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Structural determinants for specific interaction of interfaces G and NC of septins: validating the substitution rules in the assembly of the filament

Grant number: 16/04658-9
Support type:Scholarships in Brazil - Doctorate
Effective date (Start): July 01, 2016
Effective date (End): August 31, 2020
Field of knowledge:Biological Sciences - Biophysics - Molecular Biophysics
Principal researcher:Richard Charles Garratt
Grantee:Diego Antonio Leonardo Cabrejos
Home Institution: Instituto de Física de São Carlos (IFSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil
Associated research grant:14/15546-1 - Septins: comparative studies and the correlation between structure and function, AP.TEM


Septins are GTPases that play an important role in cell structure, forming the fourth component of the cytoskeleton and participating in recruitment of other proteins. Septins form heterofilaments, which are stabilized by interactions between their subunits through two types of interfaces: G and NC. These interfaces were observed in the crystallographic structure of heterocomplex SEPT2-SEPT6-SEPT7, but due to low resolution (4 Å) was not possible to analyze in detail the structural determinants responsible for their training as well as to understand the disposition of each subunit along the filament . Still, it was observed by Kinoshita in 2002, that septins belonging to the same group could occupy equivalent positions in the filament thus generating a diversity of heterocomplexes. The challenge of understanding filament polymerization specificity is based on the surprising observation of promiscuous interfaces formed by isolated septins. These interactions lead to the formation homofilaments stabilized by type G and the NC interfaces and are observed in several crystallographic structures described in the literature, even if no in physiological filaments. To address this phenomenon, our group initially worked in co-expression and co-purification of trimeric and tetrameric complexes with structural purposes, but without much success due to lack of equimolarity in the samples. For this reason, the candidate proposed in the master's project (2014) an alternative approach to study a single interface, where the study was focused on the G interface between SEPT5 and SEPT8. The results show a greater stability for SEPT5-SEPT8 complex front alone SEPT8, strong tendency toward crystallization for the heterocomplex and finally identified an interaction between single amino acid for these septins (Phe131 and Thr19 in SEPT5 and SEPT8 respectively), which proposal is as the most important for the specificity and stability of this G interface. This PhD project proposes to extend the study of the G interface interaction specificity between septins group II (SEPT8 group) and group III (SEPT5 group), quantitatively validating observation Kinoshita, and to study the interaction between Phe131 and Thr19 in order to understand its importance in the stability and specificity for the interface. It will also be explored the idea that coiled-coils belonging to group III septins are responsible for the stability of the complex higher order, where antiparallel coiled-coils can serve as cross-links between the filaments. Finally, we will be made studies of electron microscopy for SEPT3-SEPT7-SEPT8-SEPT5 complex with the purpose of establishing the willingness of each subunit in the filament. This proposal is linked to the thematic 2014/15546-1 "Septins: comparative studies to correlate structure and function."

Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
DIAS ROSA, HIGOR VINICIUS; LEONARDO, DIEGO ANTONIO; BROGNARA, GABRIEL; BRANDAO-NETO, JOSE; PEREIRA, HUMBERTO D'MUNIZ; ULIAN ARAUJO, ANA PAULA; GARRATT, RICHARD CHARLES. Molecular Recognition at Septin Interfaces: The Switches Hold the Key. Journal of Molecular Biology, v. 432, n. 21, SI, p. 5784-5801, OCT 2 2020. Web of Science Citations: 0.
SILVA DO VALE CASTRO, DANIELLE KAROLINE; DE OLIVEIRA DA SILVA, SABRINA MATOS; PEREIRA, HUMBERTO D'MUNIZ; ALVES MACEDO, JOCI NEUBY; LEONARDO, DIEGO ANTONIO; VALADARES, NAPOLEAO FONSECA; KUMAGAI, PATRICIA SUEMY; BRANDAO-NETO, JOSE; ULIAN ARAUJO, ANA PAULA; GARRATT, RICHARD CHARLES. A complete compendium of crystal structures for the human SEPT3 subgroup reveals functional plasticity at a specific septin interface. IUCRJ, v. 7, n. 3, p. 462-479, MAY 2020. Web of Science Citations: 0.

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