Characterization and structure of the vaccinia virus NF-κB antagonist A46.

Successful vaccinia virus (VACV) replication in the host requires expression of viral proteins that interfere with host immunity, such as antagonists of the activation of the proinflammatory transcription factor NF-κB. Two such VACV proteins are A46 and A52. A46 interacts with the Toll-like receptor/interleukin-1R (TIR) domain of Toll-like receptors and intracellular adaptors such as MAL (MyD88 adapter-like), TRAM (TIR domain-containing adapter-inducing interferon-ß (TRIF)-related adaptor molecule), TRIF, and MyD88, whereas A52 binds to the downstream signaling components TRAF6 and IRAK2. Here, we characterize A46 biochemically, determine by microscale thermophoresis binding constants for the interaction of A46 with the TIR domains of MyD88 and MAL, and present the 2.0 Å resolution crystal structure of A46 residues 87-229. Full-length A46 behaves as a tetramer; variants lacking the N-terminal 80 residues are dimeric. Nevertheless, both bind to the Toll-like receptor domains of MAL and MyD88 with KD values in the low µm range. Like A52, A46 also shows a Bcl-2-like fold but with biologically relevant differences from that of A52. Thus, A46 uses helices α4 and α6 to dimerize, compared with the α1-α6 face used by A52 and other Bcl-2 like VACV proteins. Furthermore, the loop between A46 helices α4-α5 is flexible and shorter than in A52; there is also evidence for an intramolecular disulfide bridge between consecutive cysteine residues. We used molecular docking to propose how A46 interacts with the BB loop of the TRAM TIR domain. Comparisons of A46 and A52 exemplify how subtle changes in viral proteins with the same fold lead to crucial differences in biological activity.

Order from disorder in the sarcomere: FATZ forms a fuzzy but tight complex and phase-separated condensates with α-actinin.

In sarcomeres, α-actinin cross-links actin filaments and anchors them to the Z-disk. FATZ (filamin-, α-actinin-, and telethonin-binding protein of the Z-disk) proteins interact with α-actinin and other core Z-disk proteins, contributing to myofibril assembly and maintenance. Here, we report the first structure and its cellular validation of α-actinin-2 in complex with a Z-disk partner, FATZ-1, which is best described as a conformational ensemble. We show that FATZ-1 forms a tight fuzzy complex with α-actinin-2 and propose an interaction mechanism via main molecular recognition elements and secondary binding sites. The obtained integrative model reveals a polar architecture of the complex which, in combination with FATZ-1 multivalent scaffold function, might organize interaction partners and stabilize α-actinin-2 preferential orientation in Z-disk. Last, we uncover FATZ-1 ability to phase-separate and form biomolecular condensates with α-actinin-2, raising the question whether FATZ proteins can create an interaction hub for Z-disk proteins through membraneless compartmentalization during myofibrillogenesis.

Structure and calcium-binding studies of calmodulin-like domain of human non-muscle α-actinin-1.

The activity of several cytosolic proteins critically depends on the concentration of calcium ions. One important intracellular calcium-sensing protein is α-actinin-1, the major actin crosslinking protein in focal adhesions and stress fibers. The actin crosslinking activity of α-actinin-1 has been proposed to be negatively regulated by calcium, but the underlying molecular mechanisms are poorly understood. To address this, we determined the first high-resolution NMR structure of its functional calmodulin-like domain (CaMD) in calcium-bound and calcium-free form. These structures reveal that in the absence of calcium, CaMD displays a conformationally flexible ensemble that undergoes a structural change upon calcium binding, leading to limited rotation of the N- and C-terminal lobes around the connecting linker and consequent stabilization of the calcium-loaded structure. Mutagenesis experiments, coupled with mass-spectrometry and isothermal calorimetry data designed to validate the calcium binding stoichiometry and binding site, showed that human non-muscle α-actinin-1 binds a single calcium ion within the N-terminal lobe. Finally, based on our structural data and analogy with other α-actinins, we provide a structural model of regulation of the actin crosslinking activity of α-actinin-1 where calcium induced structural stabilisation causes fastening of the juxtaposed actin binding domain, leading to impaired capacity to crosslink actin.

Acanthocytosis and the c.680 A>G Mutation in the PANK2 Gene: A Study Enrolling a Cohort of PKAN Patients from the Dominican Republic.

Pantothenate Kinase-Associated Neurodegeneration (PKAN) is a form of Neurodegeneration with Brain Iron Accumulation (NBIA) associated with mutations in the pantothenate kinase 2 gene (PANK2). Pantothenate kinases catalyze the rate-limiting step of coenzyme A synthesis and Pank2 is the only pantothenate kinase isoform in humans that is localized to mitochondria. Acanthocytosis, the occurrence of spiculated erythrocytes, is observed in about 10% of the PKAN patients. Therefore PKAN is also classified together with other rare neurodegenerative diseases like Chorea Acanthocytosis (ChAc) and McLeod syndrome (MLS) into the Neuroacanthocytosis (NA) syndromes. It has not been investigated yet whether acanthocytosis in PKAN is associated with a specific subset of Pank2 mutations. In this study, we analyzed acanthocytosis of a cohort of 25 PKAN patients from the Dominican Republic that are homozygous for the c.680 A>G mutation in the PANK2 gene as compared to control donors that are heterozygous or wild-type with respect to this mutation. 3D modeling of this mutation indicated that the replacement of a tyrosine by a cysteine at position 227 in Pank2 disrupts a polar interaction within the A domain of the enzyme. Mean acanthocyte count was elevated in the cohort of patients, however, acanthocytosis varied among the patients with nearly half of them showing high (>20%) or elevated acanthocytosis and the rest showing mild (6-10%) or no (<6%) acanthocytosis. Heterozygous control donors revealed a tendency to mild acanthocytosis. Based on the insight that Pank2 is a normal constituent of red blood cells and de novo biosynthesis of coenzyme A is likely to take place in the erythrocyte cytosol we propose a hypothetical model that accounts for the variability in the occurrence of acanthocytic cells in PKAN.

Structural insights into Ca2+-calmodulin regulation of Plectin 1a-integrin ß4 interaction in hemidesmosomes.

The mechanical stability of epithelial cells, which protect organisms from harmful external factors, is maintained by hemidesmosomes via the interaction between plectin 1a (P1a) and integrin α6ß4. Binding of calcium-calmodulin (Ca(2+)-CaM) to P1a together with phosphorylation of integrin ß4 disrupts this complex, resulting in disassembly of hemidesmosomes. We present structures of the P1a actin binding domain either in complex with the N-ter lobe of Ca(2+)-CaM or with the first pair of integrin ß4 fibronectin domains. Ca(2+)-CaM binds to the N-ter isoform-specific tail of P1a in a unique manner, via its N-ter lobe in an extended conformation. Structural, cell biology, and biochemical studies suggest the following model: binding of Ca(2+)-CaM to an intrinsically disordered N-ter segment of plectin converts it to an α helix, which repositions calmodulin to displace integrin ß4 by steric repulsion. This model could serve as a blueprint for studies aimed at understanding how Ca(2+)-CaM or EF-hand motifs regulate F-actin-based cytoskeleton.

Structural disorder in proteins of the rhabdoviridae replication complex.

Rhabdoviridae are single stranded negative sense RNA viruses. The viral RNA condensed by the nucleoprotein (N), the phosphoprotein (P) and the large subunit (L) of the RNA-dependent RNA polymerase are the viral components of the transcription/replication machineries. Both P and N contain intrinsically disordered regions (IDRs) that play different roles in the virus life cycle. Here, we describe the modular organization of P based on recent structural, biophysical and bioinformatics data. We show how flexible loops in N participate in the attachment of P to the N-RNA template by an induced-fit mechanism. Finally, we discuss the roles of IDRs in the mechanism of replication/transcription, and propose a new model for the interaction of the L subunit with its N-RNA template.