ABCMdb

PMID: 24231430

Iram SH, Cole SP
Differential functional rescue of Lys(513) and Lys(516) processing mutants of MRP1 (ABCC1) by chemical chaperones reveals different domain-domain interactions of the transporter.
Biochim Biophys Acta. 2014 Mar;1838(3):756-65. doi: 10.1016/j.bbamem.2013.11.002. Epub 2013 Nov 11., [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC1 p.Lys513Ala Exposure of HEK cells to the chemical chaperones glycerol, DMSO, polyethylene glycol (PEG) and 4-aminobutyric acid (4-PBA) improved levels of K513A to wild-type MRP1 levels but transport activity was only fully restored by 4-PBA or DMSO treatments. Login to comment 3 ABCC1 p.Lys513Ala Tryptic fragmentation patterns and conformation-dependent antibody immunoreactivity of the transport-deficient PEG- and glycerol-rescued K513A proteins indicated that the second nucleotide binding domain (NBD2) had adopted a more open conformation than in wild-type MRP1. Login to comment 5 ABCC1 p.Lys513Ala ABCC1 p.Lys516Ala In contrast to K513A, K516A levels in HEK cells were not significantly enhanced by chemical chaperones. Login to comment 6 ABCC1 p.Lys516Ala In more permissive insect cells, however, K516A levels were comparable to wild-type MRP1. Login to comment 7 ABCC1 p.Lys516Ala Nevertheless, organic anion transport by K516A in insect cell membranes was reduced by N80% due to reduced substrate Km. Login to comment 26 ABCC1 p.Lys513Ala ABCC1 p.Arg501Ala Thus, Ala substitution of Arg501 , Glu507 or Arg532 caused a significant reduction in organic anion transport activity while Ala substitution of Lys513 , Lys516 , Glu521 or Glu535 abrogated MRP1 expression by disrupting the folding and assembly of the transporter, targeting it for degradation by the proteasome [8,9]. Login to comment 27 ABCC1 p.Lys513Ala ABCC1 p.Glu535Ala ABCC1 p.Glu521Ala In the case of three of these misfolded CL5 mutants (K513A, E521A and E535A), plasma membrane MRP1 levels could be improved significantly by exposing transfected HEK cells to the widely used chemical chaperone, 4-phenylbutyric acid (4-PBA) [9,10]. Login to comment 28 ABCC1 p.Glu535Ala ABCC1 p.Glu521Ala Although the 4-PBA- rescued E521A and E535A mutants were stably expressed, they exhibited mechanistically distinct transport deficiencies that were associated with different structural conformations of the transporter that in turn indicated that the folding and assembly processes for MRP1 are distinct from those of its homolog CFTR [9]. Login to comment 29 ABCC1 p.Lys513Ala ABCC1 p.Glu535Ala ABCC1 p.Glu521Ala In contrast to E521A and E535A, the 4-PBA rescued K513A mutant exhibited transport properties comparable to wild-type MRP1. Login to comment 30 ABCC1 p.Lys516Ala Consequently, the failure of 4-PBA to rescue expression of the fourth processing mutant, K516A, was unexpected, particularly since Lys516 is within one turn of Lys513 in the same b1;-helix (the so-called coupling b1;-helix) that atomic homology models predict runs roughly parallel to the plasma membrane and interfaces with NBD1 and NBD2 [7,8]. Login to comment 31 ABCC1 p.Lys513Ala ABCC1 p.Lys516Ala In the present study, we have further investigated the relative abilities of several chemical chaperones to rescue the expression and/or function of K513A and K516A to better understand the distinct roles that Lys513 and Lys516 play in promoting the proper folding and assembly of MRP1, and its trafficking to the plasma membrane. Login to comment 38 ABCC1 p.Lys513Ala ABCC1 p.Lys516Ala Preparation of MRP1-enriched membrane vesicles and measurement of MRP1 levels in HEK293T and Sf9 insect cells The generation of wild-type, K513A and K516A mutant MRP1 pcDNA3.1 expression vectors has been described previously [8,16]. Login to comment 46 ABCC1 p.Lys516Ala Wild-type and K516A mutant MRP1 were also cloned into pFastBac vectors (Life Technologies Inc.) for expression in Sf9 insect cells. Login to comment 50 ABCC1 p.Lys516Ala Sf9 cells grown as monolayers were infected at 80% confluency with 1.25 ml of wild-type or K516A MRP1 baculoviruses (8 &#d7; 107 IFU ml-1 ) per 150 mm plate. Login to comment 74 ABCC1 p.Lys513Ala Photolabeling of MRP1 with 8-azido-[b1;-32 P]ATP K513A mutant and wild-type MRP1 from transfected HEK cells were photolabeled with 8-azido-[b1;-32 P]ATP essentially as described [22]. Login to comment 76 ABCC1 p.Lys513Ala After 5 min incubation on ice, the samples were cross-linked at 302 nm, washed, and then subjected to SDS-PAGE. After drying, the gel was exposed to film for 2-4 h. K513A mutant and wild-type MRP1 membrane proteins were also incubated in transport buffer containing 5 mM MgCl2, 1 mM freshly prepared sodium orthovanadate, and 5 bc;M 8-azido-[b1;-32 P]ATP for 15 - min at 37 &#b0;C to measure orthovanadate-induced trapping of 8-azido- [b1;-32 P]ADP by MRP1. Login to comment 79 ABCC1 p.Lys513Ala ABCC1 p.Lys516Ala 2.7. Limited trypsin digestions HEK or Sf9 insect cell membrane vesicles (2.0 bc;g per reaction) enriched for K513A or K516A mutant or wild-type MRP1 were digested with trypsin over a range of trypsin:protein ratios for 15 min essentially as described [9,23]. Login to comment 85 ABCC1 p.Lys513Ala ABCC1 p.Lys513Ala ABCC1 p.Lys516Ala ABCC1 p.Lys516Ala K513A and K516A mutants differ in their ability to be rescued by chemical chaperones The relative effectiveness of glycerol, DMSO, PEG and 4-PBA to rescue the processing mutants K513A and K516A was evaluated by comparing MRP1 protein levels in lysates prepared from transfected HEK293T cells treated with or without these chemical chaperones. Login to comment 86 ABCC1 p.Lys513Ala As shown in Fig. 2A, all treatments enhanced levels of the K513A mutant to a similar degree while the same treatments did not significantly alter wild-type MRP1 levels indicating that the rescue is not a result of transcriptional upregulation. Login to comment 87 ABCC1 p.Lys513Ala Confocal microscopy confirmed the plasma membrane localization of the rescued K513A mutants indicating their ability to bypass the quality control mechanisms for protein folding in the endoplasmic reticulum of HEK cells and to traffic to the plasma membrane (Supplemental Fig. S1). Login to comment 88 ABCC1 p.Lys513Ala ABCC1 p.Lys516Ala In contrast to K513A, and consistent with our earlier observations [9], exposure of K516A transfected cells to glycerol, DMSO, PEG and 4-PBA only slightly increased MRP1 levels (Fig. 2B). Login to comment 90 ABCC1 p.Lys516Ala In addition, given the multiple intra-and inter-domain interactions that appear to be involved in the optimal expression of MRP1 at the plasma membrane, it may not be reasonable to expect a single small molecule to 'correct` the conformation and transport activity of K516A. Login to comment 92 ABCC1 p.Lys516Ala However, none of the chemicals, alone or in combination, enhanced K516A levels substantially, leading to the conclusion that the folding defects introduced by mutation of Lys516 must differ from the defects introduced by mutation of Lys513 , despite the close proximity of the two basic residues in the coupling helix in CL5. Login to comment 94 ABCC1 p.Lys513Ala ABCC1 p.Lys513Ala Functional characterization of chemically rescued K513A mutant MRP1 proteins To determine whether or not the chemically rescued K513A mutants were transport competent, membrane vesicles were prepared (Fig. 3A) and vesicular transport activities were measured using the prototypical organic anion substrates E217b2;G and LTC4. Login to comment 95 ABCC1 p.Lys513Ala As shown in Fig. 3, [3 H] E217b2;G and [3 H]LTC4 uptake levels by K513A mutant membrane vesicles prepared from cells treated with 4-PBA or DMSO were comparable to wild-type MRP1. Login to comment 98 ABCC1 p.Lys513Ala These results indicate that despite restoring similar levels of the K513A mutant, the chemical chaperones altered MRP1 function to different extents. Login to comment 100 ABCC1 p.Lys513Ala ABCC1 p.Lys513Ala ABCC1 p.Lys513Ala Conformational differences in chemically rescued K513A mutant proteins To determine whether the transport properties of the K513A mutants rescued by the different chemical chaperones were associated with any differences in protein folding, membrane vesicles containing K513A were subjected to limited trypsinolysis and tryptic fragments detected by immunoblotting with mAbs directed against epitopes in the NH2-proximal (mAb MRPr1) and COOH-proximal (mAb MRPm6) halves of MRP1 (Fig. 4A) [6,29]. Login to comment 102 ABCC1 p.Lys513Ala ABCC1 p.Lys516Ala Effect of chemical chaperones on relative levels of K513A and K516A mutant MRP1 proteins in HEK293T cells. Login to comment 103 ABCC1 p.Lys513Ala ABCC1 p.Lys516Ala Shown are representative immunoblots of whole cell lysates prepared from HEK293T cells transfected with K513A or K516A mutant or wild-type (WT) MRP1 cDNA expression vectors, with or without exposure to the chemicals indicated at the top of the blots. Login to comment 106 ABCC1 p.Lys513Ala ABCC1 p.Lys516Ala (A) WT-MRP1 (left panel), K513A (right panel); (B) K516A. Login to comment 109 ABCC1 p.Lys513Ala Effect of chemical chaperones on the levels and vesicular transport activity of K513A mutant MRP1 expressed in HEK cells. Login to comment 110 ABCC1 p.Lys513Ala (A) Shown is a representative immunoblot of membrane vesicles (1 bc;g protein per lane) prepared from transfected HEK293T cells, untreated (WT-MRP1) or treated with the indicated chemicals (K513A) for 48 h. Untransfected cells were used as a negative control (ctrl). Login to comment 117 ABCC1 p.Lys513Ala The four chemically rescued K513A mutants had similar tryptic profiles, indicating that the conformation of the NH2-proximal halves of these mutant proteins were comparable to wild-type MRP1. Login to comment 118 ABCC1 p.Lys513Ala To probe for conformational changes in the COOH-proximal halves of the rescued K513A mutants, the trypsin digests were then immunoblotted with mAb MRPm6 (Fig. 4B, right panel). Login to comment 119 ABCC1 p.Lys513Ala Under these conditions, the wild-type MRP1 digests yielded first a larger 75-kDa fragment (C1, corresponding to MSD2-NBD2) which was then cleaved Fig. 4. Limited trypsin digests of chemically rescued K513A mutants and wild-type MRP1 expressed in HEK293T cells. Login to comment 121 ABCC1 p.Lys513Ala (B) Membrane vesicle proteins (2 bc;g per lane) from cells expressing wild-type MRP1 or K513A mutant MRP1 rescued with the indicated chemical chaperones were incubated at 37 &#b0;C for 15 min with trypsin at different trypsin:protein ratios (range 1:5000 to 2.5:1), and then immunoblotted with (left panel) mAb MRPr1 and (right panel) mAb MRPm6. Login to comment 125 ABCC1 p.Lys513Ala The tryptic profiles for the K513A mutants rescued by DMSO or 4-PBA were similar to wild-type MRP1, suggesting that no significant conformational changes had occurred in the COOH-proximal halves of these mutants. Login to comment 126 ABCC1 p.Lys513Ala In contrast, the tryptic profiles of the glycerol and PEG rescued K513A mutants were substantially different. Login to comment 127 ABCC1 p.Lys513Ala Most notably, the NBD2 (C2) fragments were not detectable in the glycerol or PEG rescued K513A proteins. Login to comment 128 ABCC1 p.Lys513Ala These observations indicate that NBD2 in the glycerol and PEG-rescued K513A mutants is hypersensitive to trypsin suggesting that this region has adopted a more open (less compact) conformation than is the case in wild-type MRP1. Login to comment 129 ABCC1 p.Lys513Ala Conformational changes in NBD2 in the glycerol and PEG rescued K513A proteins were further corroborated by taking advantage of two MRP1-specific conformation-dependent antibodies, mAb QCRL-2 (which recognizes a discontinuous epitope within NBD1) and mAb QCRL-4 (which recognizes a discontinuous epitope within NBD2) [15]. Login to comment 131 ABCC1 p.Lys513Ala As shown in Fig. 5A, the immunoreactivity of the glycerol and PEG rescued K513A proteins detected by mAb QCRL-4 was diminished by N50% compared to wild-type MRP1, consistent with substantial changes in the conformation of NBD2. Login to comment 133 ABCC1 p.Lys513Ala Together with the tryptic fragment analyses (Fig. 4), these results indicate that the transport deficiencies of K513A rescued by glycerol or PEG are associated with a change to a less compact conformation of NBD2 in MRP1. Login to comment 135 ABCC1 p.Lys513Ala ABCC1 p.Lys513Ala E217b2;G uptake and [3 H]LTC4 photolabeling of glycerol and PEG rescued K513A MRP1 To further explore the consequences of the conformational changes detected in the glycerol or PEG rescued K513A proteins, kinetic parameters of E217b2;G uptake were determined. Login to comment 137 ABCC1 p.Lys513Ala The glycerol and PEG rescued K513A proteins exhibited comparable Km (E217b2;G) values of 3.8 and 5.6 bc;M, respectively, but their Vmax (E217b2;G) values were decreased 3 to 4-fold relative to wild-type MRP1 (230 and 317 pmol mg-1 min-1 , respectively). Login to comment 138 ABCC1 p.Lys513Ala [3 H]LTC4 photolabeling of these two rescued K513A proteins and wild-type MRP1 were also comparable (Fig. 6A). Login to comment 139 ABCC1 p.Lys513Ala Together, these results indicated that the reduced transport levels of the PEG and glycerol rescued K513A mutant were not caused by decreased Km (E217b2;G) or reduced binding of LTC4 (at 200 nM). Login to comment 141 ABCC1 p.Lys513Ala ABCC1 p.Lys513Ala Photolabeling of glycerol and PEG rescued K513A MRP1 with 8-azido- [b1;-32 P]ATP and orthovanadate-induced trapping of 8-azido-[b1;-32 P]ADP Since the transport deficiencies of the glycerol- and PEG-rescued K513A proteins were not associated with differences in Km (E217b2;G) or reduced binding of LTC4 (at 200 nM), the nucleotide interactions of the mutants were examined. Login to comment 143 ABCC1 p.Lys513Ala As shown in Fig. 6B, photolabeling of the glycerol- and PEG-rescued K513A proteins at this single concentration of 8-azido-ATP was decreased by 50% and 30%, respectively, compared to wild-type MRP1 (after differences in MRP1 levels were taken into account). Login to comment 145 ABCC1 p.Lys513Ala ABCC1 p.Lys513Ala As shown in Fig. 6C, [32 P]ADP trapping by glycerol-rescued K513A was 60% lower than trapping by wild-type MRP1 while trapping by the PEG-rescued K513A was 40% lower. Login to comment 148 ABCC1 p.Lys513Ala Immunodot blot analyses of the transport-deficient PEG and glycerol rescued K513A mutant MRP1 with conformation-dependent mAbs against NBD1 and NBD2. Login to comment 149 ABCC1 p.Lys513Ala Serial dilutions of non-denatured membrane vesicles (corresponding to 0.5, 1 and 2 bc;g protein) prepared from transfected HEK293T cells expressing K513A mutant (treated with glycerol or PEG for 48 h) or wild-type MRP1 (untreated) were spotted on a membrane as described [18] and then probed with conformation-dependent mAbs (A) QCRL-4 (NBD2); and (B) QCRL-2 (NBD1). Login to comment 152 ABCC1 p.Lys513Ala ABCC1 p.Lys513Ala WT-MRP1 (a0;); glycerol-rescued K513A (cf;); PEG-rescued K513A (੪). Login to comment 154 ABCC1 p.Lys516Ala ABCC1 p.Lys516Ala ABCC1 p.Lys516Ala Expression and functional characterization of K516A in Sf9 insect cells Since treatment of HEK293T cells expressing MRP1 K516A with chemical chaperones alone, and in combination, failed to significantly increase levels of this mutant protein (Fig. 2C), K516A was cloned into a baculovirus expression vector and expressed in Sf9 insect cells which exert less stringent quality control mechanisms during biosynthesis of human proteins. Login to comment 155 ABCC1 p.Lys516Ala As shown in Fig. 7A, this approach was successful since K516A was expressed at levels comparable to those of wild-type MRP1 in insect cells. Login to comment 156 ABCC1 p.Lys516Ala However, the LTC4 transport activity of the heterologously expressed K516A was reduced by 80% relative to wild-type MRP1 (Fig. 7B). Login to comment 157 ABCC1 p.Lys516Ala [3 H]LTC4 labeling of the heterologously expressed K516A mutant was also diminished by 90% (Fig. 7C), indicating the LTC4 substrate binding site of MRP1 had been compromised. Login to comment 159 ABCC1 p.Lys513Ala [3 H]LTC4 photolabeling and azido-[b1;32 P]ATP interactions of transport-deficient PEG and glycerol rescued K513A mutant proteins expressed in HEK293T cells. Login to comment 171 ABCC1 p.Lys516Ala Expression and function of K516A mutant MRP1 expressed in insect cells. Login to comment 172 ABCC1 p.Lys516Ala (A) Representative immunoblot of membrane vesicles (1 bc;g protein per lane) prepared from Sf9 insect cells infected with K516A mutant and wild-type MRP1 recombinant baculoviruses. Login to comment 175 ABCC1 p.Lys516Ala (B) [3 H]LTC4 transport activity of insect cell membrane vesicles enriched for K516A mutant and wild-type MRP1. Login to comment 179 ABCC1 p.Lys516Ala Control insect cell membrane vesicles (b2;-gus) and vesicles enriched for K516A mutant and wild-type MRP1 (50 bc;g protein) were photolabeled with [3 H]LTC4 as described in the legend to Fig. 6. Login to comment 183 ABCC1 p.Lys516Ala ABCC1 p.Lys516Ala K516A mutation causes 'long range` conformational changes in MSD2 In seeking a structural explanation for the reduced substrate binding of the heterologously expressed K516A mutant, the conformation of the mutant protein was probed by limited trypsinolysis followed by immunoblotting with mAbs directed against epitopes in the NH2- and COOH-proximal halves of MRP1 as before (Fig. 8A). Login to comment 184 ABCC1 p.Lys516Ala Differences in the wild-type and K516A tryptic fragmentation patterns were detected with both antibodies. Login to comment 185 ABCC1 p.Lys516Ala Thus, when probed with mAb MRPr1 (Fig. 8B, left panel), fragments of unknown identity (50-65 kDa) accumulated in the case of the K516A mutant but not wild-type MRP1, whereas the trypsin sensitivity of the full-length, as well as the expected N1 and N3 (MSD0) fragments, were quite similar. Login to comment 186 ABCC1 p.Lys516Ala In contrast, when probed with mAb 897.2 (Fig. 8B, right panel) the tryptic fragmentation of the K516A mutant was strikingly different from wild-type MRP1. Login to comment 187 ABCC1 p.Lys516Ala Thus, the levels of the C1 fragment (MSD2-NBD2) for K516A were substantially reduced relative to wild-type MRP1, despite very similar patterns of disappearance for the two full-length proteins. Login to comment 188 ABCC1 p.Lys516Ala ABCC1 p.Lys516Ala Furthermore, the levels of C2 fragment (NBD2) were also comparable between the wild-type and K516A mutant suggesting that the trypsin hypersensitivity of the C1 fragment (MSD2-NBD2) of K516A is mainly due to conformational changes in MSD2. Login to comment 199 ABCC1 p.Lys516Ala Fig. 8. Limited trypsin digests of transport-deficient K516A mutant MRP1 in insect cell membranes. Login to comment 201 ABCC1 p.Lys516Ala (B) Membrane vesicles (2 bc;g protein) prepared from insect cells expressing transport-deficient K516A mutant and wild-type MRP1 were incubated at 37 &#b0;C for 15 min with trypsin at different trypsin:protein ratios (range 1:500 to 1:12.5) and then immunoblotted with (left panel) mAb MRPr1 and (right panel) mAb 897.2. Login to comment 203 ABCC1 p.Lys516Ala The boxes highlight significant differences between the patterns of the tryptic fragments of the wild-type and transport-deficient K516A mutant proteins. Login to comment 210 ABCC1 p.Lys513Ala ABCC1 p.Lys516Ala In the present study, we have shown that despite the proximity of the affected amino acids to one another in CL5, the MRP1 processing mutants K513A and K516A display remarkable differences in their ability to be functionally rescued by four commonly used chemical chaperones. Login to comment 212 ABCC1 p.Lys513Ala In the case of K513A, levels of the mutant protein could be improved to wild-type MRP1 levels by treatment of cells with all four chemical chaperones tested (Fig. 2A) [9]. Login to comment 213 ABCC1 p.Lys513Ala However, only the 4-PBA and DMSO-rescued K513A mutants displayed organic anion transport activity comparable to wild-type MRP1. Login to comment 214 ABCC1 p.Lys513Ala The reduced transport activity of the glycerol- and PEG-rescued K513A mutants indicated that the structural defects introduced by the Lys513 substitution were only partially restored by these two chemical chaperones. Login to comment 215 ABCC1 p.Lys513Ala ABCC1 p.Lys513Ala Differences in trypsin fragmentation patterns and immunoreactivity with conformation-dependent antibodies revealed that the NBD2 region of the glycerol and PEG-rescued K513A transport-deficient MRP1 mutants had adopted a more open, trypsin sensitive conformation than the same region of wild-type MRP1 (or the functionally intact DMSO or 4-PBA rescued K513A mutants). Login to comment 216 ABCC1 p.Lys513Ala The kinetic analyses of E217b2;G transport and the nucleotide labeling results further indicated that the transport deficiencies of the DMSO and PEG-rescued MRP1 K513A proteins were caused by changes in the binding and hydrolysis of ATP. Login to comment 220 ABCC1 p.Lys513Ala These observations support the suggestion that the K513A mutation in CL5 of MSD1 interferes with the closure of the NBD2 core subdomain making it less able to form a compact interface with CL5. Login to comment 221 ABCC1 p.Lys513Ala The biochemical properties and mode of action of 4-PBA, glycerol, DMSO, and PEG are known to vary and it is not clear here why all 4 were equally able to improve K513A levels but glycerol and PEG were unable to functionally rescue the mutant. Login to comment 223 ABCC1 p.Lys513Ala Thus, in contrast to MRP1-K513A, glycerol effectively restored the function of CFTR-ƊF508 while DMSO did not [37,39]. Login to comment 225 ABCC1 p.Lys513Ala ABCC1 p.Lys516Ala In contrast to K513A, the K516A processing mutant was remarkably resistant to rescue by the chemical chaperones tested. Login to comment 228 ABCC1 p.Lys516Arg ABCC1 p.Lys513Arg In this regard, it is of interest that the same charge K513R mutant could be expressed at levels similar to wild-type MRP1 in HEK cells, while levels of the analogous K516R mutant were 40% lower [8]. Login to comment 231 ABCC1 p.Lys516Ala Despite the inability of chemical chaperones to rescue K516A expression in HEK cells, heterologous expression in Sf9 insect cells yielded levels of the mutant transporter comparable to wild-type MRP1 which is likely attributable to the lower stringency of the protein quality control processes in these cells [44-47]. Login to comment 232 ABCC1 p.Lys516Ala However, despite the robust levels of K516A mutant achieved in this system, the transport activity of this mutant was severely diminished. Login to comment 233 ABCC1 p.Lys516Ala Thus, LTC4 transport levels were just 10% those of wild-type MRP1, and the K516A mutant could no longer be photolabeled with this substrate. Login to comment 234 ABCC1 p.Lys516Ala These observations indicated that the substrate binding pocket of MRP1 had been compromised which in turn suggested structural differences between the K516A mutant and wild-type MRP1 proteins, despite the fact that both were expressed equally well. Login to comment 235 ABCC1 p.Lys516Ala Support for this conclusion was obtained by comparing the tryptic digest patterns of the two proteins which indicate that the CL5 K516A mutation in MSD1 introduces a change in the COOH-proximal half of MRP1 (MSD2-NBD2) to a more open, trypsin-sensitive conformation. Login to comment 236 ABCC1 p.Lys516Ala Since the tryptic fragments of NBD2 (C2) in both wild-type and K516A were similar, it may be inferred that this conformational change lies predominantly in MSD2. Login to comment 237 ABCC1 p.Lys513Ala This is in contrast to the K513A mutant where NBD2 folding was mainly affected. Login to comment 238 ABCC1 p.Lys513Ala ABCC1 p.Lys516Ala According to ab initio analyses, both K513A and K516A mutations are predicted to introduce significant rigidity into the tertiary structure of the coupling helix in CL5 where these Lys residues are found. Login to comment 241 ABCC1 p.Lys513Ala ABCC1 p.Lys516Ala This difference would provide a plausible explanation for why mutation of Lys516 might be more disruptive to normal domain-domain interactions than mutation of Lys513 , which in turn, could explain why the K516A mutant is more resistant to rescue by chemical chaperones than K513A. Login to comment