Molecular Recognition at Septin Interfaces: The Switches Hold the Key

Abstract

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 Kinoshita´s postulate 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.

Insights into animal septins using recombinant human septin octamers with distinct SEPT9 isoforms

“…Upon septin co-expression in the bacteria cytoplasm, septins are expected to form stable hexamers and octamers (Kim et al, 2011;Sellin et al, 2011;Sellin et al, 2014). Other hypothetical homo-and heterosubcomplexes could also form (Kim et al, 2012;Rosa et al, 2020;Valadares et al, 2017). A first Strep-tag affinity column isolates all Strep-tagged SEPT9 complexes ("S" for Streptag).…”

“…Multiple studies have documented promiscuity in septin-septin interactions in the absence of their physiologically relevant binding partners, affecting the availability of specific structural elements for interactions with other septins or interacting proteins (Castro et al, 2020;Valadares et al, 2017). The necessity to study septins in the context of their native heteromeric complexes is highlighted by the increasing number of structural studies of the factors governing the molecular specificity that determines the correct pairing of septins during complex assembly (Kumagai et al, 2019;Rosa et al, 2020;Sala et al, 2016).…”

Cellular functions of actin- and microtubule-associated septins

“…Similarly, mammalian septins form heterooctamers with subunits from one of each of the four septin groups (Figure 1B) [25][26][27][28] . Paralogs of the same septin group can replace one another, generating septin complexes of various composition and combination 17,28,29 . In mammalian systems, smaller unit complexes (such as hexamers and tetramers) arise from non-canonical modes of assembly as a result of paralogs having different rates of GTP hydrolysis and/or expression levels [30][31][32] .…”

Biochemical characterization of a human septin octamer

“…The G domain is conserved among different species and is supposed to be loaded either with GTP or GDP. The available crystal structures of human and yeast septins (Zent, Vetter, and Wittinghofer 2011; Sirajuddin et al 2007, 2009; Brausemann et al 2016; Castro et al 2020; Fonseca Valadares et al 2017; Rosa et al 2020) confirm that the G domain contains all structural features of small GTPases like the P-loop and the distinct Switch1 and Switch2 loops including the invariant DXXG motif. GTP binding and hydrolysis was confirmed for human septins.…”