The Structure and Regulation of Human Muscle α-Actinin

SummaryThe spectrin superfamily of proteins plays key roles in assembling the actin cytoskeleton in various cell types, crosslinks actin filaments, and acts as scaffolds for the assembly of large protein complexes involved in structural integrity and mechanosensation, as well as cell signaling. α-actinins in particular are the major actin crosslinkers in muscle Z-disks, focal adhesions, and actin stress fibers. We report a complete high-resolution structure of the 200 kDa α-actinin-2 dimer from striated muscle and explore its functional implications on the biochemical and cellular level. The structure provides insight into the phosphoinositide-based mechanism controlling its interaction with sarcomeric proteins such as titin, lays a foundation for studying the impact of pathogenic mutations at molecular resolution, and is likely to be broadly relevant for the regulation of spectrin-like proteins

Estudo do envolvimento das helices N- e C-terminais na estabilidade e na via de enovelamento de mioglobina

Orientador: Carlos Henrique Inacio RamosTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A mioglobina é uma hemeproteína que tem a função de transportar e armazenar o oxigênio. Suas estruturas primária e secundária são compostas por 153 aminoácidos e oito a-hélices (nomeadas de A-H), respectivamente. Esta proteína tem sido usada como um bom modelo para estudos de estrutura, função, ligação de heme, estabilidade e enovelamento de proteínas. Em condições ligeiramente ácidas (pH em torno de 4), a apomioglobina se desenovela passando por uma forma intermediária que possui propriedades físicas entre o estado nativo (pH 7) e o estado desenovelado (pH 2). Este intermediário apresenta as hélices A, G e H protegidas como demonstrado por experimentos de troca de deutério. No presente trabalho, foram realizados estudos com mutantes de deleção e de permutações circulares de hélices da mioglobina com a finalidade de aumentar o conhecimento sobre como a estrutura terciária de uma proteína é construída. Três classes de mutantes foram criadas: 1) deleção de hélices: 1.1) um mutante de deleção da hélice H (Mb1-123) e 1.2) um mutante de deleção das hélices G e H (Mb1-99); 2) permutações circulares: 2.1) um mutante de permutação da hélice A da extremidade N-terminal para a C-terminal (Mb-B_GHA) e 2.2) um mutante de permutação das hélices A e B da extremidade N-terminal para a C-terminal (Mb- C_GHAB) e 3) permutação com deleção: um mutante de deleção da hélice G com permutação da hélice H da extremidade C-terminal para N-terminal (Mb-HAB_F). As proteínas mutantes foram purificadas diretamente na forma apo, embora estes mutantes possuam capacidade de se ligar ao grupo heme, com exceção do mutante Mb1-123. Estes mutantes possuem valores mais baixos de elipticidade molar e tendência maior à agregação do que a proteína selvagem. Quando na forma holo, os dois mutantes circularmente permutados são compactos e têm estrutura e função semelhantes à holoMbWT. Este resultado difere do resultado observado para as variantes de deleção (Mb1-99 e Mb-HAB_F) que não apresentaram restabelecimento da estrutura e nem supressão da tendência à agregação. Nossos resultados indicam que, embora a mioglobina possua um núcleo de ligação do grupo heme, as hélices A, B, G e H são necessárias para a correta arquitetura da proteína. Apesar da menor estabilidade dos mutantes de permutação circulares em relação à proteína selvagem, as extremidades N- e Cterminais da mioglobina são necessárias para que a proteína alcance uma estrutura estável e funcional semelhante à selvagem. As permuteínas estruturaram a forma intermediária mesmo em pH ao redor de 7, mostrando novamente que as extremidades N- e C- terminais de mioglobina são necessárias para que elas se estruturem como a selvagem. Acreditamos que um núcleo, que se mostrou insensível ao novelamento em meio ácido, foi formado nestes mutantes. Como estes mutantes diferem quanto à posição da hélice B, eles permitiram inferir sobre a participação da hélice B no intermediário de ApoMbWT. Duas formas de intermediários estão provavelmente presentes na via de enovelamento da mioglobina e suas maiores diferenças parecem estar na quantidade de estrutura formada pela hélice B. Nossos resultados estão de acordo com o modelo seqüencial de enovelamento para mioglobina, no qual a hélice B é incorporada após a formação do domínio AGHAbstract: Myoglobin functions as a protein for transporting and storing oxygen and is a soluble, globular heme-binding protein, comprising 153 amino acids arranged in eight helical segments (named A to H). This protein has been used as a good model to study structure, function, heme binding, stability, and folding pathway. Under mild acid conditions (pH 4), apomyoglobin unfolds through an intermediate form with physical properties intermediate between the native (neutral pH) and the unfolded (pH 2.0) states. This intermediate has helices A, G and H protected from hydrogen exchange. Here we present studies of deleted and circularly permuted mutations of helical blocks of myoglobin to add to our understanding of how protein topology is built. Three classes of mutants were constructed: 1) helix deletion: 1.1) a mutant deleted of H helix, Mb1-123, and 1.2) a mutant deleted of G and H helices, Mb1-99; 2) circularly permutation: 2.1) Mb-B_GHA where B-helix is N-terminal and A helix is C-terminal, and 2.2) Mb-C_GHAB where C-helix is N-terminal and B helix is Cterminal; 3) permutation/deletion: a deleted circularly permutation where H-helix is N-terminal, the G helix is deleted, and the F helix is C-terminal, Mb-HAB_F. The mutants were purified in the apo form, where although they have the ability to bind heme with an exception to Mb1-123, they have lower ellipticity and a larger tend ency to aggregate than the wild-type. When in the holo form, the two circularly permuted mutants are compact and have native-like function and conformation, different form the myoglobin variants of deletion (i.e. Mb1-99 and Mb- HAB_F), where the heme binding does not seem to be native-like and do not suppresses their tendency to aggregate. Our results indicate that although myoglobin has a core that is able to bind heme, the A, G, H and B helices are needed for the correct structural architecture of the protein. And, since the circularly permutations are less stable than the wild-type, the N- and C-termini of myoglobin need to be native-like for the optimum structure-function relationship of this protein. The apopermuteins resembled the intermediate form even at physiological pH, showing again that the N- and C-termini of myoglobin need to be native-like for the optimum structure-function relationship of this protein. We believe that a nucleus, mostly independent of acid unfolding, was formed by these mutants and, since these permutants differ in the position of the B helix, they gave insights about the participation of the B helix in the intermediate. Two forms of intermediates are likely to be present in the folding pathway of apomyoglobin and their major difference seems to be in the amount of structure in the B helix. Our results agreed with a model of sequencial folding for apomyoglobin where the B helix is added later in the AGH nucleusDoutoradoBioquimicaDoutor em Biologia Funcional e Molecula

Structural Insights into Ca2+\mathrm{Ca^{2+}}-Calmodulin Regulation of Plectin 1a-Integrin β4 Interaction in Hemidesmosomes

The mechanical stability of epithelial cells, whichprotect organisms from harmful external factors, ismaintained by hemidesmosomes via the interactionbetween plectin 1a (P1a) and integrin a6b4. Bindingof calcium-calmodulin (Ca 2+ -CaM) to P1a togetherwith phosphorylation of integrin b4 disrupts thiscomplex, resulting in disassembly of hemidesmo-somes. We present structures of the P1a actin bind-ing domain either in complex with the N-ter lobe ofCa 2+ -CaM or with the first pair of integrin b4 fibro-nectin domains. Ca 2+ -CaM binds to the N-ter iso-form-specific tail of P1a in a unique manner, via itsN-ter lobe in an extended conformation. Structural,cell biology, and biochemical studies suggest thefollowing model: binding of Ca 2+ -CaM to an intrinsi-cally disordered N-ter segment of plectin convertsit to an a helix, which repositions calmodulin todisplace integrin b4 by steric repulsion. This modelcould serve as a blueprint for studies aimed at under-standing how Ca 2+ -CaM or EF-hand motifs regulateF-actin-based cytoskeleton

Degree of acanthocytosis in patients and control donors.

<p>Acanthocyte counts of donors that are heterozygous (n = 36), homozygous (n = 23) and wild-type (22) with respect to the c.680 A>G mutation in the PANK2 gene were microscopically assessed as described in the Materials and Methods section. The samples were grouped in four classes of acanthocytosis (normal (acanthocyte count <6% of total cells), mild (6–10%), elevated (10–20%) and high (>20%)) and the number of samples of each class is shown as relative percent of the total number in each subset of donors.</p