15-Jul-2021
Septins are a family of filament proteins involved in many essential cellular processes including cytokinesis, exocytosis, membrane barrier formation and bacterial entrapment.
Fundamental to the structure of septins is the coiled coil, a motif based on two or more supercoiled alpha-helices present in a wide variety of proteins, primarily acting in stabilising oligomeric structures and in protein–protein interactions.
Most septins are composed of a C-Terminal extension, the C-domain, which usually includes sequences compatible with participating in coiled coils, believed to be involved in filament formation and bonding – essential to septin function. This is highlighted further by the discovery that the cleavage of the SEPT2 C-Terminal domain, involved in homodimeric coiled coils, by the Zika virus protease NS3 leads to mitotic defects in neural cells.
A research team from the University of São Paulo investigated the structure of five human septins, to better understand the role of the coiled coil regions in the formation of the Septin filament, but also in the bonding of these filaments into higher order structures. Of particular interest was the C-Terminal domain, the only known naturally occurring example of coiled coils which switch between parallel and antiparallel helical orientation.
Leonardo et al., 2021, benefitted from visits to the Instruct-ERIC UK centre, at Diamond Light Source, Oxfordshire. This visit was funded by the MICROBES project, and was part of a wider visit to multiple Instruct-ERIC centres.
The study specifically investigated the atypical coiled coils found in septin C-domains, using a combination of crystallographic, solution and theoretical approaches. Septin subunits were crystallised, and X-ray diffraction data was collected at Diamond Light Source.
The study analysed septins within the SEPT2 subgroup, finding that two septins (SEPT4 and SEPT1) formed antiparallel coiled coils, whilst SEPT5 formed a parallel structure.
The crystal structure of SEPT4CC showed an antiparallel dimeric coiled coil in which hydrophilic amino acids occupy all a positions and hydrophobic amino acids all d positions, as shown in Figure 1. This arrangement is atypical of coiled coils, and generates a mixed interface, with a hydrophobic side (stabilised by traditional coiled coil interactions) and a hydrophilic side (stabilised by hydrogen bonds running along the interface).
Figure 1. Helical wheel and axial view for antiparallel SEPT4CC. The helical wheel and the axial view show that in antiparallel CCs, a and d residues reside on different sides of the interface – where traditional coiled coil bonding (hydrophobic) and hydrogen bond interactions (hydrophilic) take place.
Notably, the study found that both parallel and antiparallel orientations were energetically accessible for the SEPT2 subgroup, and in fact different measurements of each structure could be found in different local environments. For example, an aqueous solution favoured the parallel arrangement, due to its orientation generating a hydrophobic interface.
Overall, the study deepened understanding of how septin coiled coils are formed, and that they present greater structural diversity than originally envisaged. This unusual type of coiled coil could be used to expand the toolkit currently available to protein engineers and synthetic biologists for the design of previously unforeseen coiled-coil based assemblies.
