Insights into the membrane interactions of the saposin-like proteins Na-SLP-1 and Ac-SLP-1 from human and dog hookworm

Abstract

Saposin-like proteins (SAPLIPs) from soil-transmitted helminths play pivotal roles in host-pathogen interactions and have a high potential as targets for vaccination against parasitic diseases. We have identified two non-orthologous SAPLIPs from human and dog hookworm, Na-SLP-1 and Ac-SLP-1, and solved their three-dimensional crystal structures. Both proteins share the property of membrane binding as monitored by liposome co-pelleting assays and monolayer adsorption. Neither SAPLIP displayed any significant haemolytic or bactericidal activity. Based on the structural information, as well as the results from monolayer adsorption, we propose models of membrane interactions for both SAPLIPs. Initial membrane contact of the monomeric Na-SLP-1 is most likely by electrostatic interactions between the membrane surface and a prominent basic surface patch. In case of the dimeric Ac-SLP-1, membrane interactions are most likely initiated by a unique tryptophan residue that has previously been implicated in membrane interactions in other SAPLIPs.

Figure 1. Quaternary structure in solution.

Left: Size exclusion with MALS detection reveals monomeric Na-SLP-1 and dimeric Ac-SLP-1 at pH 7.5 and 5.0 under normal salt conditions (representative chromatograms shown). Right: Ac-SLP-1 is exclusively observed as a dimer on native gels. The protein migrates at 20 kDa under native conditions (Lane 1), but at smaller sizes in the presence of SDS (Lane 2). The migration behaviour of Na-SLP-1 on native gels does not allow analysis due to smeared bands (data not shown).

Figure 2. Both SAPLIPS adopt the fold of the Sap domain.

Top: Cartoon representation of the crystal structure of Na-SLP-1 with the colour scheme highlighting the jaw-like arrangement of the five α-helices. Middle: Stereo figure of the 2F_o-F_c density around the Cys-39 - Cys-49 disulphide bond of Na-SLP-1. Bottom: Stereo figure of the 2F_o-F_c density around the corresponding region in Ac-SLP-1 (Cys-42 - Cys-56). The electron density is contoured at 1σ. Figure prepared with PyMOL .

Figure 3. The crystal structures suggest possible dimers for both hookworm SAPLIPs.

Left panel: Crystal packing analysis reveals a P2-symmetric dimer for Na-SLP-1 (571 Ų). Note the extended basic surface patch observed in the P2-symmetric dimer on the left. Right panel: Top-on (left) and face-on (right) views of the crystallographic dimer of Ac-SLP-1. A citrate molecule flanked by two HEPES molecules is bound in the acidic groove in a special position. Figure prepared with PyMOL .

Figure 4. Experimental membrane interactions.

Left panel: Liposome co-pelleting assays using multilamellar vesicles (DOPS/DOPE at molar ratio of 3∶1) at neutral pH shows binding of both SAPLIPs independent of metal ions. While for Na-SLP-1 full binding is observed, Ac-SLP-1 binding levels are ∼20%. Middle/right panels: Adsorption of both SAPLIPs to monolayers (DOPS/DOPE at molar ratio of 1∶3) was assessed in a Langmuir surface film balance at neutral (pH 7.5, blue curves) and acidic conditions (pH 4.7, red curves). While quantitatively different, three phases are observed for both proteins under all tested conditions: an immediate-onset Langmuir-like adsorption with small surface-pressure increase (phase 1, peripheral binding), followed by a large surface pressure increase (phase 2, embedding into the membrane), and a final stage with either stable (Ac-SLP-1) or decreasing (Na-SLP-1) surface pressure (phase 3).

Figure 5. Potential membrane binding mechanisms of Na-SLP-1 and Ac-SLP-1.

Top: Na-SLP-1 is expected to make contact with the membrane surface through electrostatic interactions. Once at the membrane, the protein can roll the surface of helix α1 (see view on the right) onto the membrane which may be embedded into the membrane interior. Bottom: Initial contact of the Ac-SLP-1 dimer with the membrane may be through Trp-48 in a swing-out conformation. This may cause the dimer to disassemble and expose the rather hydrophobic dimer interface (pictured on the right) which will be embedded into the membrane.