Similarly to most domain and dimer interfaces, non-polar contacts play a principal role in the association and stabilization of the N- and C-domains of GST and CLIC proteins (section 3.1.1 and Appendix, Table A). Hydrophobic interactions are nonspecific thus allowing sequence variation. Therefore, a number of different hydrophobic residues are found at non-polar hot-spot. On the other hand, polar charged contacts are more specific and primarily one type of residue is found at polar hot-spots. The proposed domain-addition evolutionary pathway of GST proteins (Ladner et al., 2004) provides an insight into why the N-domain was found to be
approximately 70 % more conserved than the C-domain. The Ladner study suggested that GST proteins diverged from a single-domain protein with a thioredoxin-like fold whose role was to recognize glutathione (GSH) and catalyze the addition of GSHs thiol group to electrophilic substrates. The subsequent need to extend this function resulted in the addition of varying C-terminal domains to the thioredoxin-like domain forming monomeric CLIC1 and Grx2, and the association of monomers to form dimeric GSTs. Hence, the structural fold of the thioredoxin-like N-domain has been generally preserved while the structure of the subsequently added C-domain is much more variable.
Hot-regions preserve highly stabilising contacts and form a continuous, cooperative network of interactions. Hence, domain interface residues within the 3 hot-regions of GST family proteins (Figure 8) were deemed as critical components in the stabilization and the association of the domain interface. One particular set of interactions that stood out was an inter-domain lock-and-key motif, first identified in class Alpha GSTs (Wallace et al., 2000). Removal of the hydrophobic side chain locking the N- and C-terminal domains of hGSTA1-1 resulted in significant destabilization and loss in cooperative folding (Wallace et al., 2000). In the present study the lock-and-key interaction (Figure 9) is investigated because the lock residue is structurally conserved in the GST family (see position 8 in Appendix, Table A). The anatomy of this inter-domain interaction fits the O-ring proposition (Bogan and Thorn, 1998), which states that in order for an amino acid to have a large impact on the free energy of binding it needs to be protected from contact with the bulk solvent (Bogan and Thorn, 1998). In the case of the GST family the side chain of the lock residue is buried in the domain interface, protruding from the N-domain into a hydrophobic pocket found at the C-domain (Figure 9). Solvent exclusion is achieved by tight packing and surrounding the side chain of the lock residue with energetically unimportant contacts formed by moderately conserved amino acids (Figure 9). In terms of the CLICs, the importance of the lock-and-key motif, based on visual inspection, is further highlighted due to the fact that the majority of the domain interface is formed through h3 contacts with the insertion of the hydrophobic side chain of Met32 (lock residue in the case of CLIC1) in the C-domain being the prominent interaction.
Although the CLICs and GSTs are structural homologues, the ability of CLIC proteins to exist in a soluble form as well as a membrane form implies that their amino acid sequences contain a unique set of contacts that, under the correct conditions, allow the soluble to membrane metamorphosis to take place. Three sets of domain-interface interactions were identified as unique to the CLIC1 family (Figure 11). The network of contacts formed by Glu81 – Arg29 and Glu85 – Lys37 spans the domain interface joining the N-terminal h1 and h3 with the C-terminal h5 as well as the domain linker. Interestingly, h1 and h3, which form the N-terminal domain interface, are malleable with shifts of up to 1.5 Å (Harrop et al., 2001). The displacement of these helices means that the domain interface of CLIC1, CLIC4, and possibly other CLIC proteins is flexible. This plasticity allows for contacts to be broken and/or formed under various environmental conditions. Consequently, the N- and C-domains can uncouple possibly leading to the formation of the membrane-competent conformation.
The contribution toward CLIC1 stability by the salt-bridges formed through Glu81 – Arg29 and Glu85 – Lys37 interactions is difficult to predict. In general buried salt- bridges, as was the case here, are found to have a stabilizing ∆∆GTOTℵ of 4.53 ± 5.13
kcal/mol (Kumar and Nussinov, 1999). This is a huge stabilizing effect especially in view of the fact that wtCLIC1 at its most stable has a ∆GH2O of ~ 10 kcal/mol
(McIntyre, 2006). However, the large standard deviation of ∆∆GTOT for buried salt-
bridges means that the strength of these types of interactions is protein specific. As previously mentioned, the salt bridges formed by both Glu81 – Arg29 and Glu85 – Lys37 are found to be buried. This implies that the desolvation penalty for the burial of the charge groups upon folding will be high. This penalty will probably be less in the case of Glu81 – Arg29 because the immediate surroundings of this salt-bridge are mostly made up of polar residues. On the other hand, the ionic interaction formed via Glu85 – Lys37 is in a more hydrophobic environment. The desolvation penalties will be counteracted by a strong electrostatic attraction force because salt-bridges in the interior of proteins are better screened against the solvent (Kumar and Nussinov,
ℵ: ∆∆G
TOT = ∆∆GDSLV +∆∆GBRD+∆∆GPRT. Where ∆∆GDSLV represents the sum of the unfavorable
desolvation incurred by the burial of a charged group from a polar to a relatively non-polar environment ∆∆GBRD represents the favorable energy from the electrostatic interactions between the
charged groups of side chain atoms. ∆∆GPRT represents the electrostatic interactions between the side
chains of the salt-bridge forming residues and the side chains of the surrounding amino acids in the folded conformation. Equation was taken from Kumar and Nussinov (1999).
1999). In addition, the presence of hydrogen bonds between the oppositely charged side-chains of Glu81 – Arg29 and Glu85 – Lys37 brings them closer to one other hence further increasing the strength of the salt-bridge. In terms of the salt-bridge geometry, the oppositely charged side-chains of Glu81 – Arg29 are more favorably oriented at approximately right angles to each other. Therefore, overall both set of interactions formed between Glu81 – Arg29 and Glu85 – Lys37 are expected to have a significant stabilizing contribution toward the soluble CLIC1 conformation, with the former probably forming the stronger contact.
Glu218, position 39 in the consensus GST-interface, forms part of the lock-and-key interaction. The charged side chain chemistry of this residue is unique to the CLICs (Figures 9, 10 and 11). At the corresponding position, most GST-family proteins possess hydrophobic amino acids (Appendix, Table A). Significantly all of the above- mentioned unique inter-domain interactions form, amongst others, charged contacts. This is important since the strength of ionic interaction is highly dependent on variations in pH. Lowering the pH has recently been shown to destabilize the conformation of CLIC1 significantly resulting in loss of helical content and the formation of an equilibrium unfolding intermediate (McIntyre, 2006). Due to the cooperative nature of protein folding it is unlikely that one set of contacts is solely responsible for the transition of CLIC1 from a soluble to a membrane-competent form. It is more likely that a set of communicating contacts, as those found at the domain interface (Figure 11), are weakened thus lowering the energy barrier between the soluble and membrane conformations of CLIC proteins.