Structure and oligomerization of the periplasmic domain of GspL from the type II secretion system of Pseudomonas aeruginosa

Abstract

The ability of bacteria to infect a host relies in part on the secretion of molecular virulence factors across the cell envelope. Pseudomonas aeruginosa, a ubiquitous environmental bacterium causing opportunistic infections in humans, employs the type II secretion system (T2SS) to transport effector proteins across its cellular envelope as part of a diverse array of virulence strategies. General secretory pathway protein L (GspL) is an essential inner-membrane component of the T2SS apparatus, and is thought to facilitate transduction of the energy from ATP hydrolysis in the cytoplasm to the periplasmic components of the system. However, our incomplete understanding of the assembly principles of the T2SS machinery prevents the mechanistic deconvolution of T2SS-mediated protein secretion. Here we show via two crystal structures that the periplasmic ferredoxin-like domain of GspL (GspL^fld) is a dimer stabilized by hydrophobic interactions, and that this interface may allow significant interdomain plasticity. The general dimerization mode of GspL^fld is shared with GspL from Vibrio parahaemolyticus suggesting a conserved oligomerization mode across the GspL family. Furthermore, we identified a tetrameric form of the complete periplasmic segment of GspL (GspL^peri) which indicates that GspL may be able to adopt multiple oligomeric states as part of its dynamic role in the T2SS apparatus.

Figure 1

Domain organization, construct design and purification of GspL. (a) Schematic representation of GspL (blue) and its positioning in the bacterial inner-membrane (IM). FLD: ferredoxin-like domain, MPD: membrane proximal domain, CD: cytoplasmic domain. Numbering corresponds to the domain boarders, which can be traced to the sequence in panel b. Except for the cytoplasmic part, GspL shares the domain organisation with GspM (grey), its well established interaction partner. (b) Protein sequence of the construct used in the study. GspL sequence is in blue, whereas purification tag sequence is in black. Serendipitously, in the course of crystallization experiment, GspL^peri was proteolytically cleaved in the middle of MPD (indicated with scissors), which lead to crystallization of the shorter construct, herein termed GspL^fld to distinguish it from the initially purified recombinant GspL^peri (c) SEC profile of GspL^peri accompanied by SDS-PAGE analysis of the indicated peak fractions. EEP: early elution peak, LEP: late elution peak.

Figure 2

GspL^peri adopts dimeric and tetrameric forms as revealed by SEC-MALLS (a) SEC-MALLS analysis of late elution peak of GspL^peri shows a dimeric form (dashed line), and the oligomeric state does not change upon concentration (solid line). (b) GspL^peri from early elution peak diversifies into two populations, of tetramers and dimers, respectively (dashed line). Concentration of the EEP GspL^peri does not enrich the tetrameric population (solid line). However, the procedure results in longer time separation between the purification and the light scattering experiment, which shifts the tetramer/dimer ratio in favor of dimer. Tetramer to dimer ratio calculated as the ratio of areas under the relevant peaks is 0.70 at lower and 0.57 at higher concentration. Molecular weight of GspL^peri calculated from the sequence is 13.95 kDa.

Figure 3

The architecture of the GspL^fld dimer and the hydrophobic nature of the interaction interface is conserved. (a) Crystal form 1 of GspL^fld dimer seen from the side and from the top. The inset reveals the details of the hydrophobic interface. The interacting residues are located on strand β1 and helix α1 and their symmetry related interaction partners. Leu327 is built in the model in two different conformations, labeled a and b in the bottom panel. (b) Side-view of crystal form 2 of the GspL^fld dimer. In contrast to crystal form 1, the dimer is present in the asymmetric unit but the interface also involves residues from strand β1 and helix α1 of chain A and strand β1′ and helix α1′ of chain B. In bottom panel, crystal form 2 is shown in surface representation. All atoms are colored according to the type (oxygens: red, nitrogens: blue) and carbon atoms are in the respective chain color. Display of chain B as it would face chain A at the GspL^fld dimer interface reveals a buried hydrophobic patch in the GspL^fld dimer (inner dotted ellipse). (c) GspL^fld dimer from Vibrio parahaemolyticus, PDB: 2w7v²¹. For details on the conservation of the interacting residues see Figure S2.

Figure 4

The GspL^fld dimer exhibits plasticity at the interface. (a) Schematic representation of the dimer of crystal form 1 viewed along the twofold axis (double headed arrow) and the dimer of crystal form 2 seen in the analogous way. Each block represents the whole ferredoxin-like domain and the depiction portrays this particular orientation of the dimer. The different proximity of interacting strands results in open (crystal form 1) and closed (crystal form 2) variant of the GspL^fld dimer. (b) The detailed view on the beta strands contributing to the interaction interfaces of crystal forms 1 and 2. The dramatically different inter-strand distances result in complementation of the interface with the extension of antiparallel β-sheet in case of crystal form 2 and lack of thereof in crystal form 1. The twofold axis is marked with a black diad symbol. The open variant of the crystal form 1 is mediated by coordination of a water molecule, indicated in dark red sphere. The two distinct scenarios can be fulfilled by the N-H of Leu327 and the C = O of Phe329′. The distances between main chains’ N and C = O groups are labeled with yellow dashed lines and reported in Å. The models are shown in the electron density 2Fo-Fc maps contoured at 2σ. (c) The plasticity of the GspL^fld reflected by interdomain tilting of 3°. (d) The dimer of crystal form 1 incorporated into the schematic representation of the GspL full-length dimer.

Figure 5

SEC-SAXS analysis validates the existence of dimers and tetramers of GspL^peri in solution. (a) The experimentally determined distance distribution functions of dimer and tetramer of GspL^peri (solid lines, blue and black) in comparison to the calculated P(r) functions of dimer and monomer (dashed lines, green and red) of crystal form 1. (b) Ab initio envelopes generated for dimer (green) and tetramer (cyan) (c) The dimeric envelope manually fitted with the dimer of crystal form 1 and an ensemble of most populous models of the N-terminus, presented as chains of spheres. (d) Kratky plot of the GspL^peri dimer.

Figure 6

A model for substrate loading to the periplasmic vestibule of the type II secretion system. The drawing encompasses periplasmic domains of GspL (blue), GspM (grey), GspC (dark grey), the secretin channel (light grey) and a molecule of a cargo protein (black) viewed from the top of the secretion machinery. In the proposed model the transient tetramerization of GspL^peri is proposed as a mechanism to open the periplasmic vestibule and to enable entry of an exoprotein (left). In contrast, the channel is closed when the GspL^peri dimers are equivalently distributed (right).