Dioxane contributes to the altered conformation and oligomerization state of a designed engrailed homeodomain variant

Our goal was to compute a stable, full-sequence design of the Drosophila melanogaster engrailed homeodomain. Thermal and chemical denaturation data indicated the design was significantly more stable than was the wild-type protein. The data were also nearly identical to those for a similar, later full-sequence design, which was shown by NMR to adopt the homeodomain fold: a three-helix, globular monomer. However, a 1.65 A crystal structure of the design described here turned out to be of a completely different fold: a four-helix, rodlike tetramer. The crystallization conditions included approximately 25% dioxane, and subsequent experiments by circular dichroism and sedimentation velocity analytical ultracentrifugation indicated that dioxane increases the helicity and oligomerization state of the designed protein. We attribute at least part of the discrepancy between the target fold and the crystal structure to the presence of a high concentration of dioxane.     

A parameterized, continuum electrostatic model for predicting protein pKa values

Recognizing the limits of trying to achieve chemical accuracy for pK(a) calculations with a purely electrostatic model, we include empirical corrections into the Poisson-Boltzmann solver macroscopic electrostatics with atomic detail (Bashford, Biochemistry 1990;29:10219-10225), to improve the reliability and accuracy of the model. The total number of parameters is kept to a minimum to maximize the robustness of the model for compounds outside of the fitting dataset. The parameters are based on: (a) the electrostatic interaction between functional groups close to the titratable site, (b) the electrostatic work required to desolvate the residue, and (c) the site-to-site interactions. These interactions are straightforward to calculate once the electrostatic field has been solved for each residue using the linearized Poisson-Boltzmann equation and are assumed to be linearly related to the intrinsic pK(a). Two hundred and eighty-six residues from 30 proteins are used to determine the empirical parameters, which result in a root mean square error (RMSE) of 0.70 for the entire set. Eight proteins with 46 experimentally known values were excluded from the parameterization to test the model. This test set had a RMSE of 1.08. We show that the parameterized model improves the results over other models, although like other models the error is strongly correlated with the degree to which a residue is buried. The parameters themselves indicate that local effects are most important for determining the pK(a), whereas site-to-site interactions are found to be less significant.     

Analysis of electrostatic coupling throughout the laboratory evolution of a designed retroaldolase

The roles of local interactions in the laboratory evolution of a highly active, computationally designed retroaldolase (RA) are examined. Partial Order Optimum Likelihood (POOL) is used to identify catalytically important amino acid interactions in several RA95 enzyme variants. The series RA95.5, RA95.5–5, RA95.5–8, and RA95.5–8F, representing progress along an evolutionary trajectory with increasing activity, is examined. Computed measures of coupling between charged states of residues show that, as evolution proceeds and higher activities are achieved, electrostatic coupling between the biochemically active amino acids and other residues is increased. In silico residue scanning suggests multiple coupling partners for the catalytic lysine K83. The effects of two predicted partners, Y51 and E85, are tested using site‐directed mutagenesis and kinetic analysis of the variants Y51F and E85Q. The Y51F variants show decreases in k _cat relative to wild type, with the greatest losses observed for the more evolved constructs; they also exhibit significant decreases in k _cat/K _M across the series. Only modest decreases in k _cat/K _M are observed for the E85Q variants with little effect on k _cat. Computed metrics of the degree of coupling between protonation states rise significantly as evolution proceeds and catalytic turnover rate increases. Specifically, the charge state of the catalytic lysine K83 becomes more strongly coupled to those of other amino acids as the enzyme evolves to a better catalyst.     

Electrostatics significantly affect the stability of designed homeodomain variants

The role of electrostatic interactions in determining the stability of designed proteins was studied by constructing and analyzing a set of designed variants of the Drosophila engrailed homeodomain. Computational redesign of 29 surface positions results in a 25-fold mutant with moderate stability, similar to the wild-type protein. Incorporating helix dipole and N-capping considerations into the design algorithm by restricting amino acid composition at the helix termini and N-capping positions yields a ninefold mutant of the initial design (a 23-fold mutant of wild-type) that is over 3 kcal mol(-1) more stable than the protein resulting from the unbiased design. Four additional proteins were constructed and analyzed to isolate the effects of helix dipole and N-capping interactions in each helix. Based on the results of urea-denaturation experiments and calculations using the finite difference Poisson-Boltzmann method, both classes of interaction are found to increase the stability of the designed proteins significantly. The simple electrostatic model used in the optimization of rotamers by iterative techniques (ORBIT) force-field, which is similar to the electrostatic models used in other protein design force-fields, is unable to predict the experimentally determined stabilities of the designed variants. The helix dipole and N-capping restrictions provide a simple but effective method to incorporate two types of electrostatic interactions that impact protein stability significantly.     

Computation of electrostatic complements to proteins: a case of charge stabilized binding.

Recent evidence suggests that the net effect of electrostatics is generally to destabilize protein binding due to large desolvation penalties. A novel method for computing ligand-charge distributions that optimize the tradeoff between ligand desolvation penalty and favorable interactions with a binding site has been applied to a model for barnase. The result is a ligand-charge distribution with a favorable electrostatic contribution to binding due, in part, to ligand point charges whose direct interaction with the binding site is unfavorable, but which make strong intra-molecular interactions that are uncloaked on binding and thus act to lessen the ligand desolvation penalty.     

Thermodynamics of a designed protein catenane

Topological linking of proteins is a new approach for stabilizing and controlling the oligomerization state of proteins that fold in an interwined manner. The recent design of a backbone cyclized protein catenane based on the p53tet domain suggested that topological cross-linking provided increased stability against thermal and chemical denaturation. However, the tetrameric structure complicated detailed biophysical analysis of this protein. Here, we describe the design, synthesis and thermodynamic characterization of a protein catenane based on a dimeric mutant of the p53tet domain (M340E/L344K). The formation of the catenane proceeded efficiently, and the overall structure and oligomerization of the domain was not affected by the formation of the topological link. Unfolding and refolding of the catenane was consistent with a two-state process. The topological link stabilized the dimer against thermal and chemical denaturation considerably, raising the apparent melting temperature by 59 degrees C and the midpoint of denaturation by 4.5M GuHCl at a concentration of 50 microM. The formation of the topological link increased the resistance of the dimer to proteolysis. However, the m value decreased by 1.7kcalmol(-1)M(-1), suggesting a decrease in accessible surface area in the unfolded state. This implies that the stabilization from the topological link is largely due to a destabilization of the unfolded state, similar to other cross-links in proteins. Topological linking therefore provides a powerful and orthogonal tool for the stabilization of peptide and protein oligomers.     

Effects of salt bridges on protein structure and design.

Theoretical calculations (Hendsch ZS & Tidor B, 1994, Protein Sci 3:211-226) and experiments (Waldburger CD et al., 1995, Nat Struct Biol 2:122-128; Wimley WC et al., 1996, Proc Natl Acad Sci USA 93:2985-2990) suggest that hydrophobic interactions are more stabilizing than salt bridges in protein folding. The lack of apparent stability benefit for many salt bridges requires an alternative explanation for their occurrence within proteins. To examine the effect of salt bridges on protein structure and stability in more detail, we have developed an energy function for simple cubic lattice polymers based on continuum electrostatic calculations of a representative selection of salt bridges found in known protein crystal structures. There are only three types of residues in the model, with charges of -1, 0, or + 1. We have exhaustively enumerated conformational space and significant regions of sequence space for three-dimensional cubic lattice polymers of length 16. The results demonstrate that, while the more highly charged sequences are less stable, the loss of stability is accompanied by a substantial reduction in the degeneracy of the lowest-energy state. Moreover, the reduction in degeneracy is greater due to charges that pair than for lone charges that remain relatively exposed to solvent. We have also explored and illustrated the use of ion-pairing strategies for rational structural design using model lattice studies.     

An orientation-dependent hydrogen bonding potential improves prediction of specificity and structure for proteins and protein-protein complexes

Hydrogen bonding is a key contributor to the specificity of intramolecular and intermolecular interactions in biological systems. Here, we develop an orientation-dependent hydrogen bonding potential based on the geometric characteristics of hydrogen bonds in high-resolution protein crystal structures, and evaluate it using four tests related to the prediction and design of protein structures and protein-protein complexes. The new potential is superior to the widely used Coulomb model of hydrogen bonding in prediction of the sequences of proteins and protein-protein interfaces from their structures, and improves discrimination of correctly docked protein-protein complexes from large sets of alternative structures.     

A de novo designed protein protein interface

As an approach to both explore the physical/chemical parameters that drive molecular self-assembly and to generate novel protein oligomers, we have developed a procedure to generate protein dimers from monomeric proteins using computational protein docking and amino acid sequence design. A fast Fourier transform-based docking algorithm was used to generate a model for a dimeric version of the 56-amino-acid beta1 domain of streptococcal protein G. Computational amino acid sequence design of 24 residues at the dimer interface resulted in a heterodimer comprised of 12-fold and eightfold variants of the wild-type protein. The designed proteins were expressed, purified, and characterized using analytical ultracentrifugation and heteronuclear NMR techniques. Although the measured dissociation constant was modest ( approximately 300 microM), 2D-[(1)H,(15)N]-HSQC NMR spectra of one of the designed proteins in the absence and presence of its binding partner showed clear evidence of specific dimer formation.     

Folding of a designed simple ankyrin repeat protein

Ankyrin repeats (AR) are 33-residue motifs containing a beta-turn, followed by two alpha-helices connected by a loop. AR occur in tandem arrangements and stack side-by-side to form elongated domains involved in very different cellular tasks. Recently, consensus libraries of AR repeats were constructed. Protein E1_5 represents a member of the shortest library, and consists of only a single consensus repeat flanked by designed N- and C-terminal capping repeats. Here we present a biophysical characterization of this AR domain. The protein is compactly folded, as judged from the heat capacity of the native state and from the specific unfolding enthalpy and entropy. From spectroscopic data, thermal and urea-induced unfolding can be modeled by a two-state transition. However, scanning calorimetry experiments reveal a deviation from the two-state behavior at elevated temperatures. Folding and unfolding at 5 degrees C both follow monoexponential kinetics with k(folding) = 28 sec(-1) and k(unfolding) = 0.9 sec(-1). Kinetic and equilibrium unfolding parameters at 5 degrees C agree very well. We conclude that E1_5 folds in a simple two-state manner at low temperatures while equilibrium intermediates become populated at higher temperatures. A chevron-plot analysis indicates that the protein traverses a very compact transition state along the folding/unfolding pathway. This work demonstrates that a designed minimal ankyrin repeat protein has the thermodynamic and kinetic properties of a compactly folded protein, and explains the favorable properties of the consensus framework.     

Action-at-a-distance interactions enhance protein binding affinity

The identification of protein mutations that enhance binding affinity may be achieved by computational or experimental means, or by a combination of the two. Sources of affinity enhancement may include improvements to the net balance of binding interactions of residues forming intermolecular contacts at the binding interface, such as packing and hydrogen-bonding interactions. Here we identify noncontacting residues that make substantial contributions to binding affinity and that also provide opportunities for mutations that increase binding affinity of the TEM1 beta-lactamase (TEM1) to the beta-lactamase inhibitor protein (BLIP). A region of BLIP not on the direct TEM1-binding surface was identified for which changes in net charge result in particularly large increases in computed binding affinity. Some mutations to the region have previously been characterized, and our results are in good correspondence with this results of that study. In addition, we propose novel mutations to BLIP that were computed to improve binding significantly without contacting TEM1 directly. This class of noncontacting electrostatic interactions could have general utility in the design and tuning of binding interactions.     

One- and two-body decomposable Poisson-Boltzmann methods for protein design calculations

Successfully modeling electrostatic interactions is one of the key factors required for the computational design of proteins with desired physical, chemical, and biological properties. In this paper, we present formulations of the finite difference Poisson-Boltzmann (FDPB) model that are pairwise decomposable by side chain. These methods use reduced representations of the protein structure based on the backbone and one or two side chains in order to approximate the dielectric environment in and around the protein. For the desolvation of polar side chains, the two-body model has a 0.64 kcal/mol RMSD compared to FDPB calculations performed using the full representation of the protein structure. Screened Coulombic interaction energies between side chains are approximated with an RMSD of 0.13 kcal/mol. The methods presented here are compatible with the computational demands of protein design calculations and produce energies that are very similar to the results of traditional FDPB calculations.     

Calculation of the total electrostatic energy of a macromolecular system: solvation energies, binding energies, and conformational analysis

In this report we describe an accurate numerical method for calculating the total electrostatic energy of molecules of arbitrary shape and charge distribution, accounting for both Coulombic and solvent polarization terms. In addition to the solvation energies of individual molecules, the method can be used to calculate the electrostatic energy associated with conformational changes in proteins as well as changes in solvation energy that accompany the binding of charged substrates. The validity of the method is examined by calculating the hydration energies of acetate, methyl ammonium, ammonium, and methanol. The method is then used to study the relationship between the depth of a charge within a protein and its interaction with the solvent. Calculations of the relative electrostatic energies of crystal and misfolded conformations of Themiste dyscritum hemerythrin and the VL domain of an antibody are also presented. The results indicate that electrostatic charge-solvent interactions strongly favor the crystal structures. More generally, it is found that charge-solvent interactions, which are frequently neglected in protein structure analysis, can make large contributions to the total energy of a macromolecular system.     

Src kinase activation: A switched electrostatic network

Src tyrosine kinases are essential in numerous cell signaling pathways, and improper functioning of these enzymes has been implicated in many diseases. The activity of Src kinases is regulated by conformational activation, which involves several structural changes within the catalytic domain (CD): the orientation of two lobes of CD; rearrangement of the activation loop (A-loop); and movement of an alpha-helix (alphaC), which is located at the interface between the two lobes, into or away from the catalytic cleft. Conformational activation was investigated using biased molecular dynamics to explore the transition pathway between the active and the down-regulated conformation of CD for the Src-kinase family member Lyn kinase, and to gain insight into the interdependence of these changes. Lobe opening is observed to be a facile motion, whereas movement of the A-loop motion is more complex requiring secondary structure changes as well as communication with alphaC. A key result is that the conformational transition involves a switch in an electrostatic network of six polar residues between the active and the down-regulated conformations. The exchange between interactions links the three main motions of the CD. Kinetic experiments that would demonstrate the contribution of the switched electrostatic network to the enzyme mechanism are proposed. Possible implications for regulation conferred by interdomain interactions are also discussed.     

Influence of electrostatic forces on the association kinetics and conformational ensemble of an intrinsically disordered protein

Recent work has revealed that the association of a disordered region of a protein with a folded binding partner can occur as rapidly as association between two folded proteins. This is the case for the phosphatase calcineurin (CaN) and its association with its activator calmodulin. Calmodulin binds to the intrinsically disordered regulatory domain of CaN. Previous studies have shown that electrostatic steering can accelerate the binding of folded proteins with disordered ligands. Given that electrostatic forces are strong determinants of disordered protein ensembles, the relationship between electrostatics, conformational ensembles, and quaternary interactions is unclear. Here, we employ experimental approaches to explore the impact of electrostatic interactions on the association of calmodulin with the disordered regulatory region of CaN. We find that estimated association rate constants of calmodulin with our chosen calmodulin-substrates are within the diffusion-limited regime. The association rates are dependent on the ionic strength, indicating that favorable electrostatic forces increase the rate of association. Further, we show that charged amino acids outside the calmodulin-binding site modulate the binding rate. Conformational ensembles obtained from computer simulations suggest that electrostatic interactions within the regulatory domain might bias the conformational ensemble such that the calmodulin binding region is readily accessible. Given the prevalence of charged residues in disordered protein chains, our findings are likely relevant to many protein-protein interactions.     

Increased detection of structural templates using alignments of designed sequences

Protein structure prediction by comparative modeling benefits greatly from the use of multiple sequence alignment information to improve the accuracy of structural template identification and the alignment of target sequences to structural templates. Unfortunately, this benefit is limited to those protein sequences for which at least several natural sequence homologues exist. We show here that the use of large diverse alignments of computationally designed protein sequences confers many of the same benefits as natural sequences in identifying structural templates for comparative modeling targets. A large-scale massively parallelized application of an all-atom protein design algorithm, including a simple model of peptide backbone flexibility, has allowed us to generate 500 diverse, non-native, high-quality sequences for each of 264 protein structures in our test set. PSI-BLAST searches using the sequence profiles generated from the designed sequences ("reverse" BLAST searches) give near-perfect accuracy in identifying true structural homologues of the parent structure, with 54% coverage. In 41 of 49 genomes scanned using reverse BLAST searches, at least one novel structural template (not found by the standard method of PSI-BLAST against PDB) is identified. Further improvements in coverage, through optimizing the scoring function used to design sequences and continued application to new protein structures beyond the test set, will allow this method to mature into a useful strategy for identifying distantly related structural templates.     

Spatial optimization of electrostatic interactions between the ionized groups in globular proteins

A model approach is suggested to estimate the degree of spatial optimization of the electrostatic interactions in protein molecules. The method is tested on a set of 44 globular proteins, representative of the available crystallographic data. The theoretical model is based on macroscopic computation of the contribution of charge–charge interactions to the electrostatic term of the free energy for the native proteins and for a big number of virtual structures with randomly distributed on protein surface charge consetellations (generated by a Monte-Carlo technique). The statistical probability of occurrence of random structures with electrostatic energies lower than the energy of the native protein is suggested as a criterion for spatial optimization of the electrostatic interactions. The results support the hypothesis that the folding process optimizes the stabilizing effect of electrostatic interactions, but to very different degree for different proteins. A parallel analysis of ion pairs shows that the optimization of the electrostatic term in globular proteins has increasingly gone in the direction of rejecting the repulsive short contacts between charges of equal sign than of creating of more salt bridges (in comparison with the statistically expected number of shortrange ion pairs in the simulated random structures). It is observed that the decrease in the spatial optimization of the electrostatic interactions is usually compensated for by an appearance of disulfide bridges in the covalent structure of the examined proteins. © 1994 Wiley-Liss, Inc.     

Molecular dynamics study of the stabilities of consensus designed ankyrin repeat proteins

Two designed ankyrin repeat (AR) proteins (E3_5 and E3_19) are high homologous (with about 87% sequence identity) and their crystal structures have a Calpha atom-positional root-mean-square difference of about 0.14 nm. However, it was found that E3_5 is considerably more stable than E3_19 in guanidinium hydrochloride and thermal denaturation experiments. With the goal of providing insights into the various factors contributing to the stabilities of the designed AR proteins and suggesting possible mutations to enhance their stabilities, homology modeling and molecular dynamics (MD) simulations with explicit solvent have been performed. Because the crystal structure of E3_19 was solved later than that of E3_5, a homology model of E3_19 based on the crystal structure of E3_5 was also used in the simulations. E3_5 shows a very stable trajectory in both crystal and solution simulations. In contrast, the C-terminal repeat of E3_19 unfolds in the simulations starting from either the modeled structure or the crystal structure, although it has a sequence identical to that of E3_5. A continuum electrostatic model was used to estimate the effect of single mutations on protein stability and to study the interaction between the internal ARs and the C-terminal capping AR. Mutations involving charged residues were found to have large effects on stability. Due to the difference in charge distribution in the internal ARs of E3_19 and E3_5, their interaction with the C-terminal capping AR is less favorable in E3_19. The simulation trajectories suggest that the stability of the designed AR proteins can be increased by optimizing the electrostatic interactions within and between the different repeats.