Quality matters: extension of clusters of residues with good hydrophobic contacts stabilize (hyper)thermophilic proteins

Abstract

Identifying determinant(s) of protein thermostability is key for rational and data-driven protein engineering. By analyzing more than 130 pairs of mesophilic/(hyper)thermophilic proteins, we identified the quality (residue-wise energy) of hydrophobic interactions as a key factor for protein thermostability. This distinguishes our study from previous ones that investigated predominantly structural determinants. Considering this key factor, we successfully discriminated between pairs of mesophilic/(hyper)thermophilic proteins (discrimination accuracy: ∼80%) and searched for structural weak spots in E. coli dihydrofolate reductase (classification accuracy: 70%).

Figure 1

PDFs obtained by kernel density estimation of residue-wise energy components: electrostatic energy (a and b), van der Waals energy (c and d), hydrogen bond energy (e and f), and hydrophobic interaction energy (g and h) for pairs of mesophilic/thermophilic (a, c, e, g), as well as mesophilic/hyperthermophilic (b, d, f, h) protomers. A normal kernel function with an optimal smoothing parameter at each data point was used for calculating the PDFs. The residue-wise energy values were trimmed to exclude values <1 percentile and >99 percentile. The statistical significance of the difference of two PDFs was calculated by a bootstrap hypothesis test of equality generating 10000 bootstrap samples as implemented in the “sm” package of the R program (http://www.r-project.org). Δ̃_E indicates the difference between median residue-wise energies for (hyper)thermophilic and mesophilic protomers calculated from the kernel estimates.

Figure 2

Discriminating mesophilic and (hyper)thermophilic proteins based on clusters of residues with good residue-wise energy components. Residues are clustered together if they are neighbors and if their values of the residue-wise energy components are below a cutoff E_C (largest clusters for selected E_C values are shown in the structures on the top as blue sticks). Residues are considered neighbors if the distance between the closest pair of atoms is less than or equal to 4 Å. E_C is increased in a stepwise manner, and the clustering is repeated. As a result, a hierarchical clustering is obtained where clusters become larger as E_C increases. For each E_C value, the fraction of residues that is part of the largest cluster with respect to all protein residues (F_LC) is calculated. As a descriptor for the discrimination, the area between the respective E_C vs F_LC curves for the (hyper)thermophilic and mesophilic proteins (black stripes) is then determined for the range of F_LC ∈ [0.2, 0.6] (gray shading). If this value is negative, clusters of equal relative size have better residue-wise energy components in the case of the (hyper)thermophilic protein than in the case of the mesophilic protein. Preliminary tests showed that using other ranges of F_LC values for determining the area between the E_C vs F_LC curves does not result in significantly different discrimination accuracies than the best discrimination accuracies obtained with F_LC ∈ [0.2, 0.6].

Figure 3

Discrimination accuracy between mesophilic and (hyper)thermophilic protomers based on clusters of residues with good residue-wise energy components. Lines connecting two bars indicate if the difference in discrimination accuracies for the two respective energy components is statistically significant. Marks at the bottom of a column indicate if the discrimination accuracy is significantly different from a random discrimination (50%). The statistical significance of the difference in discrimination accuracies is computed in both cases by a bootstrap hypothesis test of equality generating 10000 bootstrap samples. The significance levels are marked by ***: p < 0.001; **: p < 0.01; and ns: p > 0.05.

Figure 4

Predicted weak spots mapped onto the structure of E. coli DHFR. Residues are colored by a rainbow color ramp according to their hydrophobic interaction energies. The largest cluster with F_LC = 0.5 observed at a cutoff of the hydrophobic interaction energy E_C = −9.5 kcal mol^–1 is enclosed by a transparent surface. C_α atoms of weak spot residues are represented as spheres. Weak spots that have been validated in the literature are marked by a large sphere.