondria have their specific Hsp70 proteins, whereas the remaining six family members reside mainly in the cytosol and nucleus. The requirement for multiple highly homologous although different Hsp70 proteins is still far from clear, but their individual and tissue-specific expression suggests that they are assigned distinct biological tasks. This concept is supported by the fact that mice knockout for different Hsp70 genes display remarkably discrete phenotypes. Moreover, emerging data suggest that individual Hsp70 proteins can bring about non-overlapping and chaperone-independent functions essential for growth and survival of cancer cells. This review summarizes our present knowledge of the individual members of human Hsp70 family and elaborate on the functional differences between the cytosolic/nuclear representatives.