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. 2014 Sep;187(3):223-235.
doi: 10.1016/j.jsb.2014.07.006. Epub 2014 Aug 1.

Crystal structure of the full-length ATPase GspE from the Vibrio vulnificus type II secretion system in complex with the cytoplasmic domain of GspL

Connie Lu  1 , Konstantin V Korotkov  1 , Wim G J Hol  2
Affiliations

Crystal structure of the full-length ATPase GspE from the Vibrio vulnificus type II secretion system in complex with the cytoplasmic domain of GspL

Connie Lu et al. J Struct Biol. 2014 Sep.

Abstract

The type II secretion system (T2SS) is present in many Gram-negative bacteria and is responsible for secreting a large number of folded proteins, including major virulence factors, across the outer membrane. The T2SS consists of 11-15 different proteins most of which are present in multiple copies in the assembled secretion machinery. The ATPase GspE, essential for the functioning of the T2SS, contains three domains (N1E, N2E and CTE) of which the N1E domain is associated with the cytoplasmic domain of the inner membrane protein GspL. Here we describe and analyze the structure of the GspE•cyto-GspL complex from Vibrio vulnificus in the presence of an ATP analog, AMPPNP. There are three such ∼83 kDa complexes per asymmetric unit with essentially the same structure. The N2E and CTE domains of a single V. vulnificus GspE subunit adopt a mutual orientation that has not been seen before in any of the previous GspE structures, neither in structures of related ATPases from other secretion systems. This underlines the tremendous conformational flexibility of the T2SS secretion ATPase. Cyto-GspL interacts not only with the N1E domain, but also with the CTE domain and is even in contact with AMPPNP. Moreover, the cyto-GspL domains engage in two types of mutual interactions, resulting in two essentially identical, but crystallographically independent, "cyto-GspL rods" that run throughout the crystal. Very similar rods are present in previous crystals of cyto-GspL and of the N1E•cyto-GspL complex. This arrangement, now seen four times in three entirely different crystal forms, involves contacts between highly conserved residues suggesting a role in the biogenesis or the secretion mechanism or both of the T2SS.

Keywords: Assembly ATPase; EpsE; EpsL; Protein secretion; T2SS; T4PS.

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Figures

Fig. 1
Fig. 1. The V. vulnificus GspE•cyto-GspL complex
Domains are shown with N1Ein blue, cyto-GspLin green, CTE in red, and N2E in pink. AMPPNP atoms are shown as yellow spheres, the essential Zn ion (Robien et al., 2003) as an orange sphere. For nomenclature of the domains, see Supplementary Fig. S1.
Fig. 2
Fig. 2. The variability of N2E-vs-CTE orientation in GspE
Left: Overall structure of V. vulnificus N2E-CTE (N2E purple, CTE red) in complex with V. vulnificus cyto-GspL (green), shown as cartoon with bound AMPPNP (yellow spheres). Right: Comparison of V. vulnificus and V. cholerae N2E-CTE “di-domains” in the current V. vulnificus GspE•cyto-GspL structure and V. cholerae ΔN1EGspE (Robien et al., 2003). Superimposed are the CTEs from V. vulnificus (red) and V. cholerae GspE (orange). There is a dramatic change of ∼171 degrees in N2E-vs-CTE orientation in these two cases – compare the V. vulnificus N2E (purple) and the V. cholerae N2E (orange) domains. Comparison with the left panel also shows that V. vulnificus cyto-GspL in the current structure occupies approximately the same position as N2E in the V. cholerae N2E-CTE didomain structure (Robien et al., 2003).
Fig. 3
Fig. 3. Structure of the V. vulnificus N1E•cyto-GspL heterodimer
The N1E of GspE shown in blue, subdomain I of cyto-GspL in green, subdomain II of cyto-GspL in lime, subdomain III of cyto-GspL in yellow. Note how all three GspL subdomains are involved in contacting N1E with helix α2 of N1E, a major component of the interface. When compared with the V. cholerae N1E•cyto-GspL heterodimer (Abendroth et al., 2005) the r.m.s.d. is 1.4 Å with a difference of ∼11 degrees in N1E-vs-cyto-GspL orientation, and ∼48 and 53 % amino acid sequence identity for N1E and cyto-GspL, respectively (see also Supplementary Fig. S2 and S3).
Fig. 4
Fig. 4. Nucleotide binding by V. vulnificus GspE•cyto-GspL and by V. cholerae GspE
A. Superposition of V. vulnificus and V. cholerae GspE with AMPPNP bound (stereo figure). Residues that interact with AMPPNP in the V. cholerae GspE•AMPPNP complex but do not interact with AMPPNP in the V. vulnificus GspE•cyto-GspL•AMPPNP, due to a major change in conformation of the N2E-CTE linker (225-238), are shown in sticks. V. vulnificus: CTE red, N2E-CTE linker purple, cyto-GspL green, AMPPNP as sticks with yellow carbons, and Mg in cyan. V. cholerae: N2E and N2E-CTE linker orange, CTE white, and AMPPNP as white sticks (PDB 1P9W). Some CTE residues are removed for clarity. B. V. vulnificus cyto-GspL•AMPPNPinteractions. Cyto-GspL residues Gln13 and His120 interact with the PNP of AMPPNP. Specifically, the side chains of Gln13 and His120 form hydrogen bonds with the oxygen atoms of γ phosphorous atom with distances of ∼2.5-2.8 Å. The side-chain oxygen of Gln13 forms a hydrogen bond with a distance of ∼2.7 Å with the nitrogen linking the β and γ phosphor atoms. Tyr14 makes hydrophobic contacts with the ribose of AMPPNP. C. Interactions between V. vulnificus Cyto-GspL and CTE. Key residues contributing to the cyto-GspL and CTE interactions are shown in sticks. The three Arg-Asp salt bridges are indicated with dashed lines. Residues making hydrogen bonds with main chain atoms of another subunit are also labeled.
Fig. 5
Fig. 5. Linear arrangements of Vibrio cyto-GspL domains in multiple crystals
A. The α- and β-interfaces among cyto-GspL domains in the current V. vulnificus GspE•cyto-GspL crystals. The interfaces between cyto-GspL domain #3 and crystallographically related domains are depicted. The interfaces between cyto-GspL domains #1 and #2 are essentially the same (see text). The α-interface is formed mainly by side chain contacts between residues of α2 helices (orange) and the loops between strand βE and helix α2 from two domains. The domains are related by a twofold axis approximately parallel to the direction of view. (See text and Fig. 5B for further description of the contacts). In the β-interface, main chain hydrogen bonds between antiparallel βc strands (yellow) are the main contacts between two subunits, which are also related by a twofold approximately parallel to the direction of view. B. Close-ups of four similar cyto-GspL α-interfaces in three different crystal forms. Key residues are shown in sticks. Hydrogen bonds and electrostatic interactions are indicated with dashed lines. Note the completely conserved Pro70 (red) in all interfaces, twice in contact with the highly conserved Tyr83 (blue) and Leu84 (purple). Left upper: The α-interface between two neighboring cyto-GspL domains #3 in the current V. vulnificus GspE•cyto-GspL crystals. Right upper: The α-interface between neighboring cyto-GspL domains #1 and #2 in the current V. vulnificus GspE•cyto-GspL crystals. Left lower: The α-interface between neighboring cyto-GspL domains in the V. cholerae N1E•cyto-GspL crystals (PDB 2BH1) (Abendroth et al., 2005). Right lower: The α-interface between neighboring cyto-GspL domains in the V. cholerae cyto-GspL crystals (PDB 1YF5) (Abendroth et al., 2004a) C. Four similar linear arrangements of cyto-GspL domains in three different crystal forms. From top to bottom: cyto-GspL rods from: V. vulnificus GspE•cyto-GspL complex #3; V. vulnificus GspE•cyto-GspL complex#1 and #2; V. cholerae N1E•cyto-GspL; and V. cholerae cyto-GspL. The α-interface and β-interfaces are colored orange and yellow, respectively. D. Three linear arrangements of cyto-GspL domains with associated N1E domains in two different crystal structures. From top to bottom: V. vulnificus GspE•cyto-GspL complex #3; V. vulnificus GspE•cyto-GspL complex #1 and #2; and V. cholerae N1E•cyto-GspL The α-interface and β-interfaces are colored orange and yellow, respectively. N1E domains are shown in surface representation.
Fig. 6
Fig. 6. A possible pre-assembly complex of the T2SS Inner Membrane Platform
The upper views are perpendicular to the membrane. The lower views are parallel to the membrane. A. Thre separate dimers of GspL with interactions across the α-interface (orange) of the cytoplasmic domains (green colors) and, e p-interface in the periplasmic domains (blue colors). B. Multiple GspL dimers form linear arrays by β-interfaces amongst cytoplasmic domains. C. N1E domains of GspE interact with cyto-GspL domains of a linear array, yielding a pre-assembly complex. Additional proteins like GspM (not shown) might also be part of the pre-assembly complex (see text). D. After an assembly signal, six GspL and six GspE subunits form an assembly with the lower N2E-CTE didomain as a hexamer with approximate C6 symmetry, connected by N1E-N2E linker residues to the N1E•GspL complex with approximate C3 symmetry. The latter C3 axis relates three N1E•GspL dimers with each of these dimers containing an approximate C2 axis. The aforementioned approximate C6, C3 and C2 axes run parallel to each other, perpendicular to the inner membrane plane, with the C6 and C3 axes coinciding. The C2 axes have a different position, approximately related by the C3 axis.
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References

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