RESUMO
Gelsolin participates in the reorganization of the actin cytoskeleton that is required during such phenomena as cell movement, cytokinesis, and apoptosis. It consists of six structurally similar domains, G1-G6, which are arranged at resting intracellular levels of calcium ion so as to obscure the three actin-binding surfaces. Elevation of Ca(2+) concentrations releases latches within the constrained structure and produces large shifts in the relative positioning of the domains, permitting gelsolin to bind to and sever actin filaments. How Ca(2+) is able to activate gelsolin has been a major question concerning the function of this protein. We present the improved structure of the C-terminal half of gelsolin bound to monomeric actin at 3.0 A resolution. Two classes of Ca(2+)-binding site are evident on gelsolin: type 1 sites share coordination of Ca(2+) with actin, while type 2 sites are wholly contained within gelsolin. This structure of the complex reveals the locations of two novel metal ion-binding sites in domains G5 and G6, respectively. We identify both as type 2 sites. The absolute conservation of the type 2 calcium-ligating residues across the six domains of gelsolin suggests that this site exists in each of the domains. In total, gelsolin has the potential to bind eight calcium ions, two type 1 and six type 2. The function of the type 2 sites is to facilitate structural rearrangements within gelsolin as part of the activation and actin-binding and severing processes. We propose the novel type 2 site in G6 to be the critical site that initiates overall activation of gelsolin by releasing the tail latch that locks calcium-free gelsolin in a conformation unable to bind actin.
Assuntos
Actinas/metabolismo , Cálcio/metabolismo , Gelsolina/química , Gelsolina/metabolismo , Modelos Moleculares , Fatores de Despolimerização de Actina , Actinas/química , Sequência de Aminoácidos , Sítios de Ligação , Cádmio/química , Cádmio/metabolismo , Cálcio/química , Sequência Conservada , Cristalografia por Raios X , Destrina , Evolução Molecular , Humanos , Ligantes , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Modelos Químicos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Água/químicaRESUMO
Understanding how a particular cell type expresses the lamellipodial or filopodial form of the actin machinery is essential to understanding a cell's functional interactions. To determine how a cell "chooses" among these alternative modes of "molecular hardware," we tested the role of key proteins that affect actin filament barbed ends. Depletion of capping protein (CP) by short hairpin RNA (shRNA) caused loss of lamellipodia and explosive formation of filopodia. The knockdown phenotype was rescued by a CP mutant refractory to shRNA, but not by another barbed-end capper, gelsolin, demonstrating that the phenotype was specific for CP. In Ena/VASP deficient cells, CP depletion resulted in ruffling instead of filopodia. We propose a model for selection of lamellipodial versus filopodial organization in which CP is a negative regulator of filopodia formation and Ena/VASP has recruiting/activating functions downstream of actin filament elongation in addition to its previously suggested anticapping and antibranching activities.