Recapitulating_the_frataxin_activation_mechanism_i
➤ Gửi thông báo lỗi ⚠️ Báo cáo tài liệu vi phạmNội dung chi tiết: Recapitulating_the_frataxin_activation_mechanism_i
Recapitulating_the_frataxin_activation_mechanism_i
bioRxiv preprint doi: https /i'dcl ora'10.1101,2020 10.06.326603; this version pasted October 6, 2Ọ2O The L-44.„_,(tttilch was not certified by peer r Recapitulating_the_frataxin_activation_mechanism_ireview) is the authonfunder. who has granted DioRxiv a license to display a nnps://Knoinuvien.comavailable under aCC.BY.NC.ND 4.0 International license.rRecapitulating the frataxin activation mechanism in an engineered bacterial cysteine desulfurase supports the architectural switch modelRunning tit Recapitulating_the_frataxin_activation_mechanism_ile: Architectural-based activation of cysteine desulfurasesShachin Patra, Cheng-Wei Lin, Manas K. Ghosh, Steven M. Havens, Seth A. Cory,David H. RusseRecapitulating_the_frataxin_activation_mechanism_i
ll, and David p. Barondeau*Department of Chemistry, Texas A&M University, College station. TX 77842, USA.*To whom correspondence should be addressed: bioRxiv preprint doi: https /i'dcl ora'10.1101,2020 10.06.326603; this version pasted October 6, 2Ọ2O The L-44.„_,(tttilch was not certified by peer r Recapitulating_the_frataxin_activation_mechanism_ir cluster, frataxin, native mass spectrometry. Friedreich's ataxia, stop ped-flowAuthor Contributions: S.P., C.L., M.K.G., and S.M.H. performed the research; all authors were involved in the design of the research, analyzing data, and writing the manuscript.bicR-xlv preprint doi: https /i'dd ora'10. Recapitulating_the_frataxin_activation_mechanism_i1 101i2O2Ọ 10.06.326603; this version posted October 6, 2020 The L-XA-_. i~(which was not certified by peer review) is the authonfunder. who has grantRecapitulating_the_frataxin_activation_mechanism_i
ed cioRxiv a license to display 0 nups://Knoinuvien.comavailable under aCC-BY-NC.ND 4.0 International license.'ABSTRACTIron-sulfur (Fe-S) clusters havbioRxiv preprint doi: https /i'dcl ora'10.1101,2020 10.06.326603; this version pasted October 6, 2Ọ2O The L-44.„_,(tttilch was not certified by peer r Recapitulating_the_frataxin_activation_mechanism_ir biosynthetic pathway, critical questions remain unresolved. Although human NFS1 and E. coli IscS share -60% sequence identity, NFS1 exhibits low activity and requires activation by the Friedreich's ataxia protein frataxin (FXN) for in vivo function. Surprisingly, structures of the human complex re Recapitulating_the_frataxin_activation_mechanism_iveal three distinct quaternary structures with one form exhibiting the same subunit interactions as IscS. An architectural switch model has been propoRecapitulating_the_frataxin_activation_mechanism_i
sed in which evolutionarily lost interactions between NFS1 subunits results in the formation of low-activity architectures; FXN binding compensates fobioRxiv preprint doi: https /i'dcl ora'10.1101,2020 10.06.326603; this version pasted October 6, 2Ọ2O The L-44.„_,(tttilch was not certified by peer r Recapitulating_the_frataxin_activation_mechanism_iidentify three conserved residues proposed to weaken interactions between NFS1 subunits and transplanted these amino acids into IscS. Compared to native IscS, the engineered variant had a 4000-fold weaker dimer interface and diminished activity that correlated with the absence of the second catalyti Recapitulating_the_frataxin_activation_mechanism_ic subunit. Remarkably, the addition of the FXN homolog to the engineered variant stimulated the decay of the Cys-quinonoid pyridoxal 5’-phosphate inteRecapitulating_the_frataxin_activation_mechanism_i
rmediate, shifted IscS from the monomeric to dimeric form, and increased the cysteine desulfurase activity, reproducing results from the human system bioRxiv preprint doi: https /i'dcl ora'10.1101,2020 10.06.326603; this version pasted October 6, 2Ọ2O The L-44.„_,(tttilch was not certified by peer r Recapitulating_the_frataxin_activation_mechanism_ihe evolution of the eukaryotic system and provide new insights into the role of FXN.bioRxiv preprint doh https /i'Od ora' 10.1101,2020 10.06.326603; this version posteo October 6, 2020 The L£X„ _, iII,u -IL„(which was not certified by peer review) is the authorrtunder. who has granted DioRxiv a lice Recapitulating_the_frataxin_activation_mechanism_inse to display Ứ nnps://Knoinuvien.comavailable under aCC-BY-NC.NO 4.0 International license.rINTRODUCTIONIron-sulfur (Fe-S) clusters are ubiquitous pRecapitulating_the_frataxin_activation_mechanism_i
rotein cofactors that play critical roles in a variety of cellular processes such as aerobic respiration and the biosynthesis of DNA. RNA. proteins, mbioRxiv preprint doi: https /i'dcl ora'10.1101,2020 10.06.326603; this version pasted October 6, 2Ọ2O The L-44.„_,(tttilch was not certified by peer r Recapitulating_the_frataxin_activation_mechanism_ie assembly and insertion of appropriate Fe-S cluster cofactors for enzymes in these processes (2.3). The best-studied Fe-S biosynthetic pathway is the ISC system, likely due to its wide-spread occurrence in prokaryotes and its critical role in the mitochondrial matrix of eukaryotes (4). Despite the Recapitulating_the_frataxin_activation_mechanism_ibacterial and mitochondrial ISC pathways having analogous proteins with high sequence homology and similar mechanisms for the formation and distributiRecapitulating_the_frataxin_activation_mechanism_i
on of Fe-S cluster cofactors, there are some notable differences. In particular, the eukaryotic Fe-S assembly system appears to have incorporated new bioRxiv preprint doi: https /i'dcl ora'10.1101,2020 10.06.326603; this version pasted October 6, 2Ọ2O The L-44.„_,(tttilch was not certified by peer r Recapitulating_the_frataxin_activation_mechanism_iding evolutionary modifications to these systems are therefore critical for elucidating the regulatory and enzymatic mechanisms of Fe-S cluster assembly and for treating human diseases associated with defects in the ISC biosynthetic pathway, including the incurable neurodegenerative disease Friedrei Recapitulating_the_frataxin_activation_mechanism_ich’s Ataxia (FRDA) (7).The eukaryotic modifications to the ISC system are focused around the central enzyme in the pathway, the pyridoxal-5-phosphateRecapitulating_the_frataxin_activation_mechanism_i
(PLP) dependent cysteine desulfurase that converts L-cysteine into L-alanine and provides sulfur for Fe-S cluster synthesis on the scaffold protein IsbioRxiv preprint doi: https /i'dcl ora'10.1101,2020 10.06.326603; this version pasted October 6, 2Ọ2O The L-44.„_,(tttilch was not certified by peer r Recapitulating_the_frataxin_activation_mechanism_ireprint ơọi: httpsWd ora110.1101.2020 10.06.326603; this versxxi postep October 6, 2020 The IA(which was not certified by peer review) is the author.funder. who has granted bioRxiv a license to display 0 nups://Knouiuvien.comavailable under aCC-BY4VC.NO 4.0 International license.'13). In contrast, t Recapitulating_the_frataxin_activation_mechanism_ihe human cysteine desulfurase NFS1, which is 60% identical to E. coli IscS, is unstable, essentially inactive, and prone to aggregation (14). NFS1 isRecapitulating_the_frataxin_activation_mechanism_i
stabilized by forming a functional complex with the eukaryotic-specific LYRM protein ISD11 (15.16) and the mitochondrial acyl carrier protein (ACP) (5bioRxiv preprint doi: https /i'dcl ora'10.1101,2020 10.06.326603; this version pasted October 6, 2Ọ2O The L-44.„_,(tttilch was not certified by peer r Recapitulating_the_frataxin_activation_mechanism_iver an order of magnitude by the Friedreich’s ataxia protein frataxin (FXN) in a scaffold protein (ISCU2) dependent manner (18-20). This activity is further stimulated by the presence of iron. Interestingly, both FXN and the E. coli homolog CyaY stimulate the cysteine desulfurase activity of the euk Recapitulating_the_frataxin_activation_mechanism_iaryotic complex but do not impact the cysteine desulfurase activity in the bacterial system (9,21). Overall, these data point to fundamental differencRecapitulating_the_frataxin_activation_mechanism_i
es in the bacterial and mitochondrial cysteine desulfurase enzymes that engender distinct regulation for the assembly of Fe-S clusters.Three differentbioRxiv preprint doi: https /i'dcl ora'10.1101,2020 10.06.326603; this version pasted October 6, 2Ọ2O The L-44.„_,(tttilch was not certified by peer r Recapitulating_the_frataxin_activation_mechanism_iions between catalytic subunits similar to IscS, whereas two forms have distinct alternate protein interfaces. The first “Open" structure exhibited an O20Z;'2 quaternary structure with ISD11 molecules mediating interactions between two NFS1-ISD11-ACPec protomers (Fig. 1A). Unlike the extensive dimer Recapitulating_the_frataxin_activation_mechanism_i interface in the IscS structure (10,11), there are few direct interactions between the NFS1 subunits in this open architecture. As a consequence, theRecapitulating_the_frataxin_activation_mechanism_i
channel that guides the mobile S-transfer loop cysteine to the active site for the intermolecular sulfur transfer reaction is incomplete and the PLP bioRxiv preprint doi: https /i'dcl ora'10.1101,2020 10.06.326603; this version pasted October 6, 2Ọ2O The L-44.„_,(tttilch was not certified by peer r Recapitulating_the_frataxin_activation_mechanism_icS (Fig. 1B), but with a relativebioRxlv preprint doi: https .■•coi pra-10 IIOI.'ZOZO 10 06.326603; mis version posted October 6, 2020 The(which was not certified by peer review) is the authofifunde-. who has granted cioRxiv a license to display u nnps://Knoinuvien.comavailable under aCC.BY-NC.ND 4. Recapitulating_the_frataxin_activation_mechanism_i0 International license.rrotation of the catalytic subunits that move the PLP cofactors 5 A closer together and place structural elements from the othRecapitulating_the_frataxin_activation_mechanism_i
er subunit in a position to potentially inhibit the trajectory of the mobile S-transfer loop. The third "ready" architecture (Fig. 1C), which has onlybioRxiv preprint doi: https /i'dcl ora'10.1101,2020 10.06.326603; this version pasted October 6, 2Ọ2O The L-44.„_,(tttilch was not certified by peer r Recapitulating_the_frataxin_activation_mechanism_ih NFS1-NFS1 interactions similar to the IscS structure (Fig. 1D) (10,11). These different architectures raised questions as to which of these forms govern the solution behavior of the human cysteine desulfurase and result in the different biochemical properties compared to the analogous prokaryotic Recapitulating_the_frataxin_activation_mechanism_ienzymes.More recently, stopped-flow and radiolabeling experiments coupled to previous biochemical studies revealed a link between the function of theRecapitulating_the_frataxin_activation_mechanism_i
NFS1 mobile S-transfer loop cysteine and FXN-based activation of the cysteine desulfurase complex (24-26). The addition of FXN was shown to facilitateGọi ngay
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