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. 2008 Jul;17(7):1285–1290. doi: 10.1110/ps.034975.108

Cooperativity of complex salt bridges

Anzor G Gvritishvili 1, Alexey V Gribenko 1,3, George I Makhatadze 1,2
PMCID: PMC2442001  PMID: 18469176

Abstract

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 Cα atoms, Θ, is <90°. 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, Θ is close to 160°. Based on these observations, we hypothesized that complex salt bridges are cooperative for Θ < 90° and anti-cooperative for 90° < Θ < 180°. To provide a further experimental test for this hypothesis, we engineered a complex salt bridge with Θ = 150° 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.

Keywords: protein structure/folding, stability and mutagenesis, circular dichroism, forces and stability, thermodynamics, hydrodynamics

Charged residues in globular proteins frequently form salt bridges (Barlow and Thornton 1983). Some of the salt bridges form so-called complex salt bridges, in which one charged residue (anchor residue) forms salt bridges with two or more residues simultaneously (Musafia et al. 1995; Kumar and Nussinov 2002a; Sarakatsannis and Duan 2005). The energetic contribution of complex salt bridges has been suggested to have importance for protein stability (Horovitz et al. 1990; Mayne et al. 1998). Furthermore, the statistical analysis of salt bridges from mesophilic and thermophilic organisms has shown a higher frequency of complex salt bridges in thermophilic proteins, suggesting they have a special role in thermostabilization (Elcock and McCammon 1998; Xiao and Honig 1999; Kumar et al. 2000). A detailed analysis of the net energetics of complex salt bridge formation using double- and triple-mutant cycle analysis revealed interesting and conflicting results. In two cases, it was shown that complex salt bridge formation is cooperative (Horovitz et al. 1990; Mayne et al. 1998), i.e., the net strength of the complex salt bridge is greater than the sum of the energies of the individual pairs. In one case, it was reported that complex salt bridge formation is anti-cooperative (Iqbalsyah and Doig 2005), i.e., the net strength of the complex salt bridge is less than the sum of the energies of the individual pairs.

To resolve these different findings, we performed an analysis of the geometries of salt bridges in a representative nonredundant set of over 1500 protein structures from the Protein Data Base (PDB). In addition to the different statistical parameters describing complex salt bridges reported by others (Barlow and Thornton 1983; Musafia et al. 1995; Kumar and Nussinov 1999, 2002b; Sarakatsannis and Duan 2005), we evaluated the distribution of the angle at the anchor residue, Θ, formed by the Cα atoms of the residues that form the complex salt bridges. We found that a complex salt bridge anchored by Arg/Lys has a preferred geometry that is also observed in the two reported instances when the energetics of complex bridge formation is cooperative. Based on these observations, we hypothesized that the value of Θ defines whether a complex salt bridge is cooperative or anti-cooperative. To provide a further experimental test for this hypothesis, we engineered a complex salt bridge into a model protein, the activation domain of human procarboxypeptidase A2 (ADA2h). The experimentally derived stabilities of the ADA2h variants allowed us to estimate the net stabilities of the individual salt bridges making up the complex bridge and compare those to the net energy of interactions among residues making up the complex salt bridge. In agreement with the hypothesis, we found that the complex salt bridge in ADA2h is anti-cooperative.

Results and Discussion

The criterion for the existence of a salt bridge was the same as originally proposed by Barlow and Thornton (Barlow and Thornton 1983), which is that the distance between the heavy atoms of the ionizable groups of charged residues is <4 Å. We identified 9836 simple salt bridges that contained 3877 lysines (39.4%), 5959 arginines (60.6%), 4635 aspartates (47.1%), and 5201 glutamates (52.9%). Of these salt bridges only 644 (6.5%) were found to be complex salt bridges. We further divided the complex salt bridges depending on the charge of the anchor residue. There were 377 complex salt bridges anchored by Arg/Lys and 267 anchored by Asp/Glu. For all of the complex salt bridges, we calculated the angle at the anchor residue, Θ, formed by the three Cα atoms of the participating residues. Figure 1 shows the diagram of the distribution of the Θ angles. There is a major difference between the geometries of complex salt bridges anchored by Arg/Lys and those anchored by Asp/Glu. For the Asp/Glu anchored salt bridges, all Θ angles are equally populated (the small preference at 40° is not statistically significant): 60% of Θ angles are between 0° and 90° and 40% between 90° and 180°. This is dramatically different than what is observed for the Arg/Lys anchored complex salt bridges, for which >87% of the Θ angles are between 0° and 90° and only 13% between 90° and 180°. Thus, the Arg/Lys anchored complex salt bridges show a clear preference toward the 20° to 90° range for the Θ values.

Figure 1.

Figure 1.

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The distribution of angles between pairs of salt bridges making a complex salt bridge. Black line: anchored by Arg/Lys; gray line: anchored by Asp/Glu.

Interestingly, the two salt bridges that were found to be cooperative—a complex salt bridge in barnase studied by the Fersht group (Horovitz et al. 1990) and a complex salt bridge in a helical peptide studied by the Kallenbach group (Mayne et al. 1998)—have Θ values of 39° and 35°, respectively (see Fig. 2A,B). Moreover, the anti-cooperative salt bridge in a helical peptide studied by the Doig group (Iqbalsyah and Doig 2005) has a Θ value of 164° (see Fig. 2C). It is possible that the difference in the Θ angle for these complex salt bridges defines their cooperativity. To have another example for a complex salt bridge with a Θ value in the infrequent range (90°–180°), we engineered a complex salt bridge by making amino acid substitutions at positions 5, 32, and 42 of the activation domain of human procarboxypeptidase A2 (ADA2h) (Fig. 3). The structure of ADA2h allows the placement of Lys as an anchor residue at position 42 to form salt bridges with the two negatively charged Glu residues placed at positions 5 and 32. The Θ angle for such an arrangement is 150° (Fig. 2D), similar to that of the anti-cooperative salt bridge in the helical peptide studied by Iqbalsyah and Doig (2005).

Figure 2.

Figure 2.

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Cartoon representation of the spatial arrangement of complex salt bridges in (A) barnase, studied by Horovitz et al. (1990), (B) helical peptide, studied by Mayne et al. (1998), (C) helical peptide, studied by Iqbalsyah and Doig (2005), and (D) ADA2h (this work). The values for the Θ angle and distances between Cα carbons are also given.

Figure 3.

Figure 3.

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Cartoon representation of the structure of activation domain of human procarboxypeptidase with the side chains of the amino acid residues in the positions 5, 32, and 42, shown in a CPK representation. The figure was generated using PDB entry 1AYE (García-Saez et al. 1997).

In order to estimate the energetics of interactions between the residues at these three positions, eight separate variants of ADA2h were constructed. These include three variants with single site substitutions, three with double substitutions, and one with a triple substitution, as required by the triple-mutant cube analysis originally proposed by Horovitz and Fersht (1992) and used by others (Marqusee and Sauer 1994; Perl and Schmid 2001). The thermodynamic stability of these variants was determined from temperature-induced unfolding experiments, where changes in the ellipticity at 222 nm were monitored as a function of temperature. The melting profiles were analyzed using a two-state model of unfolding (for details, see the Materials and Methods section), and the results are presented in Table 1. The data allows for the construction of several double-mutant cycles (DMC). The DMC shown in Figure 4A estimates that, when position 32 is neutral, the energy of interactions between the charged residues in positions 42 and 5 is 1.6 kJ/mol. Similarly, the DMC shown in Figure 4C estimates the energy of interactions between charged residues in positions 42 and 32, when position 5 is neutral, to be 0.5 kJ/mol. Therefore, as expected, the salt bridges contribute favorably to the stability of the protein (Serrano et al. 1990; Elcock 1998; Xiao and Honig 1999; Kumar and Nussinov 2002a; Makhatadze et al. 2003). Moreover, these estimates compare well with the DMC energies for salt bridges in other globular proteins (see compilation in Makhatadze et al. 2003).

Table 1.

Thermodynamic parameters characterizing the stabilities of the ADA2h variants

graphic file with name 1285tbl1.jpg

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Figure 4.

Figure 4.

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Analysis of the interaction energies between residues in positions 5, 42, and 32 of ADA2h: double-mutant cycles (A–D) and triple-mutant cube (E). The numbers represent the energy of interactions between corresponding pairs of residues (or triplet in the case of panel E) and are derived from experimental values listed in Table 1 as: ΔΔG +,− = ΔG(+,−) + ΔG(o,o) − ΔG(+,o) − ΔG(+,o) for simple salt bridges and ΔΔΔG −,+,− = ΔG(−,+,−) + ΔG(o,o,−) + ΔG(o,+,o) + ΔG(−,o,o) − ΔG(o,+,−) − ΔG(−,o,−) − ΔG(−,+,o) − ΔG(o,o,o) for the complex salt bridge.

The DMCs shown in Figure 4B and D estimate the energy of interactions between two charged residues (42 and 5 or 42 and 32, respectively) when the residue in the third position (32 or 5, respectively) is also charged. For the 42 and 5 pair this energy is 3.3 kJ/mol, while for the pair of charged residues in positions 42 and 32 it is 2.2 kJ/mol. Thus, adding an ionic interaction to a residue already involved in a salt bridge (i.e., forming complex salt bridge from) has higher energetic contribution than forming a single salt bridge. This enhancement can be rationalized in the following way. The formation of a simple salt bridge “freezes” the side chain of residues involved. As a result, the energy of the simple salt bridge formation in a first approximation will include favorable Coulombic interactions and an unfavorable entropic cost of restricting the rotation of the side chains. However, when a second ionic interaction is added to the existing salt bridge, only approximately half of the entropic term will be lost because the other side chain already lost its conformational entropy when the first salt bridge was formed.

The net energy of interactions in the complex salt bridge was estimated to be 1.7 kJ/mol using a triple-mutant cube as shown in Figure 4E. Again, as in the case of simple salt bridges, the complex salt bridges are favorable for the stability of native proteins, in agreement with previous observations (Serrano et al. 1990; Elcock 1998; Xiao and Honig 1999; Kumar and Nussinov 2002a; Makhatadze et al. 2003). However, the net energy of formation of the complex salt bridge is less than the sum of the energies of the individual simple salt bridges, 1.6 + 0.5 = 2.1 kJ/mol. This suggests that the complex salt bridge formation between residues 5, 42, and 32 in ADA2h is anti-cooperative.

The experimental data presented here, combined with the three data sets published previously, suggest that the cooperativity of a complex salt bridge depends on the geometric arrangement of the participating amino acid residues. This arrangement can be characterized by the angle Θ, which is a two-dimensional angle between the Cα atoms of the residues involved in the formation of the complex salt bridge. If the anchor residue is Arg/Lys, and this angle is <90°, the salt bridge is cooperative, while if the angle is >90°, the respective complex salt bridge will show anti-cooperativity. What is the possible molecular mechanism of this difference in the cooperativity of complex salt bridges? We propose that it is related to the preferential position of the side chains (see Fig. 5). In both cases (i.e., when Θ is small and large), the entropic cost of fixing side chains in a complex salt bridge is largely the same. However, when the Θ angle is <90°, the anchoring residue does not move much upon switching from a simple to a complex salt bridge (Fig. 5). This results in almost equal distances from the anchor residue to each of the oppositely charged residues, and thus, the individual salt bridges will have approximately the same pairwise Coulombic energy of interactions. As a result, the complex salt bridge with Θ < 90° is cooperative.

Figure 5.

Figure 5.

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Cartoon illustrating a possible source of cooperativity in complex salt bridges.

The situation is dramatically different when the Θ angle is >90° (see Fig. 5). The anchoring residue undergoes much larger movement upon switching from a simple to a complex salt bridge. The entropic changes are similar to the case when a complex salt bridge is formed between the residues having Θ < 90°. However, the Coulombic energy of interactions for the complex salt bridge will be lower than the sum of Coulombic energies of interactions for individual simple salt bridges because the average distances between charged residues will be significantly different in each type of salt bridge. This will result in an anti-cooperative complex salt bridge formation.

It is important to note that complex salt bridges anchored by Arg or Lys are more frequently found to have the Θ angle that favors the cooperative complex salt bridge formation (Fig. 1). Our statistical analysis of a representative non-homologous data set of structures also shows that complex salt bridges are not very common in proteins. Nevertheless, the comparative statistical analysis of complex salt bridges in mesophilic and thermophilic proteins shows that the complex salt bridges appear to be used to stabilize the proteins from thermophilic organisms (Kumar and Nussinov 1999, 2004; Xiao and Honig 1999; Kumar et al. 2000; Eijsink et al. 2004).

Finally, the conclusion about the cooperativity of the complex salt bridges is based on, and valid only for, the instances when Arg or Lys residues are anchoring the complex salt bridge. This is the consequence of the geometric properties of these side chains (length and flexibility). For the complex salt bridges anchored by Asp or Glu, the observed cooperativity effect might be very different, because Asp/Glu have shorter and less flexible side chains. Actually, based on the different geometries of Asp/Glu and on the observed distribution of the Θ angle for these residues (Fig. 1), we predict that Asp/Glu anchored salt bridges are unlikely to have any (i.e., positive or negative) cooperativity.

Materials and Methods

Protein cloning, expression, and purification

The coding sequences for the activation domain of human procarboxypeptidase A2 (ADA2h) variants were constructed using a stepwise polymerase chain reaction (PCR). The final PCR products were cloned into the T7-based expression plasmid with an additional N-terminal 6xHis tag (Strickler et al. 2006). All variants contained an additional substitution, M70A, which was included to eliminate possible secondary interactions at the sites of interest (i.e., E5, K32, and H42 in the wild-type sequence). The presence of mutations was confirmed by sequencing the entire gene using an ABI50 genetic analyzer. Expression of protein variants was performed in Escherichia coli BL21 (DE3) or BL21 (DE3)pLys strains at 37°C. Protein expression was induced by adding 1 mM IPTG when the optical density of the cells reached ∼1.2 units at 600 nm. After 4 h, cells were harvested by centrifugation at 4000g for 20 min at 4°C. The cell pellet was resuspended in ∼20 mL of 8 M urea, 0.01 M Tris-HCl, pH 8.0 buffer. Cells were lysed by passing the cell suspension through the French pressure cell three times. Cell lysate was diluted with an equal volume of the resuspension buffer. The cell debris was removed by centrifugation at 14,000g for 40 min at 4°C. Purification of the ADA2h variants was done using a two-step procedure in which protein fractions were first separated on Ni-NTA His-bind resin (Novagen) under denaturing conditions, followed by gel filtration on a Sephadex G-50 column (2.5 × 100 cm) equilibrated with 5% acetic acid. Protein fractions were combined, lyophilized, and stored at −20°C. The purity of the protein variants was assayed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The identity of protein variants was confirmed by determining the molecular mass using MALDI-TOF mass spectroscopy (Voager DE-PRO, Perseptive Biosystems). The concentration of the ADA2h variants was measured spectrophotometrically using a molar extinction coefficient of E 280nm = 6970 o.u./M (Pace et al. 1995). Correlation for light scattering was taken into account as previously described (Winder and Gent 1971).

Circular dichroism (CD) spectroscopy

All CD measurements were done with a JASCO-715 spectropolarimeter. The far-UV CD spectra were obtained in the range 190–260 nm at 10°C. The structural changes upon substitutions for all ADA2h variants were tested by comparing far-UV CD spectra and found to be insignificant by this criterion. A water- jacketed 1-mm cylindrical quartz cuvette was used in all experiments. The temperature was controlled by the external programmable thermostated water bath. The temperature induced unfolding of protein variants was monitored by following the changes in ellipticity at 222 nm at a scan rate of ∼30 deg/h. The concentration of the ADA2h variants was 0.04–0.06 mg/mL in 50 mM sodium phosphate pH 7.5 containing 2 M urea, 1 M sucrose. Reversibility of thermal unfolding transition was verified by comparing the spectra before and after the temperature melts. All experiments were done in duplicate. The unfolding profiles were globally fitted to a two-state transition model using the nonlinear regression software, NLREG, as described previously (Gribenko and Makhatadze 2007).

Statistical analysis of salt bridges in protein structure

Statistical analysis of the frequencies and geometries of salt bridges in PDB was performed on the March 2006 PDB_select release data subset containing 1558 nonredundant proteins structures solved by X-ray crystallography with a resolution better than 2.5 Å (Hobohm and Sander 1994). There were 256,758 total amino acid residues, of which 28,071 were positively charged (12,678 of Arg and 15,393 of Lys) and 32,293 were negatively charged (15,151 Asp and 17,142 of Glu). A list of all PDB filenames with chain IDs as well as the list of identified simple and complex salt bridges is given in the Supplemental material.

Acknowledgments

This work was supported by a grant (MCB-0110396 to G.I.M.) from the National Science Foundation. We thank Katrina Schweiker for useful comments on the manuscript.

Footnotes

Supplemental material: see www.proteinscience.org

Reprint requests to: George Makhatadze, Department of Biology, Rensselaer Polytechnic Institute, CBIS Room 3244A, 110 8th Street, Troy, NY 12180-3590, USA; e-mail: makhag@rpi.edu; fax: (518) 276-2955.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.034975.108.

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