Structure and Protein-Protein Interactions of Ice Nucleation Proteins Drive Their Activity

Abstract

Microbially-produced ice nucleating proteins (INpro) are unique molecular structures with the highest known catalytic efficiency for ice formation. Airborne microorganisms utilize these proteins to enhance their survival by reducing their atmospheric residence times. INpro also have critical environmental effects including impacts on the atmospheric water cycle, through their role in cloud and precipitation formation, as well as frost damage on crops. INpro are ubiquitously present in the atmosphere where they are emitted from diverse terrestrial and marine environments. Even though bacterial genes encoding INpro have been discovered and sequenced decades ago, the details of how the INpro molecular structure and oligomerization foster their unique ice-nucleation activity remain elusive. Using machine-learning based software AlphaFold 2 and trRosetta, we obtained and analysed the first ab initio structural models of full length and truncated versions of bacterial INpro. The modeling revealed a novel beta-helix structure of the INpro central repeat domain responsible for ice nucleation activity. This domain consists of repeated stacks of two beta strands connected by two sharp turns. One beta-strand is decorated with a TxT amino acid sequence motif and the other strand has an SxL[T/I] motif. The core formed between the stacked beta helix-pairs is unusually polar and very distinct from previous INpro models. Using synchrotron radiation circular dichroism, we validated the β-strand content of the central repeat domain in the model. Combining the structural model with functional studies of purified recombinant INpro, electron microscopy and modeling, we further demonstrate that the formation of dimers and higher-order oligomers is key to INpro activity. Using computational docking of the new INpro model based on rigid-body algorithms we could reproduce a previously proposed homodimer structure of the INpro CRD with an interface along a highly conserved tyrosine ladder and show that the dimer model agrees with our functional data. The parallel dimer structure creates a surface where the TxT motif of one monomer aligns with the SxL[T/I] motif of the other monomer widening the surface that interacts with water molecules and therefore enhancing the ice nucleation activity. This work presents a major advance in understanding the molecular foundation for bacterial ice-nucleation activity.

Keywords: atmospheric bacteria; ice-nucleating proteins; protein activity; protein structure; protein–protein interactions.

Figure 1

The central repeat region (CRD) is an elongated template for ice nucleation (A) Four INpro constructs were produced to investigate the role of CRD size for the nucleation activity. Purple signifies the N-terminal domain and the yellow signifies the C-terminal domain. The colors of the CRD are used to differentiate the recombinant INpro constructs. (B) Ice nucleation spectra of INpro constructs expressed as ice nucleation site density per molecule $(N_n)$ as a function of temperature. Each color represents a protein of different length. Averages are shown with maximal and minimal values presented as shaded areas. More detailed figures showing the frozen fractions and the ice nucleation spectra of each protein construct can be found in the SI (Supplementary Figures S8, S9). The quality with which the steepness of the increase was captured depends on the concentration of the purified INpro molecules in each sample. (C,D,E) A sketch of the ice nucleation template (3) and the ice cluster (2) in supercooled water (1) for cuboid geometry proposed based on the ab initio β-helical INpro model (C,D) and spherical cap geometry previously used to model INpro activity (E). The interfaces are defined via the respective interfacial free energies ${\sigma }_{\mathrm{ij}}$ of the adjoining phases. The cuboid is determined by three axes (a, b, c), whereas the spherical cap can be described by the critical radius ${\mathrm{r}}_{\mathrm{crit}}$ and the contact angle $\theta .$ (F) Comparison of the relationship between the characteristic length (a or r in this figure) and the temperature predicted from the cuboid and spherical cap models according to CNT and the experimental measurements of characteristic nucleation temperatures and lengths of INpro dimers determined from the ab initio β-helical model of the INpro.

Figure 2

Ab initio model of the first 16 repeats of the INpro CRD. (A) The ab initio model predicted using machine-learning-based algorithms in AlphaFold. The model consists of a β-helix with one extended β-sheet on each side. The β-strands have a rotation along the longitudinal axis of approximately 40 degrees when comparing N- to C-terminal. The highly conserved tyrosine ladder (annotated) is solvent-exposed along the side (shown with stick-representation). (B) Stick representation of repeat 13 which displays the best match to the consensus sequence (shown in C). The two putative ice-nucleation active sites are annotated as TxT and SxL[T/I], respectively. (C) The consensus sequence of the INpro CRD. The predicted structural features are annotated above the sequence corresponding to the color scheme in (A) and (B).

Figure 3

Representative synchrotron radiation circular dichroism spectra for INpro-9R and INpro-16R. The spectra show beta strand/coil/turn structure features when deconvoluted with the SMP180 reference datasets at the DichroWeb portal. Data are shown for the wavelength range 182–280 nm.

Figure 4

Modelled homodimer structure of the initial 16-repeats of the INpro CRD domain. (A) The proposed homodimer structure of the INpro CRD. The tyrosine ladder comprises the dimerization interface. The monomers are parallel, and the TxT motif of one monomer is aligned with the SxL[T/I] motif of another monomer (blue and orange, respectively). The tyrosine ladder forms the dimerization interface. Approximate dimensions of the dimer surface are indicated. (B) End-view of the dimer model. The longitudinal rotation in the β-helix causes the dimer to form a saddle-like surface.

Figure 5

Two types of INpro oligomers are responsible for the ice-nucleation activity of two INpro classes. (A) Ice nucleation activity of E. coli expressing INpro-67R as well as of purified INpro-67R and INpro-16R proteins measured by the LINA setup. Presumed class A and class C characteristics and structures are indicated for E. coli and Snomax® ice nucleation spectra, but are also seen in INpro-16R spectra. Snomax® data were obtained from previous studies (Wex et al., 2015; Knackstedt et al., 2018). Data from WISDOM ice-nucleation measurements of INpro-16R and INpro-67R shown for comparison. (B) Representative negative stain TEM image of the INpro-67R showing highly oligomerized, filamentous structures. Individual filamentous structures, marked with yellow arrows, seem to assemble in an end-to-end manner into longer, higher order oligomeric assemblies. (C) Representative negative stain TEM image of an individual filamentous structure formed by INpro-67R.