A Frame-by-Frame Glance at Membrane Fusion Mechanisms: From Viral Infections to Fertilization.

Viral entry and fertilization are distinct biological processes that share a common mechanism: membrane fusion. In viral entry, enveloped viruses attach to the host cell membrane, triggering a series of conformational changes in the viral fusion proteins. This results in the exposure of a hydrophobic fusion peptide, which inserts into the host membrane and brings the viral and host membranes into close proximity. Subsequent structural rearrangements in opposing membranes lead to their fusion. Similarly, membrane fusion occurs when gametes merge during the fertilization process, though the exact mechanism remains unclear. Structural biology has played a pivotal role in elucidating the molecular mechanisms underlying membrane fusion. High-resolution structures of the viral and fertilization fusion-related proteins have provided valuable insights into the conformational changes that occur during this process. Understanding these mechanisms at a molecular level is essential for the development of antiviral therapeutics and tools to influence fertility. In this review, we will highlight the biological importance of membrane fusion and how protein structures have helped visualize both common elements and subtle divergences in the mechanisms behind fusion; in addition, we will examine the new tools that recent advances in structural biology provide researchers interested in a frame-by-frame understanding of membrane fusion.

SPACA6 ectodomain structure reveals a conserved superfamily of gamete fusion-associated proteins.

SPACA6 is a sperm-expressed surface protein that is critical for gamete fusion during mammalian sexual reproduction. Despite this fundamental role, little is known about how SPACA6 specifically functions. We elucidated the crystal structure of the SPACA6 ectodomain at 2.2-Å resolution, revealing a two-domain protein containing a four-helix bundle and Ig-like ß-sandwich connected via a quasi-flexible linker. This structure is reminiscent of IZUMO1, another gamete fusion-associated protein, making SPACA6 and IZUMO1 founding members of a superfamily of fertilization-associated proteins, herein dubbed the IST superfamily. The IST superfamily is defined structurally by its distorted four-helix bundle and a pair of disulfide-bonded CXXC motifs. A structure-based search of the AlphaFold human proteome identified more protein members to this superfamily; remarkably, many of these proteins are linked to gamete fusion. The SPACA6 structure and its connection to other IST-superfamily members provide a missing link in our knowledge of mammalian gamete fusion.

Structural Basis of Ligand Selectivity by a Bacterial Adhesin Lectin Involved in Multispecies Biofilm Formation.

Carbohydrate recognition by lectins governs critical host-microbe interactions. MpPA14 (Marinomonas primoryensis PA14 domain) lectin is a domain of a 1.5-MDa adhesin responsible for a symbiotic bacterium-diatom interaction in Antarctica. Here, we show that MpPA14 binds various monosaccharides, with l-fucose and N-acetylglucosamine being the strongest ligands (dissociation constant [Kd ], ∼150 µM). High-resolution structures of MpPA14 with 15 different sugars bound elucidated the molecular basis for the lectin's apparent binding promiscuity but underlying selectivity. MpPA14 mediates strong Ca2+-dependent interactions with the 3,4-diols of l-fucopyranose and glucopyranoses, and it binds other sugars via their specific minor isomers. Thus, MpPA14 only binds polysaccharides like branched glucans and fucoidans with these free end groups. Consistent with our findings, adhesion of MpPA14 to diatom cells was selectively blocked by l-fucose, but not by N-acetyl galactosamine. The MpPA14 lectin homolog present in a Vibrio cholerae adhesin was produced and was shown to have the same sugar binding preferences as MpPA14. The pathogen's lectin was unable to effectively bind the diatom in the presence of fucose, thus demonstrating the antiadhesion strategy of blocking infection via ligand-based antagonists.IMPORTANCE Bacterial adhesins are key virulence factors that are essential for the pathogen-host interaction and biofilm formation that cause most infections. Many of the adhesin-driven cell-cell interactions are mediated by lectins. Our study reveals for the first time the molecular basis underlying the binding selectivity of a common bacterial adhesin lectin from the marine bacterium Marinomonas primoryensis, homologs of which are found in both environmental and pathogenic species. The lectin-ligand interactions illustrated at the atomic level guided the identification of a ligand that serves as an inhibitor to block bacterium-host adhesion. With conventional bactericidal antibiotics losing their potency due to resistance, our work gives critical insight into an antiadhesion strategy to treat bacterial infections.

Essential role of calcium in extending RTX adhesins to their target.

RTX adhesins are long, multi-domain proteins present on the outer membrane of many Gram-negative bacteria. From this vantage point, adhesins use their distal ligand-binding domains for surface attachment leading to biofilm formation. To expand the reach of the ligand-binding domains, RTX adhesins maintain a central extender region of multiple tandem repeats, which makes up most of the proteins' large molecular weight. Alignments of the 10-15-kDa extender domains show low sequence identity between adhesins. Here we have produced and structurally characterized protein constructs of four tandem repeats (tetra-tandemers) from two different RTX adhesins. In comparing the tetra-tandemers to each other and already solved structures from Marinomonas primoryensis and Salmonella enterica, the extender domains fold as diverse beta-sandwich structures with widely differing calcium contents. However, all the tetra-tandemers have at least one calcium ion coordinated in the linker region between beta-sandwich domains whose role appears to be the rigidification of the extender region to help the adhesin extend its reach.

Bacterial sugar-binding protein as a one-step affinity purification tag on dextran-containing resins.

Marinobacter hydrocarbonoclasticus is an oil-eating bacterium that possesses a large adhesion protein (MhLap) with the potential to bind extracellular ligands. One of these ligand-binding modules is the ~20-kDa PA14 domain (MhPA14) that has affinity for glucose-based carbohydrates. Previous studies showed this sugar-binding domain is retained on dextran-based size-exclusion resins during chromatography, requiring the introduction of glucose or EDTA to remove the protein from the column. Given the ready availability of such size-exclusion resins in biochemistry laboratories, this study explores the use of MhPA14 as an affinity tag for recombinant protein purification. Two different fusion proteins were tested: 1) Green fluorescent protein (GFP) linked to the N-terminus of the MhPA14 tag; and 2) the ice-binding domain from the Marinomonas primoryensis ice-binding protein (MpIBD) linked to the MhPA14 C-terminus by a TEV cut site. The GFP_MhPA14 fusion visibly bound to Superdex, Sephadex, and Sephacryl resins, but did not bind to Sepharose. Using Superdex resin, dextran-affinity purification proved to be an effective one-step purification strategy for both proteins, superior to even nickel-affinity chromatography. Dextran-affinity chromatography was also the most effective method of separating the MhPA14 tag from MpIBD following TEV proteolysis, as compared to both nickel-affinity and ice-affinity methods. These results indicate that MhPA14 has potential for widespread use in recombinant protein purification.

Correction: Structure and functional analysis of a bacterial adhesin sugar-binding domain.

[This corrects the article DOI: 10.1371/journal.pone.0220045.].

Structure and functional analysis of a bacterial adhesin sugar-binding domain.

Bacterial adhesins attach their hosts to surfaces through one or more ligand-binding domains. In RTX adhesins, which are localized to the outer membrane of many Gram-negative bacteria via the type I secretion system, we see several examples of a putative sugar-binding domain. Here we have recombinantly expressed one such ~20-kDa domain from the ~340-kDa adhesin found in Marinobacter hydrocarbonoclasticus, an oil-degrading bacterium. The sugar-binding domain was purified from E. coli with a yield of 100 mg/L of culture. Circular dichroism analysis showed that the protein was rich in beta-structure, was moderately heat resistant, and required Ca2+ for proper folding. A crystal structure was obtained in Ca2+ at 1.2-Å resolution, which showed the presence of three Ca2+ ions, two of which were needed for structural integrity and one for binding sugars. Glucose was soaked into the crystal, where it bound to the sugar's two vicinal hydroxyl groups attached to the first and second (C1 and C2) carbons in the pyranose ring. This attraction to glucose caused the protein to bind certain polysaccharide-based column matrices and was used in a simple competitive binding assay to assess the relative affinity of sugars for the protein's ligand-binding site. Fucose, glucose and N-acetylglucosamine bound most tightly, and N-acetylgalactosamine hardly bound at all. Isothermal titration calorimetry was used to determine specific binding affinities, which lie in the 100-µM range. Glycan arrays were tested to expand the range of ligand sugars assayed, and showed that MhPA14 bound preferentially to branched polymers containing terminal sugars highlighted as strong binders in the competitive binding assay. Some of these binders have vicinal hydroxyl groups attached to the C3 and C4 carbons that are sterically equivalent to those presented by the C1 and C2 carbons of glucose.

Ice-binding proteins and the 'domain of unknown function' 3494 family.

Ice-binding proteins (IBPs) control the growth and shape of ice crystals to cope with subzero temperatures in psychrophilic and freeze-tolerant organisms. Recently, numerous proteins containing the domain of unknown function (DUF) 3494 were found to bind ice crystals and, hence, are classified as IBPs. DUF3494 IBPs constitute today the most widespread of the known IBP families. They can be found in different organisms including bacteria, yeasts and microalgae, supporting the hypothesis of horizontal transfer of its gene. Although the 3D structure is always a discontinuous ß-solenoid with a triangular cross-section and an adjacent alpha-helix, DUF3494 IBPs present very diverse activities in terms of the magnitude of their thermal hysteresis and inhibition of ice recrystallization. The proteins are secreted into the environments around the host cells or are anchored on their cell membranes. This review covers several aspects of this new class of IBPs, which promise to leave their mark on several research fields including structural biology, protein biochemistry and cryobiology.

RTX Adhesins are Key Bacterial Surface Megaproteins in the Formation of Biofilms.

Gram-negative bacteria produce repeats-in-toxin adhesion proteins (RTX adhesins) to facilitate microbial adhesion. These large, multidomain proteins share a common architecture comprised of four regions. First to emerge from the bacterium, C terminal end leading, is the RTX export sequence that directs the protein through the type 1 secretion system (T1SS). This is followed by the ligand-binding region responsible for host adhesion and cohesion, which contains diverse ligand-binding domains. These serve a zip code function to direct bacteria to a particular environmental niche. Thereafter is a large extension region consisting of tens to hundreds of tandem bacterial immunoglobulin-like (BIg) domains, whose function is to extend the reach of the ligand-binding domains away from the bacterial surface. Lastly, there is a conserved N terminal cell-membrane-anchor region that retains the adhesin within the secretion system. This is also a site of in situ proteolysis, when nutrients are scarce, that enables the bacterium to leave the biofilm. In this review, the four regions of RTX adhesins are presented in the order in which they emerge from the cell during synthesis and retention.

An ice-binding and tandem beta-sandwich domain-containing protein in Shewanella frigidimarina is a potential new type of ice adhesin.

Out of the dozen different ice-binding protein (IBP) structures known, the DUF3494 domain is the most widespread, having been passed many times between prokaryotic and eukaryotic microorganisms by horizontal gene transfer. This ~25-kDa ß-solenoid domain with an adjacent parallel α-helix is most commonly associated with an N-terminal secretory signal peptide. However, examples of the DUF3494 domain preceded by tandem Bacterial Immunoglobulin-like (BIg) domains are sometimes found, though uncharacterized. Here, we present one such protein (SfIBP_1) from the Antarctic bacterium Shewanella frigidimarina. We have confirmed and characterized the ice-binding activity of its ice-binding domain using thermal hysteresis measurements, fluorescent ice plane affinity analysis, and ice recrystallization inhibition assays. X-ray crystallography was used to solve the structure of the SfIBP_1 ice-binding domain, to further characterize its ice-binding surface and unique method of stabilizing or 'capping' the ends of the solenoid structure. The latter is formed from the interaction of two loops mediated by a combination of tandem prolines and electrostatic interactions. Furthermore, given their domain architecture and membrane association, we propose that these BIg-containing DUF3494 IBPs serve as ice-binding adhesion proteins that are capable of adsorbing their host bacterium onto ice. DATABASE: Submitted new structure to the Protein Data Bank (PDB: 6BG8).

Structure of a 1.5-MDa adhesin that binds its Antarctic bacterium to diatoms and ice.

Bacterial adhesins are modular cell-surface proteins that mediate adherence to other cells, surfaces, and ligands. The Antarctic bacterium Marinomonas primoryensis uses a 1.5-MDa adhesin comprising over 130 domains to position it on ice at the top of the water column for better access to oxygen and nutrients. We have reconstructed this 0.6-µm-long adhesin using a "dissect and build" structural biology approach and have established complementary roles for its five distinct regions. Domains in region I (RI) tether the adhesin to the type I secretion machinery in the periplasm of the bacterium and pass it through the outer membrane. RII comprises ~120 identical immunoglobulin-like ß-sandwich domains that rigidify on binding Ca2+ to project the adhesion regions RIII and RIV into the medium. RIII contains ligand-binding domains that join diatoms and bacteria together in a mixed-species community on the underside of sea ice where incident light is maximal. RIV is the ice-binding domain, and the terminal RV domain contains several "repeats-in-toxin" motifs and a noncleavable signal sequence that target proteins for export via the type I secretion system. Similar structural architecture is present in the adhesins of many pathogenic bacteria and provides a guide to finding and blocking binding domains to weaken infectivity.

Unusually high mechanical stability of bacterial adhesin extender domains having calcium clamps.

To gain insight into the relationship between protein structure and mechanical stability, single molecule force spectroscopy experiments on proteins with diverse structure and topology are needed. Here, we measured the mechanical stability of extender domains of two bacterial adhesins MpAFP and MhLap, in an atomic force microscope. We find that both proteins are remarkably stable to pulling forces between their N- and C- terminal ends. At a pulling speed of 1 µm/s, the MpAFP extender domain fails at an unfolding force Fu = 348 ± 37 pN and MhLap at Fu = 306 ± 51 pN in buffer with 10 mM Ca2+. These forces place both extender domains well above the mechanical stability of many other ß-sandwich domains in mechanostable proteins. We propose that the increased stability of MpAFP and MhLap is due to a combination of both hydrogen bonding between parallel terminal strands and intra-molecular coordination of calcium ions.

Multivalent Display of Antifreeze Proteins by Fusion to Self-Assembling Protein Cages Enhances Ice-Binding Activities.

Antifreeze proteins (AFPs) are small monomeric proteins that adsorb to the surface of ice to inhibit ice crystal growth and impart freeze resistance to the organisms producing them. Previously, monomeric AFPs have been conjugated to the termini of branched polymers to increase their activity through the simultaneous binding of more than one AFP to ice. Here, we describe a superior approach to increasing AFP activity through oligomerization that eliminates the need for conjugation reactions with varying levels of efficiency. A moderately active AFP from a fish and a hyperactive AFP from an Antarctic bacterium were genetically fused to the C-termini of one component of the 24-subunit protein cage T33-21, resulting in protein nanoparticles that multivalently display exactly 12 AFPs. The resulting nanoparticles exhibited freezing point depression >50-fold greater than that seen with the same concentration of monomeric AFP and a similar increase in the level of ice-recrystallization inhibition. These results support the anchored clathrate mechanism of binding of AFP to ice. The enhanced freezing point depression could be due to the difficulty of overgrowing a larger AFP on the ice surface and the improved ice-recrystallization inhibition to the ability of the nanoparticle to simultaneously bind multiple ice grains. Oligomerization of these proteins using self-assembling protein cages will be useful in a variety of biotechnology and cryobiology applications.

Ca2+-stabilized adhesin helps an Antarctic bacterium reach out and bind ice.

The large size of a 1.5-MDa ice-binding adhesin [MpAFP (Marinomonas primoryensis antifreeze protein)] from an Antarctic Gram-negative bacterium, M. primoryensis, is mainly due to its highly repetitive RII (Region II). MpAFP_RII contains roughly 120 tandem copies of an identical 104-residue repeat. We have previously determined that a single RII repeat folds as a Ca2+-dependent immunoglobulin-like domain. Here, we solved the crystal structure of RII tetra-tandemer (four tandem RII repeats) to a resolution of 1.8 Å. The RII tetra-tandemer reveals an extended (~190-Å × ~25-Å), rod-like structure with four RII-repeats aligned in series with each other. The inter-repeat regions of the RII tetra-tandemer are strengthened by Ca2+ bound to acidic residues. SAXS (small-angle X-ray scattering) profiles indicate the RII tetra-tandemer is significantly rigidified upon Ca2+ binding, and that the protein's solution structure is in excellent agreement with its crystal structure. We hypothesize that >600 Ca2+ help rigidify the chain of ~120 104-residue repeats to form a ~0.6 µm rod-like structure in order to project the ice-binding domain of MpAFP away from the bacterial cell surface. The proposed extender role of RII can help the strictly aerobic, motile bacterium bind ice in the upper reaches of the Antarctic lake where oxygen and nutrients are most abundant. Ca2+-induced rigidity of tandem Ig-like repeats in large adhesins might be a general mechanism used by bacteria to bind to their substrates and help colonize specific niches.