RESUMO
Centrins are calcium-binding proteins associated with microtubules organizing centers. Members of two divergent subfamilies of centrins were found in the aquatic fungus Blastocladiella emersonii, contrasting with the occurrence of only one member known for the better explored terrestrial fungi. BeCen1 shows greatest identity with human centrins HsCen1, HsCen2 and green algae centrin CrCenp, while BeCen3 records largest identity with human centrin HsCen3 and yeast centrin Cdc31p. Following the discovery of this unique feature, BeCen1 and BeCen3 centrins were produced to study whether these proteins had distinct features upon calcium binding. Circular dichroism showed opposite calcium binding effects on the α-helix arrangement of the secondary structure. The spectra indicated a decrease in α-helix signal for holo-BeCen1 contrasting with an increase for holo-BeCen3. In addition, only BeCen1 refolds after being de-natured. The fluorescence emission of the hydrophobic probe ANS increases for both proteins likely due to hydrophobic exposure, however, only BeCen1 presents a clear blue shift when calcium is added. ITC experiments identified four calcium binding sites for both proteins. In contrast to calcium binding to BeCen1, which is mainly endothermic, binding to BeCen3 is mainly exothermic. Light-scattering evidenced the formation of large particles in solution for BeCen1 and BeCen3 at temperatures above 30°C and 40°C, respectively. Atomic force microscopy confirmed the presence of supramolecular structures, which differ in the compactness and branching degree. Binding of calcium leads to different structural changes in BeCen1 and BeCen3 and the thermodynamic characteristics of the interaction also differ.
Assuntos
Blastocladiella/química , Cálcio/química , Proteínas Fúngicas/química , Dobramento de Proteína , Combinação Trimetoprima e Sulfametoxazol/química , Blastocladiella/metabolismo , Cálcio/metabolismo , Dicroísmo Circular , Proteínas Fúngicas/metabolismo , Humanos , Microscopia de Força Atômica , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Termodinâmica , Combinação Trimetoprima e Sulfametoxazol/metabolismoRESUMO
The molecular mechanisms responsible for protein structural changes in the central nervous system leading to Alzheimer's disease are unknown, but there is evidence that a family of proteins known as septins may be involved. Septins are a conserved group of GTP-binding proteins which participate in various cellular processes, including polarity determination and membrane dynamics. SEPT1, SEPT4, and SEPT2 have been found in deposits known as neurofibrillary tangles and glial fibrils in Alzheimer's disease. In this study, we provide molecular-level information for the interaction of SEPT2 with Langmuir monolayers at the air/water interface, which are used as simplified membrane models. The high surface activity of SEPT2 causes it to adsorb onto distinct types of lipid Langmuir monolayers, namely dipalmitoylphosphatidylcholine and PtdIns(4,5)P2. However, the interaction with PtdIns(4,5)P2 is much stronger, not only leading to a higher adsorption, but also to SEPT2 remaining inserted within the membrane at high surface pressures. Most importantly, in situ polarization-modulated infrared reflection absorption spectroscopy results indicated that the native secondary structure of SEPT2 is preserved upon interacting with PtdIns(4,5)P2, but not when dipalmitoylphosphatidylcholine is at the air/water interface. Taken together, the results presented here suggest that the interaction between SEPT2 and the cell membrane may play an important role in the assembly of SEPT2 into amyloid-like fibers.
Assuntos
Lipídeos de Membrana/química , Membranas Artificiais , Septinas/química , Espectrofotometria Infravermelho/métodos , 1,2-Dipalmitoilfosfatidilcolina/química , Adsorção , Varredura Diferencial de Calorimetria , Cinética , Lipídeos de Membrana/metabolismo , Fosfatidilinositol 4,5-Difosfato/química , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Septinas/metabolismo , Propriedades de SuperfícieRESUMO
The human genome codes for 13 members of a family of filament-forming GTP-binding proteins known as septins. These have been divided into four different subgroups on the basis of sequence similarity. The differences between the subgroups are believed to control their correct assembly into heterofilaments which have specific roles in membrane remodelling events. Many different combinations of the 13 proteins are theoretically possible and it is therefore important to understand the structural basis of specific filament assembly. However, three-dimensional structures are currently available for only three of the four subgroups. In the present study we describe the crystal structure of a construct of human SEPT3 which belongs to the outstanding subgroup. This construct (SEPT3-GC), which includes the GTP-binding and C-terminal domains, purifies as a nucleotide-free monomer, allowing for its characterization in terms of GTP-binding and hydrolysis. In the crystal structure, SEPT3-GC forms foreshortened filaments which employ the same NC and G interfaces observed in the heterotrimeric complex of human septins 2, 6 and 7, reinforcing the notion of 'promiscuous' interactions described previously. In the present study we describe these two interfaces and relate the structure to its tendency to form monomers and its efficiency in the hydrolysis of GTP. The relevance of these results is emphasized by the fact that septins from the SEPT3 subgroup may be important determinants of polymerization by occupying the terminal position in octameric units which themselves form the building blocks of at least some heterofilaments.