ABCMdb

PMID: 26324772

Geng J, Pogozheva ID, Mosberg HI, Raghavan M
Use of Functional Polymorphisms To Elucidate the Peptide Binding Site of TAP Complexes.
J Immunol. 2015 Oct 1;195(7):3436-48. doi: 10.4049/jimmunol.1500985. Epub 2015 Aug 31., [PubMed]

Sentences

No. Mutations Sentence Comment

129 ABCB3 p.Leu266Phe As shown in the peptide transport analysis (Fig. 2A, 2B), rTAP1/TAP2a(Q262R), rTAP1/TAP2a(S265P), and rTAP1/TAP2a (L266F) displayed transport efficiencies quite similar to the wild-type rTAP1/TAP2a complex for both peptides. Login to comment 133 ABCB3 p.Leu266Phe To address whether other polymorphic rTAP2 residues have synergistic effects, we introduced a combination of four mutations within TM2 and TM3, which resulted in the quadruple mutant construct rTAP1/TAP2a (E218M/Q262R/S265P/L266F). Login to comment 136 ABCB3 p.Leu266Phe Similar results were obtained for the effects of the single (E218M) and quadruple (E218M/Q262R/S265P/L266F) mutations in rTAP2a upon transport of a pair of decapeptides, RYWANATK*SR (RR) and RYWANATK*SF (RF). Login to comment 154 ABCB3 p.Cys362Ala To further investigate which cysteine residue within the internal cavity of the wild-type rTAP becomes conjugated with TC6R, two mutant constructs (rTAP1[C273A] and rTAP2a[C362A]) were generated and tested for cross-linking to the TC6R peptide. Login to comment 157 ABCB3 p.Cys362Ala In contrast, the rTAP1(C175A)/TAP2a and the rTAP1/TAP2a(C362A) mutants retained the ability to crosslink with the TC6R peptide (Fig. 3D). Login to comment 186 ABCB3 p.Leu266Phe The quadruple mutant of rat TAP, TAP1/TAP2a(E218M/Q262R/ S265P/L266F), is indicated as EQSL/MPRF. Login to comment 213 ABCB3 p.Cys362Ala Top panel, Cross-linking between TC2R or TC6R (1 mM) and wild-type or mutants, rTAP1(C175A)/TAP2a (T1C175A/T2), rTAP1/TAP2a(C362A) (T1/T2C362A), and rTAP1(C273A)/TAP2a (T1C273A/T2). Login to comment 227 ABCB3 p.Cys362Ala To avoid cross-linking to other cysteine residues in the vicinity of the cavity, the cysteine mutants were generated on the rTAP1(C273A)/TAP2a(C362A) background (named rTAP1*/TAP2a*) to remove the other cysteines that face the internal cavity of the TAP heterodimer (Fig. 3A). Login to comment 228 ABCB3 p.Ser251Cys We found that the TC2R peptide could be conjugated via BMOE only to rTAP1*(E436C)/TAP2a* but not to rTAP1*(S263C)/ TAP2a* or rTAP1*/TAP2a* (S251C) (Fig. 6B). Login to comment 232 ABCB3 p.Ser251Cys ABCB3 p.Ser251Ala In contrast, the lack of cross-linking of rTAP1*/TAP2a* (S251C) or rTAP1*(S263C)/TAP2a* mutants to TC2R could not be explained by the impaired binding of these mutants because transport and binding of the TR peptide by rTAP1/TAP2(S251A) and rTAP1(S263A)/TAP2 complexes were not reduced relative to the wild-type TAP (Supplemental Fig. 2). Login to comment 245 ABCB3 p.Leu266Phe Binding of indicated TR (A) or TV (B) peptides to wild-type rTAP1/TAP2a (shown as WT), single mutant rTAP1/TAP2a(E218M) and quadruple mutant rTAP1/TAP2a(E218M/Q262R/S265P/L266F) (shown as EQSL/MRPF) was assessed using BMOE-mediated cross-linking assays, performed as described in Fig. 3. Login to comment 295 ABCB3 p.Cys362Ala All constructs shown in the figure are on the rTAP1(C273A)/TAP2a(C362A) background (labeled as T1*/T2*). Login to comment