RESUMO
The mitotic kinesin motor protein KIF14 is essential for cytokinesis during cell division and has been implicated in cerebral development and a variety of human cancers. Here we show that the mouse KIF14 motor domain binds tightly to microtubules and does not display typical nucleotide-dependent changes in this affinity. It also has robust ATPase activity but very slow motility. A crystal structure of the ADP-bound form of the KIF14 motor domain reveals a dramatically opened ATP-binding pocket, as if ready to exchange its bound ADP for Mg·ATP. In this state, the central ß-sheet is twisted ~10° beyond the maximal amount observed in other kinesins. This configuration has only been seen in the nucleotide-free states of myosins-known as the "rigor-like" state. Fitting of this atomic model to electron density maps from cryo-electron microscopy indicates a distinct binding configuration of the motor domain to microtubules. We postulate that these properties of KIF14 are well suited for stabilizing midbody microtubules during cytokinesis.
Assuntos
Cinesinas/química , Microtúbulos/química , Difosfato de Adenosina/química , Adenosina Trifosfatases/química , Sequência de Aminoácidos , Animais , Domínio Catalítico , Cristalografia por Raios X , Cinética , Camundongos , Microtúbulos/ultraestrutura , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Multimerização Proteica , Estabilidade Proteica , Estrutura Secundária de ProteínaRESUMO
The periplasmic alginate lyase AlgL is essential for the synthesis and export of the exopolysaccharide alginate in Pseudomonas sp. and also plays a role in its depolymerization. P. aeruginosa PAO1 AlgL has been overexpressed and purified and diffraction-quality crystals were grown using the hanging-drop vapour-diffusion method. The crystals grew as thin plates, with unit-cell parameters a = 56.4, b = 59.6, c = 102.1â Å, α = ß = γ = 90°. The AlgL crystals exhibited the symmetry of space group P2(1)2(1)2(1) and diffracted to a minimum d-spacing of 1.64â Å. Based on the Matthews coefficient (V(M) = 2.20â Å(3)â Da(-1)), one molecule is estimated to be present in the asymmetric unit.