Figure 2
Surface-exposed Trp114 links TrxR1 dimers into tetramers, communicates with the FAD and is required for full enzyme activity. (a) Crystal structure of the TrxR1 tetramer, showing two dimers (yellow/purple and green/blue) linked together by the interaction of two modified Trp114 residues (middle, surface between the yellow and blue subunits of the two separate dimers; here with their modified side chains indicated by the composite omit electron density map). (b) Close-up of the final 2Fo-Fc map at 1σ of the region where two modified Trp114 side chains link the two TrxR1 dimers in the tetramer structure (one dimer here colored green and the other yellow). The electron density spanning the linkage could not be modeled with any known modifications of Trp residues and the Trp114 side chains are therefore only shown as electron density. Neighboring residues from each dimer are indicated with their three letter codes. (c) NBS irreversibly inhibits TrxR1. For this experiment, wild-type dimeric TrxR1 was incubated in the dark with NBS at the indicated concentrations for 60 min, desalted and subsequently enzyme activities were measured with either DTNB or Trx-dependent insulin reduction. (d) Free Trp in solution protects TrxR1 from inhibition by incubation for 120 min with 50 μM NBS. (e) 3D fluorescence excitation/emission spectra reveal long-range communication between Trp114 and FAD. Typical enzyme-bound flavin fluorescence53 at Emmax≈520 nm has three excitation peaks of Exmax≈280 nm, ≈370 nm and ≈470 nm as seen in wild-type TrxR1, whereas one of these fluorescence peaks, Exmax≈470 nm/Emmax≈520 nm (block arrows), was virtually absent in the Trp114 variants, where instead a new peak at Exmax≈370 nm/Emmax≈440 nm was seen (thin arrows). (f) Representative EPR analysis of wild-type TrxR and the W114R variant. For these spectra, TrxR (300 μM dimer) was incubated with 900 μM NADPH for 80 s. In the absence of NADPH, the oxidized enzymes did not show this signal, nor did NADPH without enzyme. The bar graph at right shows the relative EPR signal intensities of wild-type TrxR (wtTrxR) and the W114R variant incubated with an excess of NADPH to generate the reduced (EH4) state of the enzyme, or with a limiting amount of NADPH to generate a partially reduced (EH2) state. *P<0.05 or **P<0.01 for W114R versus wtTrxR. EPR instrument settings are described in the Materials and methods section. (g) Immunoblot detection of different TrxR1 species with antibodies against either TrxR1 (top) or the FLAG-tag (bottom) as resolved on reducing SDS-PAGE analyses with lysates from HCT116 cells transfected for expression of FLAG-tagged wild-type TrxR1 (WT), the W114R mutant or non-transfected controls, treated with either RITA, RITA and NDGA or using non-treated control cells (as indicated)
