Novel dimeric β-helical model of an ice nucleation protein with bridged active sites
© Garnham et al; licensee BioMed Central Ltd. 2011
Received: 23 June 2011
Accepted: 27 September 2011
Published: 27 September 2011
Ice nucleation proteins (INPs) allow water to freeze at high subzero temperatures. Due to their large size (>120 kDa), membrane association, and tendency to aggregate, an experimentally-determined tertiary structure of an INP has yet to be reported. How they function at the molecular level therefore remains unknown.
Here we have predicted a novel β-helical fold for the INP produced by the bacterium Pseudomonas borealis. The protein uses internal serine and glutamine ladders for stabilization and is predicted to dimerize via the burying of a solvent-exposed tyrosine ladder to make an intimate hydrophobic contact along the dimerization interface. The manner in which Pb INP dimerizes also allows for its multimerization, which could explain the aggregation-dependence of INP activity. Both sides of the Pb INP structure have tandem arrays of amino acids that can organize waters into the ice-like clathrate structures seen on antifreeze proteins.
Dimerization dramatically increases the 'ice-active' surface area of the protein by doubling its width, increasing its length, and presenting identical ice-forming surfaces on both sides of the protein. We suggest that this allows sufficient anchored clathrate waters to align on the INP surface to nucleate freezing. As Pb INP is highly similar to all known bacterial INPs, we predict its fold and mechanism of action will apply to these other INPs.
Two extraordinary families of proteins have evolved to influence ice growth in opposite ways: antifreeze proteins (AFPs) that irreversibly adsorb to the surface of ice crystals to prevent their further growth ; and ice-nucleation proteins (INPs) that cause ice to form in solution at high sub-zero temperatures [2, 3]. Whereas AFPs are small (Mr 3,000 - 35,000), and generally monomeric proteins, INPs are large (Mr >100,000) and function as multimers .
The tertiary structures of many AFPs are known, but none has been experimentally determined for an INP. Most INPs contain three distinct domains, with the majority of their mass residing within a highly repetitive central domain . This domain consists of a variable number (ca. 50-80) of tandem 16-amino-acid (aa) repeats, with each repeat following the general consensus sequence of GYGSTxTAxxxSxLxA . NMR and CD studies of synthetic bacterial INP peptides have not revealed a basic folding unit [6–9]. Molecular models of INP from the bacterium Pseudomonas syringae have included a planar array of anti-parallel β-strands  and a left-handed β-helix . The latter was modelled on UDP-acetylglucosamine acyltransferease as an AFP-like β-helix with the β-stranded TxT motifs located within each 16-aa repeat aligning down one side of the protein and functioning as the site of ice nucleation. Interestingly, a 96-aa recombinantly-expressed fragment of Ps INP was shown to produce moderate levels of AFP activity , hinting that INPs and AFPs share a similar mechanism of action.
The Gram-negative bacterium P. borealis produces a 1244-aa INP (Pb INP) similar to all other known bacterial INPs . Here we have predicted the structure of a 128-aa segment of the protein using a combination of homology-based modelling and molecular dynamics (MD) simulations. The right-handed β-helical model is stabilized by internal serine and glutamine ladders, and is predicted to dimerize via a highly conserved solvent-exposed tyrosine ladder. ach chain of the dimer contains two putative ice-nucleation sites, located opposite one another, and comprised of repetitive TQTA and SLTA β-strands. Each surface is flat and relatively hydrophobic, but also replete with hydrogen bond donors and acceptors; hallmarks of a typical AFP ice-binding site (IBS). MD simulations show each site is capable of ordering water molecules into an ice-like lattice, indicating that INPs use the same anchored clathrate water (ACW) mechanism of action that was recently elucidated for all AFPs . Indeed, ACWs align across the entire width of both sides of the INP dimer, dramatically increasing the 'active' surface area of the protein and further strengthening the idea that size, and not fold or mechanism of action, is the primary discriminating factor between the two families of proteins with diametrically opposite functions regarding ice growth.
Results and Discussion
PbINP contains two ice-nucleating motifs per 16-aa repeat
PbINP folds as a parallel β-helix
Pb INP was modelled as a right-handed β-helix due to the right-handedness of the modelling template. Mp AFP_RIV was previously modelled as a right-handed β-helix using the same β-roll template from alkaline protease and proved to be right-handed based on its crystal structure [13, 18]. However, Pb INP could just as likely be left-handed, and the handedness of an ice-binding protein does not affect its ability to bind ice. Both L- and D-enantiomers of type I fish AFP and snowflea AFP display identical levels of antifreeze activity [19, 20], while the non-homologous β-helical insect AFPs produced by Tenebrio molitor and Choristoneura fumiferana are right- and left-handed respectively, yet display near identical ice-binding sites and activity levels. Therefore, while we modelled Pb INP as a right-handed β-helix, we think that either a left- or right-handed version of the protein would be equally capable of efficiently nucleating ice.
The glutamine ladder is formed by the inward-pointing Gln residue of each TQ TA motif (Figure 3B). Glutamines typically repeat two out of every three loops at this position, with the third loop usually having a glycine substitution. Glutamine side chains form hydrogen bonds with main-chain carbonyl oxygens and amide nitrogens of the turn immediately preceding the TQTA motif, as shown in stereo in Figure 4B. The void produced by the regularly-spaced glycine substitutions is filled by an internal water molecule that bridges adjacent glutamines and also forms hydrogen bonds to the main chain of the protein (see Additional file 1, Figure S3). The hyperactive β-helical Tm AFP uses internal water molecules to stabilize its structure . Here it should be noted that these proteins have evolved to function at sub-zero temperatures where hydrogen bonding is relatively strong. While no structure in the Protein Data Bank contains a glutamine ladder, several β-helical proteins use very similar asparagine ladders to stabilize their folds , and TibA, a 104-kDa bacterial glycoprotein with both adhesin and invasin properties, is also predicted to contain an internal glutamine ladder .
PbINP dimerizes via a solvent-exposed tyrosine ladder
The tightly packed core of the protein left no room for the tyrosine residue of the GY GS motifs, and as such, they were forced to adopt a solvent-exposed orientation during initial model building. During the course of the MD simulation, the tyrosine residues stacked on top of one another (Figure 3B, Figure 4C), and each tri-peptide YGS motif adopted a β-stranded conformation. Stacking of solvent-exposed aromatic residues has been observed within various proteins, and they typically reside in areas involved in receptor binding and/or dimerization. For example, InIJ, a β-helical leucine-rich repeat produced by the bacterium Listeria monocytogenes, contains multiple solvent-exposed and stacked aromatic residues within its predicted receptor recognition domain . Most interestingly, engineered tyrosine ladders have been shown to promote the flatness  and dimerization  of OspA, a single-layer-β-sheet containing protein from the bacterium Borrelia burgdorferi. These results hinted at the possibility that Pb INP's tyrosine ladder might play a role in dimerization.
To test the stability of the dimer, 10-ns MD simulations were performed on the energy-minimized average dimeric structure at the elevated temperatures of 298 K and 310 K (Figure 5C, Additional file 1 - Figure S4). In each case, the dimer remained stable throughout the trajectory, with no dissociation of individual chains, and complete exclusion of water molecules at the dimerization interface. Identical MD simulations were performed on the Pb INP monomer, and in each case, a partial unravelling of the C terminus of the protein occurred (at the 8-ns mark of the 298 K simulation and the 4-ns mark of the 310 K simulation) (Figure 5C, see Additional file 1 - Figure S4). These results demonstrate that dimerization increases the stability of Pb INP.
The ice nucleating surfaces of PbINP are highly conserved
A s previously mentioned, when Pb INP is modelled as a parallel dimer, the SLTA surface of one chain resides on the same side of the dimer as the TQTA surface of the other chain (Figure 6A). This creates a flat surface on both sides of the dimer, each spanning the entire width of the structure. The amino acid composition of both the TQTA and SLTA surfaces is highly conserved, as is the area in between them created by the dimerization interface. More specifically, the solvent-exposed first and third position of the T QT A motifs have threonines present at these positions 87% and 92% of the time respectively. The solvent-exposed first and third position of the S LT A motifs show greater variation, however, serines and threonines are present at the first position 71% of the time (48% serine, 23% threonine), while threonine and isoleucine are present at the third position 98% of the time (60% threonine, 38% isoleucine). The rare substitutions at these positions are aspartate, valine and leucine. The dimerization interface is the most highly conserved portion of the whole protein, with the intercalated hydroxyl groups of the tyrosine and serine residues present 90% and 98% of the time, respectively.
PbINP orders water molecules via the anchored clathrate water mechanism
This high degree of amino acid conservation endows the Pb INP dimer with two sides that are flat, and relatively hydrophobic, but also contain many hydrogen bond donors and acceptors. These characteristics define the IBS's of all AFPs, and as such, raise the possibility that INPs function by the ACW mechanism of AFP action . The ACW mechanism states that the relative hydrophobicity of an AFP's IBS orders water molecules into an ice-like lattice, and this lattice is then anchored to the surface of the protein via hydrogen bonds.
To investigate the water ordering potential of Pb INP, a 2-ns MD simulation was performed on the energy-minimized average dimeric structure at a temperature of 273 K. The TIP5P water model was used as it accurately represents the behaviour of water during an MD simulation [27, 28]. Waters within 10 Å of Pb INP's surface were extracted at 20-ps intervals throughout the course of the simulation and their crystallographic structure factors were calculated followed by a Fourier transform, giving the electron density of all extracted waters. Strong electron density with ice-like spacing was present across the entire width of the protein's surface (Figure 7A). Waters built into this density aligned down the troughs created by the outward projecting residues of the T QT A and S LT A motifs, down the flat areas immediately preceding and following these motifs, and across the dimerization interface as well (Figure 7B). These waters closely match the 4.5 Å × 7.35 Å spacing of waters on the primary prism plane of ice, and as such, have revealed a potential orientation of a nascent ice crystal on the surface of the INP (Figures 7C,D).
Dimerization increases the active surface area of PbINP
The temperature at which an INP nucleates ice is dependent upon its oligomerization state . Approximately one INP monomer is capable of nucleating ice at a temperature of -12°C, while an aggregate of at least 50 INP monomers is required for ice nucleation in the -2°C to -3°C temperature range. However, due to the lack of an experimentally-determined INP structure, the mechanism by which an INP oligomerizes has remained a mystery. Previously, there have been only two attempts to predict how an INP might oligomerize. Wu et al. speculated that overlapping protein-protein interactions between the two flat IBS motifs could generate 'stairs' of INPs, facilitating ice growth along the discontinuities. Prior to that, however, Kajava and Lindow  modelled Ps INP as an array of interdigitating anti-parallel β-strands that formed a flat ice-nucleating array upon oligomerization. While intriguing, the model was created prior to the structural determination of several β-helical AFPs [13–15], and as such, it overlooked the potential water-organizing capabilities of the TQTA and SLTA motifs when aligned as parallel β-strands in a flat β-sheet. As previously mentioned, Graether and Jia  modelled an INP as a β-helix, but the potential for oligomerization was not discussed. In that model, the serine of the GYGS motif pointed inwards and was not solvent exposed as predicted in this study. This would prevent formation of the intercalated hydrogen-bond network between the serine and tyrosine residues of the GYGS motifs from opposing chains in the dimer. Nevertheless, both β-helical models predict a solvent-exposed orientation for the tyrosine residue of the GYGS motif, and this argues against INP peptide studies that suggest it points towards the interior of the structure [8, 9]. It is likely that short peptides adopt conformations in solution that are not representative of the full-length protein.
Even without oligomerization, the size of Pb INP is significantly larger than any known AFP (Figure 9). This fact, combined with an INP's ability to order water molecules via the ACW mechanism, further strengthens the idea that size, and not a particular protein fold or mechanism of action, is the primary discriminating factor between the two families of proteins with diametrically opposite functions.
This paper demonstrates through the use of homology modelling and MD simulations that bacterial INPs are able to fold as novel β-helical dimers. De-solvation of solvent-exposed tyrosine ladders drives INP dimerization, and this allows the ice nucleation sites of the protein to extend as a continuum across the width of the dimer. Both sides of the dimer order water molecules into an ice-like lattice using the anchored clathrate water mechanism of action. Offset dimerization can allow INP oligomerization without active site occlusion. This increases the active surface area of the INP, therefore raising the temperature at which ice nucleates.
Initial model building
All model building was performed using a combination of the programs PyMOL  and SYBYL (version 6.4, Tripos Associates, St. Louis, MO). PyMOL was used to excise the β-roll from alkaline protease (PDB 1KAP), remove the roll's Ca2+-binding turns, and change its tri-peptide xux β-strands (where x is any amino acid and u is a hydrophobic residue) to the appropriate TQTA and SLTA motifs of Pb INP. SYBYL was used to manually build and energetically minimize the xxxS and GYGS loops that connect the TQTA and SLTA β-strands. Finally, PyMOL was used to copy and expand the 16-aa loop until an 8-loop β-helix was completed (corresponding to residues 217-345 of Pb INP).
Molecular Dynamics simulations of initial model
All MD simulations were performed using the program Gromacs v. 4.5.3 . Prior to all full-scale MD simulations described below, energy minimization and a 50-ps position-restrained MD simulation was performed to relax the solvent around the protein. Berendsen temperature and pressure coupling were applied in all cases, and the GROMOS96 43a1 force field and SPC water model were used unless stated otherwise. The initial model of Pb INP (residues 217-345) was solvated in a box containing 6239 water molecules and 14 Na+ ions to offset the charge of the protein. A full-scale 5-ns MD simulation was then performed at 277 K. The average structure of the final 3 ns of the simulation was calculated and energy minimized in vacuo. A Ramachandran plot of the protein was generated using the program PROCHECK .
PbINP dimer construction and MD simulations
The dimer of Pb INP was built by duplicating the protein, rotating it 180° about the long axis of the structure, and then placing the duplicated chain's tyrosine ladder in close proximity to the tyrosine ladder of the original chain. This parallel arrangement of the helices placed their N termini at the same end of the dimer. The dimer was solvated in a box containing 13,401 waters and 28 Na+ ions to offset the charge of the protein. The protein was subjected to a 10-ns full-scale MD simulation. An average structure of the protein from the final 5 ns of the simulation was calculated and energy minimized in the same manner as previously mentioned. This structure was then re-solvated and subjected to a 10-ns full-scale MD simulation at 298 K to test the stability of the dimeric protein. A single chain from the dimer was also subjected to the same simulation to test its stability at the elevated temperature of 298 K.
PbINP dimer hydration studies
A 2-ns MD simulation was performed at the temperature of 273 K on the energy-minimized average dimeric structure of Pb INP to investigate the hydration of the protein. As a positive control, chain B from region IV of the AFP produced by the Antarctic bacterium Marinomonas primoryensis (Mp AFP_RIV) (PDB 3P4G) was subjected to the same simulation. The OPLS-aa force field [35, 36] along with the TIP5P water model  were used in each case. To determine the probability density of water molecules, the following protocol was followed for each MD simulation. Coordinates of the system were extracted at 20-ps intervals throughout the course of the simulation (100 coordinate sets total/simulation). After superimposing these 100 structures by performing a least-squares fit on the protein Cα atoms, the waters within 10 Å of the protein were extracted and their electron densities were determined by calculating crystallographic structure factors followed by a Fourier transform using the programs SFall  and FFT  respectively of the CCP4 software suite . The electron densities were calculated with the protein and water in a P1 unit cell equivalent to the box size used for the MD simulation. The FFT calculation used a grid spacing of 0.33 Å and a resolution of 1 Å. Waters were then manually built into the density using the program Coot .
anchored clathrate water
ice nucleation protein
- Mp AFP_RIV:
Marinomonas primoryensis region IV antifreeze protein
- Pb INP:
Pseudomonas borealis ice nucleation protein
- Ps INP:
Pseudomonas syringae ice nucleation protein
spruce budworm antifreeze protein
snow flea antifreeze protein
- Tm AFP:
Tenebrio molitor antifreeze protein.
We are grateful to Dr. John Allingham for access to his servers. This work was funded by grants from the CIHR and NSERC to PLD and VKW, respectively. CPG was the recipient of an NSERC-PGSD3 scholarship and an R. Samuel McLaughlin fellowship. PLD holds a Canada Research Chair in Protein Engineering and VKW is a Queen's University Research Chair.
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