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Septin C-Terminal Domain Interactions: Implications for Filament Stability and Assembly

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Abstract

Septins form a conserved family of filament forming GTP binding proteins found in a wide range of eukaryotic cells. They share a common structural architecture consisting of an N-terminal domain, a central GTP binding domain and a C-terminal domain, which is often predicted to adopt a coiled-coil conformation, at least in part. The crystal structure of the human SEPT2/SEPT6/SEPT7 heterocomplex has revealed the importance of the GTP binding domain in filament formation, but surprisingly no electron density was observed for the C-terminal domains and their function remains obscure. The dearth of structural information concerning the C-terminal region has motivated the present study in which the putative C-terminal domains of human SEPT2, SEPT6 and SEPT7 were expressed in E. coli and purified to homogeneity. The thermal stability and secondary structure content of the domains were studied by circular dichroism spectroscopy, and homo- and hetero-interactions were investigated by size exclusion chromatography, chemical cross-linking, analytical ultracentrifugation and surface plasmon resonance. Our results show that SEPT6-C and SEPT7-C are able to form both homo- and heterodimers with a high α-helical content in solution. The heterodimer is elongated and considerably more stable than the homodimers, with a K D of 15.8 nM. On the other hand, the homodimer SEPT2-C has a much lower affinity, with a K D of 4 μM, and a moderate α-helical content. Our findings present the first direct experimental evidence toward better understanding the biophysical properties and coiled-coil pairings of such domains and their potential role in filament assembly and stability.

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Abbreviations

SPR:

Surface plasmon resonance

NTA:

Nitrilotriacetate

IPTG:

Isopropyl-β-d-thiogalactopyranoside

EGS:

Ethylene glycol-bis

SDS/PAGE:

Sodium dodecylsulfate/polyacrylamide gel electrophoresis

EDC:

1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride

NHS:

N-hydroxysuccinimide

SEC:

Size exclusion chromatography

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Acknowledgments

We acknowledge the financial support of FAPESP (via its CEPID program to the Centro de Biotecnologia Molecular Estrutural) and CNPq (via the INCT initiative). This work was further supported by a collaborative CSIC-CNPq grant for international exchange awarded to JMA and RCG as well as by grant MCINN BFU2008-00013 to JMA. IAM received a CAPES studentship and NFV, WG, JCPD and JNAM held bursaries from FAPESP.

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Authors and Affiliations

  1. Centro de Biotecnologia Molecular Estrutural, Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador são-carlense 400, São Carlos, SP, 13560-970, Brazil

    Ivo de Almeida Marques, Napoleão Fonseca Valadares, Julio Cesar Pissuti Damalio, Joci Neuby Alves Macedo, Ana Paula Ulian de Araújo & Richard Charles Garratt

  2. Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Rua Santa Adélia 166, Santo André, SP, 09210-170, Brazil

    Wanius Garcia

  3. Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu, 9, 28040, Madrid, Spain

    Carlos Alfonso Botello & José Manuel Andreu

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  1. Ivo de Almeida Marques
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  2. Napoleão Fonseca Valadares
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Correspondence to Richard Charles Garratt.

Additional information

Ivo de Almeida Marques and Napoleão Fonseca Valadares contributed equally to this work.

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de Almeida Marques, I., Valadares, N.F., Garcia, W. et al. Septin C-Terminal Domain Interactions: Implications for Filament Stability and Assembly. Cell Biochem Biophys 62, 317–328 (2012). https://doi.org/10.1007/s12013-011-9307-0

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