While most attention has been focused on the aberrant biosynthetic processing and trafficking through the secretory pathway of ΔF508 CFTR10
, the mutant channel had been observed to run-down rapidly at physiological temperture13
and it more recently has become apparent that the primary impact of the mutation is to decrease the thermodynamic stability of the channel protein13; 14; 19; 20
. Despite virtually no change from the WT NBD1 fold at the low temperature at which the isolated domain was crystallized36
, the mutant domain exhibits increased sensitivity to both thermal and chemical denaturation7; 8
. Similarly, the ion channel activity of full-length ΔF508 CFTR is lost at temperatures well below the physiological range19
. Thus, the major challenge now is to restore a level of stability sufficient to provide near normal ion channel function and life time at the cell surface.
Although the effects of known second site changes (in and proximal to the signature sequence and at I539) have since been studied extensively26; 27; 33,37
their mechanisms of action are still not understood. Additional substitutions, primarily on the surface of NBD1, also empirically discovered to improve solubility during a crystallization project36
were found to improve the maturation of full-length ΔF508 CFTR in cells38
. Similarly, deletion of the RI peptide, that enhanced solubility and dimer formation by isolated NBD139
, not only greatly improved the maturation and life-time of the full length mutant, but also restored its thermostability, providing near-normal channel activity at 37°C and above19
In the current work we sought a rational approach to discover other allosteric sites in NBD1 where changes might have similar or stronger stabilizing effects. Modulation of protein thermodynamic stability a priori
remains a general problem in protein chemistry, with approaches generally limited to amino acid substitutions that enhance favorable or remove unfavorable electrostatics or decrease the conformational entropy of the unfolded state40
. Amino acid substitutions to achieve the latter effect are essentially limited to the introduction of pairs of cysteines to form disulfides or rigid proline residues. The judicious introduction of prolines at multiple sites has been found to increase the thermal stability of many proteins, due to the restriction of the residue by its pyrrolidine ring to fewer conformations than other amino acids 41
Earlier work comparing evolutionary conservation of prolines in enzymes from mesophilic and thermophilic organisms detected an increased frequency of the residue at certain sites in the latter42
. Extensive studies by Mathews and coworkers43; 44
and others45; 46; 47
with a variety of proteins of known 3D structures demonstrated that replacement of residues with prolines could either stabilize or destabilize depending on the sites of their introduction. Clearly, if the residue being replaced contributes to the folded structure, its removal will be destabilizing. The prolines introduced can be more easily accommodated at positions of higher than average mobility, where rearrangements that the rigid proline may cause can occur with minimal energetic cost. As a consequence, prolines are generally well accommodated in turns, coils, and at the N-termini of α-helices and β-strands43
. Nevertheless, in practice, despite these guidelines, the selection of residues to replace with prolines in a sizeable protein or domain remains largely empirical.
However, when we compared the sequences of CFTR orthologs that remained quite thermostable when the ΔF508 mutation was introduced, the presence of prolines at several sites was apparent (). Each of these locations appeared to satisfy the criteria of stabilizing prolines at mobile unstructured sites, i.e., the RI, the Q-loop and the I539 loop within the SDR (Fig. S5
). We recently have shown that the RI has a major influence on ΔF508 CFTR thermal instability19
. The Q-loop or γ-phosphate switch has variable configurations in different X-ray structures that were observed to influence folding in computational simulations48; 49
, as does the I539 loop. Thus it is not surprising that a rigidifying effect of prolines in these locations might diminish the thermal instability of ΔF508 NBD1. The apparent additive effects of several substituted prolines acting independently also has been observed in other proteins47
Molecular dynamics simulations reveal that the SDR of NBD1 is stabilized by the S492P substitution and the stability further increases with each proline substitution. Clustering of the different configurations assumed by the protein during the simulation at different temperatures revealed that the smaller peaks in the temperature range of ~280–300K represent configurations where the RI is flipped out and/or the α-subdomain of NBD1 is partially unfolded (Fig. S6
). The first major peak in the Cv profile ( – dashed lines) corresponds to the transition from the folded to the unfolded state. A shift in this peak towards lower temperatures represents destabilization while a shift towards higher temperatures suggests stabilization of the protein. The major peak for ΔF508 NBD1 (at ~313K) ( – red dashed line) shifts significantly to the right with four proline substitutions (to~335K) ( – blue dashed line). The appearance of an additional peak at ~342 K is a consequence of high entropy of the system at high temperatures. From the simulation trajectories, we observed that the configurations corresponding to the final peak represent transient formation of secondary structural elements that cannot collapse into a folded form due to high temperatures.
A striking feature of the strong stabilizing effect of the proline substitutions was the essentially absolute dependence on the I539T substitution. This dependence contrasts the positive effects on ΔF508 CFTR maturation of other second site changes that are not wholly dependent on I539T, such as those near the NBD1 signature sequence (G550E/R553M/R555K) and the RI. I539T has an additive effect with the 550/553/555 set33
. The I539T substitution alone promotes a low level of human ΔF508 CFTR maturation and its normal presence in murine CFTR probably contributes to the partial maturation of the mouse ΔF508 variant15;26
as well as that of the rabbit (). However, this degree of maturation is quite limited compared to the orthologs also containing the proline substitutions described here. The vital role of the hydrophilic threonine residue at the 539 position in determining the fate of ΔF508 CFTR in cells is most clearly demonstrated by the complete prevention of maturation of chicken ΔF509 CFTR by the T540I substitution. Since the residue occupying the 539/540 position has virtually no influence on the wild-type human or chicken CFTR, there is clearly a tight coupling between the 508 (509) and 539 (540) positions. Having the hydroxyl amino acid at 539 (540) enables a modest level of maturation by an unknown mechanism26; 15
but this effect is permissive of major stabilization by the key proline substitutions. Their impact is unambiguously confirmed by the fact that their removal completely destabilizes chicken ΔF509 CFTR.
An important finding in this study was the requirement for an appropriate balance between thermostability and CFTR channel function. Thus, as increasing numbers of stabilizing prolines were introduced, channel activity of ΔF508 CFTR diminished at lower temperatures before reaching near wild-type open probability and gating rates at physiological temperature. This has important implications for understanding the influence of ΔF508 on channel gating. On the basis of the short term behavior of the mutant channel on the surface of cells grown at <30°C, it has generally been described as operating with a Po
approximately 30% of that of the wild-type50
. However, as shown in our present and previous19; 20
work, and that of others14
this is not the key functional defect that needs to be remedied. Instead, the rapid essentially complete loss of activity as temperature is raised to the physiological range, and the need to restore a level of thermodynamic stability to avoid this loss, is an essential requirement for the development of more effective molecular therapeutic approaches. Significantly, there are now at least three different experimental manipulations that can do this: the combined Teem second site missense mutations14
and proline introductions (this study).
All of these manipulations are within NBD1, raising the question of whether stabilizing that domain alone is sufficient to restore maturation and function of the mutant full-length protein. Although the question cannot be definitively answered as yet, the levels of maturation of the chicken ΔF509 CFTR and its human mimic, the lifetime of their mature products, as well as their channel activities at physiological temperature suggests that repair of NBD1 might be adequate to correct the stability of the full-length mutant protein. With the F508 residue still absent, the important interface of NBD1 with cytoplasmic loop 4 would remain non-native, and it is known that modification of this interface by substitution of residues on either side, for example by the V510D51
substitutions, without NBD1 stabilization, improves maturation of ΔF508 CFTR. Nevertheless, the near wild-type behavior of ΔF508/4PT suggests that the interface may be slightly rearranged by the stabilized NBD1 such that the connection between domains is adequate for the essential gating-supporting signal transduction across it23; 24
. Biochemical evidence of a restored interface was provided by the cross linking of cysteine residues placed on either side (). Thus, we have now accumulated strong evidence that thermostabilization of NBD1 has a major restorative influence on full-length ΔF508 CFTR, whereas so-called rescue of cells in which it is expressed by keeping them at reduced temperature (<30°C) or exposure of known small molecule correctors does not. This provides strong motivation for searches for NBD1 stabilizing agents as potential starting points for the development of complementary ΔF508 CFTR corrective therapeutics.