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

Abstract

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.

Empirical power laws for the radii of gyration of protein oligomers

“…proteins [PDB entries 4p99 (Vance et al, 2014), 4wve (Gruszka et al, 2015) and 2ww8 (Izoré et al, 2010)], a fragment of nonmuscle myosin 2C (PDB entry 2ycu) and the EphA4 ectodomain (PDB entry 4m4p; Xu et al, 2013). The adhesion proteins share a common feature of tandem domain repeats, as shown for PDB entry 4wve (Fig.…”

Interaction of ice binding proteins with ice, water and ions

“…Other IBPs structure ice, 6,7 inhibit ice recrystallization, 8,9 or promote ice adhesion. 10,11 Ice nucleating proteins (INPs) stimulate ice nucleation at high subzero temperatures. 12,13 The unique ability of IBPs to modify ice crystal growth also holds great promise for a range of application areas including food technology, materials science, and biomedicine.…”

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

“…Three Ca 2+ ions reside within each Mp AFP RII monomer, while one Ca 2+ ion is bound between monomer repeats. [20] Furthermore, the thermal stability of RII repeats is affected by calcium (see Figure H in S1 Supporting Information). Since Mp AFP RII monomers fold in a Ca 2+ -dependent manner, we investigated the impact of calcium on the mechanical stability of the protein.…”

“…RII comprises 90% of the mass of Mp AFP and consists of 120 identical 104-amino acid repeats of an immunoglobulin-like β-sandwich, which fold in a Ca 2+ -dependent manner. [20, 25] Recent small angle X-ray scattering measurements on a RII tetra-tandemer demonstrate that calcium also rigidifies the RII domain of Mp AFP. [20] RII of Mh Lap comprises 25 repeats of a 97-amino-acid domain with on average 76% sequence identity between repeats at the protein level (Fig 1).…”

“…[20, 25] Recent small angle X-ray scattering measurements on a RII tetra-tandemer demonstrate that calcium also rigidifies the RII domain of Mp AFP. [20] RII of Mh Lap comprises 25 repeats of a 97-amino-acid domain with on average 76% sequence identity between repeats at the protein level (Fig 1). For both adhesins, region II forms the link between the bacterium and its natural substrate, and as such is predicted to require high stability and strength to combat environmental shear forces.…”