Study On Structure And Dynamics Of The Pathogenic Mutant G131V Of Human Prion Protein

Human Prion Diseases is a class of fatal neurodegenerative diseases that can cause degeneration of the central nervous system(CNS)of the Human brain.The pathogenic process is related to the conformation of Cellular form human prion protein(HuPrPc)into the Scrapie isoform human prion protein(Hu PrPSc).Pathogenic point mutations of prion proteins typically facilitate conformational conversion.Previous studies have demonstrated that the pathogenic G131V mutation of human prion protein(HuPrP)brings in Gerstmann-Straussler-Scheinker syndrome.Additionally,our previous work reported profound differences in 2D 1H-15N HSQC spectra between HuPrPc(G131V)and WT HuPrPC proteins,which implied that the G131 mutation-induced significant conformational changes in HuPrP.However,the pathogenic molecular mechanism of mutant G131V is still unclear.Expectedly,elucidation the pathogenic molecular mechanism of G131V mutant could facilitate the development of novel methods for the treatment and diagnosis of human prion diseases.In order to reveal the pathogenic molecular mechanism of HuPrPC(G131V),the solution structure of human prion pathogenic mutant G131V(amino acid residues of Gln91-Ser231,abbreviated as HuPrP G131V(91-231))was analyzed by heteronuclear multidimensional nuclear magnetic resonance(NMR)techniques.Through NMR methods,we analyzed the backbone dynamics properties of HuPrP G131V(91-231)and mapped the binding sites on it for binding GN8,an intermediate product in the organic synthesis pathway of GN8.The main results in this study are listed as follows:(1)We determined the solution structure of HuPrP G131V(91-231),and the RMSD of backbone atom(125-228)RMSD was 1.19±0.28 A.In the Ramachandran plot,89.7%of the amino acid residues are in the most favored region,and 9.3%are in the additional allowed region.1.0%is in the generously allowed regions,and no amino acid residue is in the disallowed regions.The evidences indicat that the overall structure is better.(2)The solution structure of HuPrP G131V(91-231)was significantly different from that of the wild type,and other known disease-resistant or pathogenic mutants of human prions,primarily characterized by the fragments of Tyr128-Val131 and Val161-Arg164.The solution structure of HuPrP G131V(91-231)displayed the following distinctions:Firstly,after the mutation of Gly131 to a hydrophobic valine(Val131),the atomic distances between Val131 and both the hydrophilic residues Tyr163 and Gln217 increased significantly compared to those in the wild type,preventing fragment Tyr128-Val131 from forming hydrogen bonds with fragment Val161-Arg164 and reducing the former fragment into a flexible Loop;secondly,due to the lack of the hydrogen bond restriction and steric hindrance from the Tyr128-Val131 fragment,the gap between the Val161-Arg164 fragment and the α3 helix increases,leading to the increase in the αl-α2 gap and in the interatomic distance betweenal and α3.Compared to WT HuPrP(91-231),these changes resulted in a looser interhelical stacking in HuPrP G131V(91-231);finally,fragments Tyr128-Val131 and Val161-Arg164 exhibited neutral surface electrostatics,a rare case among human prion proteins and mutants.(3)The NMR dynamic data of HuPrP G131V(91-231)showed that the N-terminal region and C-terminal region showed obvious flexibility.Val189 and Tyr128 of HuPrP G131V(91-231)have higher transverse relaxation rates(R2)and lower spectral density function J(0).CPMG experiments showed that Val189 and Tyr128 exhibited on μs-ms timescales conformational chemical exchange.The slow conformational chemical exchange of Tyr128 may have caused the fragment Tyr128-Val131 to form a flexible Loop rather than a β-fold or Stretch-Strands(SSs)pattern.(4)The affinity and binding site of HuPrP G131V(91-231)with molecular chaperone GN8(2-pyrrolidin-1-The affinity and binding site of yl-N-[4-[4-(2-pyrrolidin-1-yl-acetylamino)-benzyl]-phenyl]-acetamide)was analyzed by nuclear magnetic resonance technique.The results showed that HuPRP G131V(91-231)had a moderate interaction with small fraction GN8,and its potential binding sites were 19 such as L125,M134,H140,H155,1184,Q186,N197,V203,V209,and E219.In this study,the solution structure,dynamics properties,and mapping of the binding sites by GN8 and GN8 of HuPrP G131V(91-231)were studied in detail,and the potential reasons for the conformational transformation of HuPrP were elucidated.These results provide a structural basis for further study of the conformational transformation process of PrPC,and also provide a theoretical basis for revealing the pathogenic molecular mechanism and drug target of Prion.