In this report, we have characterized in considerable detail the interactions of FSBA with P-gp. The main reason for this is that FSBA is an ATP analog and it has been used extensively to chemically modify and probe the nucleotide-binding sites of proteins that bind and hydrolyze nucleotides.14
This could thus also be a useful reagent to study the NBDs of P-gp as well as other ABC transporters.
The reports in the literature primarily consider FSBA as a nucleotide analog14
for kinases and other ATP-binding proteins. However, our results show that FSBA interacts with NBDs and also has an effect on the substrate-binding sites of P-gp (, , and ). In intact cells, FSBA, similar to other P-gp inhibitors (XR9576), blocked the efflux of rhodamine 123 (), suggesting that FSBA inhibits P-gp mediated transport in intact cells by interacting at the transport-substrate sites in the TMDs rather than in the NBDs, as one would expect that the NBD sites are occupied by ATP under physiological conditions with the intracellular concentration of ATP in the range of 3–5 mM. Most nucleotides (and nucleotide analogs) are not transport-substrates of P-gp and the transport-substrates do not directly interact with the NBDs.10
This is consistent with a large body of work that shows that ATP hydrolysis and drug-substrate transport occur at different domains.9
The TMDs are anchored in the plasma membrane and interact with the transport-substrates which are generally hydrophobic, while the NBDs, which are physically located in the cytoplasm, interact with hydrophilic nucleotides.9
In FSBA, the benzoylsulfonylfluoride replaces the tri-phosphate moiety of ATP (). As a result, unlike ATP, which is negatively charged, FSBA is neutral15
and there is the addition of an aromatic benzoyl group. These changes make FSBA more hydrophobic (Log P = 2.64), thus penetrating the membrane domains and providing it with structural features that characterize transport substrates of P-gp. This can explain how FSBA is able to interact with the drug-substrate-binding sites of P-gp. We provide evidence for the effect of FSBA on the substrate-binding sites in . The prazosin analog IAAP has been used extensively as a photoaffinity probe for the substrate-binding sites of P-gp.47
The concentration-dependent inhibition of IAAP binding to P-gp by FSBA () demonstrates that FSBA interacts directly with the substrate-binding site(s). It has been pointed out previously that the replacement of the phosphate groups of ATP with the sulfonylfluoride group makes FSBA uncharged and a less desirable ATP analog;15
however, these modifications make FSBA more accessible to the substrate-binding site(s) (see above). In addition, there appear to be different energetic requirements for binding to the ATP sites and chemical crosslinking. The former can be demonstrated by the virtually instantaneous displacement of 8-azido-[α-32
P]-ATP by FSBA at 4°C. On the other hand, the irreversible FSBA-mediated inhibition of ATP hydrolysis (as a consequence of chemical crosslinking) has a t1/2
of approximately 4 min (), and this inhibition is strongly temperature-dependent (). It is clear that while the reversible binding of FSBA to NBDs is temperature-independent, the chemical crosslinking to residues within and nearby NBDs is temperature-dependent.
The sulfonyl fluoride group of FSBA, which replaces the phosphate groups of ATP, is a reactive functional group that can react with several amino acids.16–18
It has been argued that as the sulfonyl fluoride participates in a broad range of reactions, there is a reasonable probability of reaction within any particular active site.15
To distinguish between covalent and non-covalent interactions, P-gp was incubated for 30 min in the absence or presence of FSBA. Reactions were carried out either at 4°C or 37°C and the excess unreacted FSBA was removed by dilution with cold buffer followed by centrifugation. The P-gp samples that were incubated with FSBA at 37°C showed a strong inhibition of ATP hydrolysis (: compare third and fourth bars), while those incubated at 4°C showed no inhibition (: compare first and second bars). The control (incubation at 4°C) shows that un-reacted FSBA can be effectively removed by centrifugation and it is only the chemically-crosslinked FSBA that inhibits ATP hydrolysis. The binding of the transport substrate IAAP to wild-type P-gp or mutant Y401A/1044A (that cannot bind to ATP) is not affected by the formation of an FSBA-P-gp adduct (), suggesting that FSBA is not crosslinked to the IAAP binding site. Thus, the interaction of FSBA at the drug-substrate binding pocket in TMDs is reversible and it does not depend on the functional status of ATP sites ().
The covalent crosslinking of FSBA to P-gp is further verified by crosslinking of [14C]-labeled FSBA to purified P-gp reconstituted into proteoliposomes (). In addition, we used mass spectrometry to identify sites on P-gp labeled with FSBA (). Seven peptides were labeled with FSBA, of which four were protected by pretreatment with 5 mM ATP (). This is consistent with the observation that incubation with a saturating concentration of ATP prior to addition of FSBA provides only partial protection to labeling by [14C]-FSBA (). Identification of the peptides labeled with FSBA further suggests that FSBA crosslinks mainly to residues within or near the NBDs (five out of seven fragments are within or near the ATP sites; , and and ), despite interrogating both the nucleotide- and substrate-binding sites (, ).
Previous studies have shown that FSBA participates in covalent reactions with various amino acids (for review see 15
), including tyrosine, lysine, histidine, serine, and cysteine. The X-ray crystal structures of the NBD domains of several ABC transporters (e.g. MJ0796-1L2T.pdb; Hly-B -1XEF.pdb) show that the ATP molecule is sandwiched between the A-loop, the Walker A & B domains and the H-loop of one NBD and the signature sequence and D-loop of the opposing NBD.39, 51
Docking studies of FSBA at both ATP sites show the sulfonyl fluoride moiety of FSBA (which is the reactive group) is located close to the position normally occupied by the β- and γ-phosphate of ATP (see ). Therefore, residues such as the cysteine, serine and lysine of Walker A, the histidine of the H-loop and the serine of the signature motif are potential targets for FSBA. However, all those residues (except the H loop-histidine) are not properly oriented for chemical reaction to the partially positively charged sulfur of FSBA. The H loop-histidine seems to be suitably oriented for reaction, but is too far (more than 5Å) from the reactive fluorosulfonyl group. In addition, LC/MS/MS analysis of P-gp labeled with FSBA did not yield any fragment with residues responsible for binding ATP ( and ). On the other hand, modeling studies yielded a reasonable explanation for the origin of fragment #2, and further structural analysis revealed that just small conformational changes at loops close to the ATP sites are necessary to explain the origin of fragments #4, #5, and #6. However, the fact that ATP does not prevent crosslinking of three fragments out of seven suggests that FSBA is also binding to P-gp at other sites near the NBDs (see comments column in ).
Tyrosine kinase inhibitors, which are developed based on their ability to bind to the catalytic site and prevent ATP binding (gleevec, nilotinib, etc.), interact at the drug-substrate sites of P-gp and ABCG2 but not at the ATP sites.52
FSBA is, to our knowledge, the first ATP analog that has an effect on the both the drug-substrate and ATP sites of P-gp, providing a unique and useful tool for biochemical studies of other ABC drug transporters including ABCG2, ABCC1. ABCC2, ABCC4 and ABCC7.