Structural insights into functional overlapping and differentiation among myosin V motors

Orientador: Mário Tyago MurakamiTexto em português e inglêsTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: As miosina de classe V, amplamente distribuídas em sistemas eucarióticos desde leveduras até vertebrados, são um dos mais caracterizados motores moleculares da superfamília das miosinas e desempenham um papel chave no transporte intracelular de vesículas, organelas e RNA mensageiro. As miosinas V consistem de duas cadeias pesadas idênticas que se dimerizam através da formação de uma estrutura coiled-coil e sua arquitetura tridimensional pode ser dividida em três distintos domínios: a porção N-terminal ou domínio motor que apresenta os sítios de interação com ATP e actina; a porção central ou pescoço formada por 6 domínios IQ, responsável pela regulação e interação com calmodulina; e a porção C-terminal que inclui a região coiled-coil e o domínio de ligação de cargas celulares também conhecido como domínio cauda globular (GTD). Uma das mais importantes questões pertinentes até hoje é como ocorre a sinalização e a interação entre as vesículas/organelas e o domínio globular C-terminal das miosinas V. Neste estudo, foram resolvidas as estruturas dos domínios cauda globular das miosinas Va, Vb e Vc humanas, revelando pequenas mudanças estruturais que levam a diferenciação funcional e, ainda, um novo mecanismo redox que controla a dimerização do GTD de forma independente do coiled-coil, que é exclusivo para a miosina Vc. As alterações estruturais induzidas pela fosforilação do GTD também foram exploradas, mostrando que o estado fosforilado possuí uma flexibilidade menor, podendo estar envolvido na regulação do estado inibido e/ou reconhecimento de cargas nucleares. Além disso, os sítios de ligação a carga e ao domínio motor foram estruturalmente anotados, indicando uma conservação de resíduos envolvidos na interação com adaptadores para o transporte de peroxissomos e proporcionando detalhes da inibição da atividade motora pelo GTD. Estes resultados contribuem para a compreensão dos determinantes estruturais para o transporte de carga, autoinibição e mecanismos de regulação dos motores de miosina V. Além dos resultados obtidos com a cauda globular, alguns problemas cristalográficos como o problema da fase, pseudosimetria e ordem-desordem cristalina foram abordados (descrito nos Apêndices 9.3 e 9.4). Patologias cristalinas como ordem-desordem parcial ou total podem estar relacionadas à valores elevados de Rfactor e Rfree, mesmo após a conclusão do refinamento, ou mesmo à dificuldade de resolução da estrutura. Problemas de ordem-desordem rotacional parcial e pseudosimetria foram observados em cristais de uma hidrolase glicosídica (TpAbn). Nesse caso, valores satisfatórios de Rfactor e Rfree foram obtidos somente após um minucioso processamento dos dados e redução da simetria. Além disso, os dados dos GTDs das miosinas de classe V foram utilizados como caso teste para o desenvolvimento de novas metodologias de faseamento ab initio em média e baixa resolução (2 ¿ 3 Å) em colaboração com o grupo de Prof. Dr. Isabel Usón (IBMB, Barcelona, Espanha). Utilizando o programa ARCIMBOLDO foi possível resolver a estrutura do GTD-MioVb a 2,1 Å utilizando apenas duas hélices de poli-Ala de 22 resíduos (7,5% do conteúdo da unidade assimétrica), mostrando o grande potencial desta metodologia para dados de média a baixa resoluçãoAbstract: The class V myosins (MyoVs) are widely distributed in eukaryotic organisms from yeast to vertebrates, being one of the most characterized molecular motors of the myosin superfamily. MyoVs play a central role in the intracellular transport of vesicles, organelles, messenger RNA and proteins. MyoVs consist of a coiled-coil-stabilized dimer of two identical heavy chains and their general structure can be divided into three distinct domains: the N-terminal portion or motor domain which binds both actin and ATP; the central portion or neck formed by 6 IQ domains, responsible for the regulation and interaction with calmodulin; and the C-terminal portion that includes the coiled-coil region and the cargo-binding domain also known as globular tail domain (GTD). One of the most important issues still obscure so far is how occurs the interaction between the cargoes and the globular C-terminal domain of myosin V. Here, we have solved the globular tail domain structures of the three human MyoV paralogs (Va, Vb and Vc), revealing subtle structural changes that drive functional differentiation and a novel redox mechanism controlling the GTD dimerization process, which is unique for the MyoVc subclass. The structural changes induced by the phosphorylation of GTD have also been explored, showing that the phosphorylated state is less flexible and may be involved in the regulation of the auto-inhibition mechanism and/or in the recognition of nuclear cargoes. Moreover, the cargo- and motor-binding sites were structurally assigned indicating the conservation of residues involved in the recognition of adaptors for peroxisome transport and providing high-resolution insights into motor domain inhibition by GTD. These results contribute to the understanding of the structural requirements for cargo transport, auto-inhibition and regulatory mechanisms in myosin V motors. In addition to the results obtained with the GTD structures, some crystallographic problems, such as the phase problem, pseudosymmetry and lattice order-disorder were discussed (described in Appendices 9.3 and 9.4). Crystal pathologies such as partial or total order-disorder may be related to high values of Rfactor and Rfree, even at late stages of crystallographic refinement, or even hindering the structure determination. Problems of partial rotational order-disorder and pseudosymmetry were found in TpAbn crystals where only after a careful data processing and symmetry reduction was possible to obtain satisfactory values of residuals (Rfactor and Rfree). Moreover, data from MyoV GTDs were used as a test case for the development of new methodologies for ab initio phasing at medium and low resolution (2 ¿ 3 Å) in collaboration with the group of Prof. Dr. Isabel Usón (IBMB, Barcelona, Spain). Using the program ARCIMBOLDO we have solved the GTD-MioVb structure at 2.1 Å using only two 22-residue-long poly-Ala helix fragments (7.5% of asymmetric unit content), showing the great potential of this methodology for data at medium to low resolutionDoutoradoBioquimicaDoutor em Biologia Funcional e Molecula

Crystal structure and regulation of the citrus pol III repressor MAF1 by auxin and phosphorylation

MAF1 is the main RNA polymerase (Pol) III repressor that controls cell growth in eukaryotes. The Citrus ortholog, CsMAF1, was shown to restrict cell growth in citrus canker disease but its role in plant development and disease is still unclear. We solved the crystal structure of the globular core of CsMAF1, which reveals additional structural elements compared with the previously available structure of hMAF1, and explored the dynamics of its flexible regions not present in the structure. CsMAF1 accumulated in the nucleolus upon leaf excision, and this translocation was inhibited by auxin and by mutation of the PKA phosphorylation site, S45, to aspartate. Additionally, mTOR phosphorylated recombinant CsMAF1 and the mTOR inhibitor AZD8055 blocked canker formation in normal but not CsMAF1-silenced plants. These results indicate that the role of TOR on cell growth induced by Xanthomonas citri depends on CsMAF1 and that auxin controls CsMAF1 interaction with Pol III in citrusThis work was supported by Sa˜ o Paulo Research Foundation (FAPESP grant 2011/20468-1). C.E.B. and A.F.Z.N. received a fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).Peer reviewe

Exploiting distant homologues for phasing through the generation of compact fragments, local fold refinement and partial solution combination

Macromolecular structures can be solved by molecular replacement provided that suitable search models are available. Models from distant homologues may deviate too much from the target structure to succeed, notwithstanding an overall similar fold or even their featuring areas of very close geometry. Successful methods to make the most of such templates usually rely on the degree of conservation to select and improve search models. ARCIMBOLDO_ SHREDDER uses fragments derived from distant homologues in a brute-force approach driven by the experimental data, instead of by sequence similarity. The new algorithms implemented in ARCIMBOLDO_SHREDDER are described in detail, illustrating its characteristic aspects in the solution of new and test structures. In an advance from the previously published algorithm, which was based on omitting or extracting contiguous polypeptide spans, model generation now uses three-dimensional volumes respecting structural units. The optimal fragment size is estimated from the expected log-likelihood gain (LLG) values computed assuming that a substructure can be found with a level of accuracy near that required for successful extension of the structure, typically below 0.6 A ° root-mean-square deviation (r.m.s.d.) from the target. Better sampling is attempted through model trimming or decomposition into rigid groups and optimization through Phaser’s gyre refinement. Also, after model translation, packing filtering and refinement, models are either disassembled into predetermined rigid groups and refined (gimble refinement) or Phaser’s LLGguided pruning is used to trim the model of residues that are not contributing signal to the LLG at the target r.m.s.d. value. Phase combination among consistent partial solutions is performed in reciprocal space with ALIXE. Finally, density modification and main-chain autotracing in SHELXE serve to expand to the full structure and identify successful solutions. The performance on test data and the solution of new structures are described.MS and CM received financial support from CCP4 for a research stay in the group of RJR. CM is grateful to MINECO for her BES-2015-071397 scholarship associated with the Structural Biology Maria de Maeztu Unit of Excellence. AFZN received a fellowship from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´ gico (CNPq), Brazil. GP acknowledges the Generalitat de Catalunya for an Industrial Doctorate predoctoral fellowship at Biochemize S.L. This work was supported by grants BIO2015-64216-P, BIO2013- 49604-EXP, BFU2014-59389-P and MDM2014-0435-01 from the Spanish Ministry of Economy and Competitiveness and 2014SGR-997 from Generalitat de Catalunya. This research was supported by the Wellcome Trust (Principal Research Fellowship to RJR, grant 082961/Z/07/Z) and by grant BB/ L006014/1 from the BBSRC, UK. The research was facilitated by Wellcome Trust Strategic Award 100140 to the Cambridge Institute for Medical Research. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 635595 (CarbaZymes).Peer Reviewe

Staphylococcus aureus Exfoliative Toxin E, Oligomeric State and Flip of P186: Implications for Its Action Mechanism

Staphylococcal exfoliative toxins (ETs) are glutamyl endopeptidases that specifically cleave the Glu381-Gly382 bond in the ectodomains of desmoglein 1 (Dsg1) via complex action mechanisms. To date, four ETs have been identified in different Staphylococcus aureus strains and ETE is the most recently characterized. The unusual properties of ETs have been attributed to a unique structural feature, i.e., the 180° flip of the carbonyl oxygen (O) of the nonconserved residue 192/186 (ETA/ETE numbering), not conducive to the oxyanion hole formation. We report the crystal structure of ETE determined at 1.61 Å resolution, in which P186(O) adopts two conformations displaying a 180° rotation. This finding, together with free energy calculations, supports the existence of a dynamic transition between the conformations under the tested conditions. Moreover, enzymatic assays showed no significant differences in the esterolytic efficiency of ETE and ETE/P186G, a mutant predicted to possess a functional oxyanion hole, thus downplaying the influence of the flip on the activity. Finally, we observed the formation of ETE homodimers in solution and the predicted homodimeric structure revealed the participation of a characteristic nonconserved loop in the interface and the partial occlusion of the protein active site, suggesting that monomerization is required for enzymatic activity