Cooperativity of complex salt bridges

The energetic contribution of complex salt bridges, in which one charged residue (anchor residue) forms salt bridges with two or more residues simultaneously, has been suggested to have importance for protein stability. Detailed analysis of the net energetics of complex salt bridge formation using double- and triple-mutant cycle analysis revealed conflicting results. In two cases, it was shown that complex salt bridge formation is cooperative, i.e., the net strength of the complex salt bridge is more than the sum of the energies of individual pairs. In one case, it was reported that complex salt bridge formation is anti-cooperative. To resolve these different findings, we performed analysis of the geometries of salt bridges in a representative set of structures from the PDB and found that over 87% of all complex salt bridges anchored by Arg/Lys have a geometry such that the angle formed by their Calpha atoms, Theta, is <90 degrees . This preferred geometry is observed in the two reported instances when the energetics of complex salt bridge formation is cooperative, while in the reported anti-cooperative complex salt bridge, Theta is close to 160 degrees . Based on these observations, we hypothesized that complex salt bridges are cooperative for Theta < 90 degrees and anti-cooperative for 90 degrees < Theta < 180 degrees . To provide a further experimental test for this hypothesis, we engineered a complex salt bridge with Theta = 150 degrees into a model protein, the activation domain of human procarboxypeptidase A2 (ADA2h). Experimentally derived stabilities of the ADA2h variants allowed us to show that the complex salt bridge in ADA2h is anti-cooperative.     

Statistical characterization of salt bridges in proteins

The structure and folding mechanism of a given protein are determined by many factors, including the electrostatic interactions between charged residues of protein molecules known in general as salt bridges. In this study, analyses were conducted on 10,370 salt bridges in 2017 proteins and the results compared to previous statistical surveys of 36 protein structures. Although many of the general trends remained consistent with other studies, more detailed information was illuminated by the larger dataset. In particular, it was shown that there is a strong correlation between secondary structure and salt bridge formation, and that salt bridges display preferential formation in an environment of about 30% solvent accessible surface area.     

Contribution of surface salt bridges to protein stability

The role of surface salt bridges in protein stabilization has been a source of controversy. Here we present the NMR structure of a hyperthermophilic rubredoxin variant (PFRD-XC4) and the thermodynamic analysis of two surface salt bridges by double mutant cycles. This analysis shows that the surface side chain to side chain salt bridge between Lys 6 and Glu 49 does not stabilize PFRD-XC4. The main chain to side chain salt bridge between the N-terminus and Glu 14 was, however, found to stabilize PFRD-XC4 by 1. 5 kcal mol(-)(1). The entropic cost of making a surface salt bridge involving the protein's backbone is reduced, since the backbone has already been immobilized upon protein folding.     

Cooperative helix stabilization by complex Arg-Glu salt bridges

Among the interactions that stabilize the native state of proteins, the role of electrostatic interactions has been difficult to quantify precisely. Surface salt bridges or ion pairs between acidic and basic side chains have only a modest stabilizing effect on the stability of helical peptides or proteins: estimates are roughly 0.5 kcal/mol or less. On the other hand, theoretical arguments and the occurrence of salt bridge networks in thermophilic proteins suggest that multiple salt bridges may exert a stronger stabilizing effect. We show here that triads of charged side chains, Arg(+)-Glu(-)-Arg(+) spaced at i,i+4 or i,i+3 intervals in a helical peptide stabilize alpha helix by more than the additive contribution of two single salt bridges. The free energy of the triad is more than 1 kcal/mol in excess of the sum of the individual pairs, measured in low salt concentration (10 mM). The effect of spacing the three groups is severe; placing the charges at i,i+4 or i,i+3 sites has a strong effect on stability relative to single bridges; other combinations are weaker. A conservative calculation suggests that interactions of this kind between salt bridges can account for much of the stabilization of certain thermophilic proteins.     

Contribution of arginine-glutamate salt bridges to helix stability

Peptide side chain interactions were studied by molecular dynamics simulation using explicit solvent on a peptide with the sequence AAARAAAAEAAEAAAARA. Three different protonation states of the glutamic acid side chains were simulated for four 20 ns runs each, a total simulation time of 240 ns. Two different salt bridge geometries were observed and the preferred geometry was found to depend on Glu — Arg residue spacing. Stable charge clusters were also observed, particularly in the fully charged peptide. Salt bridges were selectively interrupted upon protonation, with concomitant changes in secondary structure. The fully charged peptide was highly helical between residues 9 and 13, although protonation increased helicity near the N-terminus. The contribution of salt bridges to helix stability therefore depends on both position and relative position of charged residues within a sequence.     

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.     

Surface salt bridges stabilize the GCN4 leucine zipper.

We present a study of the role of salt bridges in stabilizing a simplified tertiary structural motif, the coiled-coil. Changes in GCN4 sequence have been engineered that introduce trial patterns of single and multiple salt bridges at solvent exposed sites. At the same sites, a set of alanine mutants was generated to provide a reference for thermodynamic analysis of the salt bridges. Introduction of three alanines stabilizes the dimer by 1.1 kcal/mol relative to the wild-type. An arrangement corresponding to a complex type of salt bridge involving three groups stabilizes the dimer by 1.7 kcal/ mol, an apparent elevation of the melting temperature relative to wild type of about 22 degrees C. While identifying local from nonlocal contributions to protein stability is difficult, stabilizing interactions can be identified by use of cycles. Introduction of alanines for side chains of lower helix propensity and complex salt bridges both stabilize the coiled-coil, so that combining the two should yield melting temperatures substantially higher than the starting species, approaching those of thermophilic sequences.     

Composition-dependent energetic contribution of complex salt bridges to collagen stability

Complex salt bridges, on which three or more charged residues interplay simultaneously, cannot be considered as addition of individual salt bridges. This is still an intriguing problem in protein folding and stability. Here, we used an obligated ABC-type collagen heterotrimer as a platform to study the relationship between energetic contributions and conformational details of three-body complex salt bridges anchored by positively charged residues, K and R. Eight complex salt bridges were constructed by engineering point mutations in the heterotrimer. The circular dichroism measurements showed that the K-anchored complex salt bridges were stronger than the R-anchored ones. The molecular dynamics simulation revealed that both types of salt bridges had distinct dynamic features. The energetic contribution of K-anchored salt bridges was mainly determined by strong single bridges. In the R-anchored complex salt bridges, both side-chain electrostatic interactions and side-chain-backbone hydrogen bonding were involved. An empirical equation was proposed to predict the energetic contributions with high accuracy (R² = 0.93). This work could help us take insights into the mechanisms of composition-dependent behaviors of the complex salt bridges on protein surface.     

Strength and co-operativity of contributions of surface salt bridges to protein stability

Many of the interactions that stabilize proteins are co-operative and cannot be reduced to a sum of pairwise interactions. Such interactions may be analysed by protein engineering methods using multiple thermodynamic cycles comprising wild-type protein and all combinations of mutants in the interacting residues. There is a triad of charged residues on the surface of barnase, comprising residues Asp8, Asp12 and Arg110, that interact by forming two exposed salt bridges. The three residues have been mutated to alanine to give all the single, double and triple mutants. The free energies of unfolding of wild-type and the seven mutant proteins have been determined and the results analysed to give the contributions of the residues in the two salt bridges to protein stability. It is possible to isolate the energies of forming the salt bridges relative to the solvation of the separated ions by water. In the intact triad, the apparent contribution to the stabilization energy of the protein of the salt bridge between Asp12 and Arg110 is -1.25 kcal mol-1, whereas that of the salt bridge between Asp8 with Arg110 is -0.98 kcal mol-1. The strengths of the two salt bridges are coupled: the energy of each is reduced by 0.77 kcal mol-1 when the other is absent. The salt-linked triad, relative to alanine residues at the same positions, does not contribute to the stability of the protein since the favourable interactions of the salt bridges are more than offset by other electrostatic and non-electrostatic energy terms. Salt-linked triads occur in other proteins, for example, haemoglobin, where the energy of only the salt-bridge term is important and so the coupling of salt bridges could be of general importance to the stability and function of proteins.     

Electrostatic strengths of salt bridges in thermophilic and mesophilic glutamate dehydrogenase monomers

Here we seek to understand the higher frequency of occurrence of salt bridges in proteins from thermophiles as compared to their mesophile homologs. We focus on glutamate dehydrogenase, owing to the availability of high resolution thermophilic (from Pyrococcus furiosus) and mesophilic (from Clostridium symbiosum) protein structures, the large protein size and the large difference in melting temperatures. We investigate the location, statistics and electrostatic strengths of salt bridges and of their networks within corresponding monomers of the thermophilic and mesophilic enzymes. We find that many of the extra salt bridges which are present in the thermophilic glutamate dehydrogenase monomer but absent in the mesophilic enzyme, form around the active site of the protein. Furthermore, salt bridges in the thermostable glutamate dehydrogenase cluster within the hydrophobic folding units of the monomer, rather than between them. Computation of the electrostatic contribution of salt bridge energies by solving the Poisson equation in a continuum solvent medium, shows that the salt bridges in Pyrococcus furiosus glutamate dehydrogenase are highly stabilizing. In contrast, the salt bridges in the mesophilic Clostridium symbiosum glutamate dehydrogenase are only marginally stabilizing. This is largely the outcome of the difference in the protein environment around the salt bridges in the two proteins. The presence of a larger number of charges, and hence, of salt bridges contributes to an electrostatically more favorable protein energy term. Our results indicate that salt bridges and their networks may have an important role in resisting deformation/unfolding of the protein structure at high temperatures, particularly in critical regions such as around the active site.     

Effects of engineered salt bridges on the stability of subtilisin BPN'

Variants designed using PROTEUS have been produced in an attempt to engineer stabilizing salt bridges into subtilisin BPN'. All the mutants constructed by site-directed mutagenesis were secreted by Bacillus subtilis, except L75K. Q19E, expressed as a single variant and also in a double variant, Q19E/Q271E, appears to form a stabilizing salt bridge based on X-ray crystal structure determination and differential scanning calorimeter measurements. Although the double mutant was found to be less thermodynamically stable than the wild-type, it did exhibit an autolytic stability about two-fold greater under hydrophobic conditions. Four variants, A98K, S89E, V26R and L235R, were found to be nearly identical to wild-type in thermal stability, indicative of stable structures without evidence of salt bridge formation. Variants Q271E, V51K and T164R led to structures that resulted in varying degrees of thermodynamic and autolytic instability. A computer-modeling analysis of the PROTEUS predictions reveals that the low percentage of salt bridge formation is probably due to an overly simplistic electrostatic model, which does not account for the geometry of the pairwise interactions.     

Role of Bohr group salt bridges in cooperativity in hemoglobin.

Possible problems in measuring the first Adair constant, K1, from accurate oxygen equilibrium curves have been investigated. Of these only the presence of small amounts of CO-hemoglobin or hemoglobin dimers had a significant effect. The former can be eliminated by treatment with oxygen, the latter by measuring the concentration-dependence of K1 or working at high protein concentrations. K1 values have been measured for normal hemoglobin at pH 7 and 9, hemoglobin specifically reacted with cyanate at Val 1alpha (alphac2beta2) and des(His 146beta) hemoglobin at pH 7. K1 is equal to KT, the oxygen affinity of the T state of hemoglobin, for all these hemoglobins and was increased in all of them when compared to normal hemoglobin at pH 7. This shows that the breakage of the Bohr group salt bridges by increasing pH or specific modification changes KT. Hence the Bohr group salt bridges break on ligation of the T state and are partially responsible for the free energy of cooperativity.     

ESBRI: A web server for evaluating salt bridges in proteins

Salt bridges can play important roles in protein structure and function and have stabilizing and destabilizing effects in protein folding. ESBRI is a software available as web tool which analyses the salt bridges in a protein structure, starting from the atomic coordinates. In the case of protein complexes, the salt bridges between protein chains can be evaluated, as well as those among specific charged amino acids and the different protein subunits, in order to obtain useful information regard the protein-protein interaction.     

Role of salt bridges in homeodomains investigated by structural analyses and molecular dynamics simulations

Homeodomains are a class of helix-turn-helix DNA-binding protein motifs that play an important role in the control of cellular development in eukaryotes. They fold in a three alpha-helix structural module, where the third helix is the recognition helix that fits into the major groove of DNA. Structural analysis of the members of the homeodomain family led to the identification of interactions likely to stabilize the protein domains. Linking the helices pairwise, three salt bridges were found to be well preserved within the family. Also well conserved were two cation-pi interactions between aromatic and positively charged side chains. To analyze the structural role of the salt bridges, molecular dynamics simulations (MD) were carried out on the wild-type homeodomain from the Drosophila paired protein (1fjl) and on three mutants, which lack one or two salt bridges and mimic natural mutations in other homeodomains. Analysis of the trajectories revealed only small structural rearrangements of the three helices in all MD simulations, thereby suggesting that the salt bridges have no essential stabilizing role at room temperature, but rather might be important for improving thermostability. The latter hypothesis is supported by a good correlation between the melting midpoint temperatures of several homeodomains and the number of salt bridges and cation-pi interactions that connect secondary structures.     

Formation of salt bridges mediates internal dimerization of myosin VI medial tail domain

The unconventional motor protein, myosin VI, is known to dimerize upon cargo binding to its C-terminal end. It has been shown that one of its tail domains, called the medial tail domain, is a dimerization region. The domain contains an unusual pattern of alternating charged residues and a few hydrophobic residues. To reveal the unknown dimerization mechanism of the medial tail domain, we employed molecular dynamics and single-molecule experimental techniques. Both techniques suggest that the formation of electrostatic-based interhelical salt bridges between oppositely charged residues is a key dimerization factor. For the dimerization to occur, the two identical helices within the dimer do not bind in a symmetric fashion, but rather with an offset of about one helical repeat. Calculations of the dimer-dissociation energy find the contribution of hydrophobic residues to the dimerization process to be minor; they also find that the asymmetric homodimer state is energetically favorable over a state of separate helices.     

Oligomerization of an archaeal group II chaperonin is mediated by N-terminal salt bridges

Group II chaperonins (Cpns) are essential mediators of cellular protein folding in eukaryotes and archaea. They consist of two back-to-back rings forming symmetrical cavities in which non-native substrates undergo appropriate folding, but the primary structural basis for the double ring formation remains unclear. To address this, we carried out systematic mutagenesis on the Cpn from the hyperthermophilic archaeon Pyrococcus furiosus, which is assembled from identical subunits. In our study, ²¹GRDAQRMNIL³⁰ was found to be a critical domain for double ring formation. Deletion of this section stepwise beyond residue 20 resulted in failure to assemble double-ring oligomers and the progressive loss of chaperone function. A key domain spanning the residues 21–50 that is essential for the formation of tetramers that appear to be the intermediates for double ring assembly. Mutation of either Arg22 to Ala22 or Glu37 to Ala37 resulted in similar defects in double-ring assembly and functional deficits. A mutant with Arg22 and Glu37 switched assembled double rings efficiently and exhibited chaperone functions similar to the wild-type. Therefore, Arg22 and Glu37 could form inter-ring salt bridges critical for double ring formation. In addition, Asn28 and Ile29 were found to contribute significantly to ring formation. Sequence alignment revealed that these four residues are highly conserved among group II Cpns. This is the first report of a comprehensive N-terminal mutational analysis for elucidating the oligomerization of group II Cpns.