Molecular recognition at septin interfaces: the switches hold the key

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>The assembly of a septin filament requires that homologous monomers must distinguish between one another in establishing appropriate interfaces with their neighbours. To understand this phenomenon at the molecular level, we present the first four crystal structures of heterodimeric septin complexes. We describe in detail the two distinct types of G-interface present within the octameric particles which must polymerize to form filaments. These are formed between SEPT2 and SEPT6 and between SEPT7 and SEPT3, and their description permits an understanding of the structural basis for the selectivity necessary for correct filament assembly. By replacing SEPT6 by SEPT8 or SEPT11, it is possible to rationalize <jats:italic>Kinoshita’s postulate</jats:italic> which predicts the exchangeability of septins from within a subgroup. Switches I and II, which in classical small GTPases provide a mechanism for nucleotide-dependent conformational change, have been repurposed in septins to play a fundamental role in molecular recognition. Specifically, it is switch I which holds the key to discriminating between the two different G-interfaces. Moreover, residues which are characteristic for a given subgroup play subtle, but pivotal, roles in guaranteeing that the correct interfaces are formed.</jats:p><jats:sec><jats:title>HIGHLIGHTS</jats:title><jats:list list-type="bullet"><jats:list-item><jats:p>High resolution structures of septin heterodimeric complexes reveal new interactions</jats:p></jats:list-item><jats:list-item><jats:p>Switches of small GTPases are repurposed in septins to play key roles in interface contacts</jats:p></jats:list-item><jats:list-item><jats:p>The GTP present in catalytically inactive septins participates in molecular recognition</jats:p></jats:list-item><jats:list-item><jats:p>Conservation of interface residues allows for subunit exchangeability from within septin subgroups</jats:p></jats:list-item><jats:list-item><jats:p>Specific residues for each septin subgroup provide selectivity for proper filament assembly</jats:p></jats:list-item></jats:list></jats:sec><jats:sec><jats:title>GRAPHICAL ABSTRACT</jats:title><jats:fig id="ufig1" position="float" fig-type="figure" orientation="portrait"><jats:graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="161463v1_ufig1" position="float" orientation="portrait" /></jats:fig></jats:sec>

Human septins in cells organize as octamer-based filaments mediating actin-membrane anchoring

“…We reasoned that in the absence of exogenous SEPT9, the slightest excess of SEPT7 leads to ectopic hexamer-based bundles, also reducing the availability of SEPT7 for forming octamers to bind SFs. SEPT7-SEPT7 interfaces being more stable than SEPT7-SEPT9 ones (Rosa et al, 2020) would facilitate this process. Exogenous co-expression of SEPT9, on the other hand, would cause incorporation of the exogenous SEPT7 into octamers, thus preventing the formation of ectopic hexamer bundles.…”

“…It is also possible that the actin/membrane binding of hexamers observed in vitro does not happen in cells. The observation that SEPT7 is the septin whose localization and assembly are most sensitive to SEPT7 and SEPT9 expression levels, and the fact that SEPT7-SEPT7 interactions are stronger than SEPT7-SEPT9 ones (Rosa et al, 2020) might suggest that SEPT9, and possibly the other SEPT3 group septins, helps prevent SEPT7 from forming ectopic bundles. It is intriguing that Drosophila does not have any SEPT3 group septins and thus contains only hexamers (Field et al, 1996).…”

“…Biochemical isolation of native septins from budding yeast, Drosophila and mammalian cells and tissues revealed that septins exist as stable heteromeric complexes that can polymerize into filaments and higher-order filament assemblies (Field et al, 1996; Frazier et al, 1998; Hsu et al, 1998; Kim et al, 2011; Kinoshita et al, 2002; Sellin et al, 2011). The isolation of recombinant septin complexes helped establish that septin complexes are palindromes, with each septin in two copies and in a specific position within the complex, with each monomer interacting with its neighbors by alternating interfaces, named NC (from the N- and C-terminal domains) and G (from the GTP-binding domain) (Bertin et al, 2008; DeRose et al, 2020; Farkasovsky et al, 2005; Garcia et al, 2011; Huijbregts et al, 2009; Iv et al, 2021; John et al, 2007; Kinoshita et al, 2002; Kumagai et al, 2019; Mavrakis et al, 2014; Mendonca et al, 2019; Rosa et al, 2020; Sala et al, 2016; Sirajuddin et al, 2007; Soroor et al, 2021; Versele and Thorner, 2004). Human septins are classified in four homology groups, namely the SEPT2 group (SEPT1, 2, 4, and 5), SEPT6 group (SEPT6, 8, 10, 11, and 14), SEPT7 group (SEPT7), and SEPT3 group (SEPT3, 9, and 12) (Kinoshita, 2003).…”

Septins and flaviviral proteases: a structural analysis

“…Again, the definitions of the flanking regions of each septin domain were evaluated based on structural alignments). 68 The amplification of the nucleotide sequence referring to the desired regions of each protein was performed using the polymerase chain reaction (PCR), using oligonucleotide primers, based on the sequences deposited in GenBank, which are shown in Table 3.1. The PCR cycles were performed in a Veritii 96-well Thermal Cycler (Applied Biosystems) using the high-fidelity polymerase Phusion (Cellco) under conditions described by the manufacturer.…”

“…Recombinant human heterodimers SEPT2NGC-SEPT6G (more stable than the SEPT2NGC homodimer) were purified following similar protocols described by Rosa et al 68 for SEPT2G-SEPT6G. Recombinant WT NS2B3 from ZIKV and YFV were purified following the same protocol as stated above for their inactive mutants.…”