Ser170 controls the conformational multiplicity of the loop 166–175 in prion proteins: implication for conversion and species barrier (original) (raw)
Related papers
Journal of Molecular Graphics and Modelling, 2001
Inherited forms of transmissible spongiform encephalopathy, e.g. familial Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker syndrome and fatal familial insomnia, segregate with specific point mutations of the prion protein. It has been proposed that the pathologically relevant Asp178Asn (D178N) mutation might destabilize the structure of the prion protein because of the loss of the Arg164-Asp178 salt bridge. Molecular dynamics simulations of the structured C-terminal domain of the murine prion protein and the D178N mutant were performed to investigate this hypothesis. The D178N mutant did not deviate from the NMR conformation more than the wild type on the nanosecond time scale of the simulations. In agreement with CD spectroscopy experiments, no major structural rearrangement could be observed for the D178N mutant, apart from the N-terminal elongation of helix 2. The region of structure around the disulfide bridge deviated the least from the NMR conformation and showed the smallest fluctuations in all simulations in agreement with hydrogen exchange data of the wild type prion protein. Large deviations and flexibility were observed in the segments which are ill-defined in the NMR conformation. Moreover, helix 1 showed an increased degree of mobility, especially at its N-terminal region. The dynamic behavior of the D178N mutant and its minor deviation from the folded conformation suggest that the salt bridge between Arg164 and Asp178 might not be crucial for the stability of the prion protein.
The role of the 132-160 region in prion protein conformational transitions
Protein Science, 2005
The native conformation of host-encoded cellular prion protein (PrP C ) is metastable. As a result of a post-translational event, PrP C can convert to the scrapie form (PrP Sc ), which emerges as the essential constituent of infectious prions. Despite thorough research, the mechanism underlying this conformational transition remains unknown. However, several studies have highlighted the importance of the N-terminal region spanning residues 90-154 in PrP folding. In order to understand why PrP folds into two different conformational states exhibiting distinct secondary and tertiary structure, and to gain insight into the involvement of this particular region in PrP transconformation, we studied the pressure-induced unfolding/ refolding of recombinant Syrian hamster PrP expanding from residues 90-231, and compared it with heat unfolding. By using two intrinsic fluorescent variants of this protein (Y150W and F141W), conformational changes confined to the 132-160 segment were monitored. Multiple conformational states of the Trp variants, characterized by their spectroscopic properties (fluorescence and UV absorbance in the fourth derivative mode), were achieved by tuning the experimental conditions of pressure and temperature. Further insight into unexplored conformational states of the prion protein, likely to mimic the in vivo structural change, was obtained from pressure-assisted cold unfolding. Furthermore, salt-induced conformational changes suggested a structural stabilizing role of Tyr150 and Phe141 residues, slowing down the conversion to a -sheet form. Article and publication are at http://www.proteinscience.org/cgi/doi/ 10.1110/ps.04989405. Protein Science (2005), 14:956-967.
Unique Structural Characteristics of the Rabbit Prion Protein
Journal of Biological Chemistry, 2010
Rabbits are one of the few mammalian species that appear to be resistant to transmissible spongiform encephalopathies (TSEs) due to the structural characteristics of the rabbit prion protein (RaPrP C ) itself. Here we determined the solution structures of the recombinant protein RaPrP C -(91-228) and its S173N variant, and detected the backbone dynamics of their structured C-terminal domains-(121-228). In contrast to many other mammalian PrP C s, loop 165-172 that connects β-sheet-2 and α-helix-2 is well-defined in RaPrP C . For the first time, order parameters S 2 are obtained for residues in this loop region, indicating that loop 165-172 of RaPrP C is highly ordered. Compared with the wild-type RaPrP C , less hydrogen bonds form in the S173N variant. The NMR dynamics analysis reveals a distinct increase in the structural flexibility of loop 165-172 and helix-3 after the S173N substitution, implying that the S173N substitution disturbs the long-range interaction of loop 165-172 with helix-3, which further leads to a marked decrease in the global conformational stability. Significantly, RaPrP C possesses a unique charge distribution, carrying a continuous area of positive charges on the surface, which is distinguished from other PrP C s. The S173N substitution causes visible changes of the charge distribution around the recognition sites for the hypothetical protein X. Our results suggest that the ordered loop 165-172 and its interaction with helix-3, together with the unique distribution of surface electrostatic potential, significantly contribute to the unique structural characteristics of RaPrP C .
Cellular prion protein conformation and function
Proceedings of the National Academy of Sciences, 2011
In the otherwise highly conserved NMR structures of cellular prion proteins (PrP C ) from different mammals, species variations in a surface epitope that includes a loop linking a β-strand, β2, with a helix, α2, are associated with NMR manifestations of a dynamic equilibrium between locally different conformations. Here, it is shown that this local dynamic conformational polymorphism in mouse PrP C is eliminated through exchange of Tyr169 by Ala or Gly, but is preserved after exchange of Tyr 169 with Phe. NMR structure determinations of designed variants of mouse PrP(121-231) at 20°C and of wild-type mPrP(121-231) at 37°C together with analysis of exchange effects on NMR signals then resulted in the identification of the two limiting structures involved in this local conformational exchange in wild-type mouse PrP C , and showed that the two exchanging structures present characteristically different solvent-exposed epitopes near the β2-α2 loop. The structural data presented in this paper provided a platform for currently ongoing, rationally designed experiments with transgenic laboratory animals for renewed attempts to unravel the so far elusive physiological function of the cellular prion protein.
PLoS pathogens, 2017
Prion diseases are infectious neurodegenerative disorders of humans and animals caused by misfolded forms of the cellular prion protein PrPC. Prions cause disease by converting PrPC into aggregation-prone PrPSc. Chronic wasting disease (CWD) is the most contagious prion disease with substantial lateral transmission, affecting free-ranging and farmed cervids. Although the PrP primary structure is highly conserved among cervids, the disease phenotype can be modulated by species-specific polymorphisms in the prion protein gene. How the resulting amino-acid substitutions impact PrPC and PrPSc structure and propagation is poorly understood. We investigated the effects of the cervid 116A>G substitution, located in the most conserved PrP domain, on PrPC structure and conversion and on 116AG-prion conformation and infectivity. Molecular dynamics simulations revealed structural de-stabilization of 116G-PrP, which enhanced its in vitro conversion efficiency when used as recombinant PrP sub...
Science, 1996
The fundamental event in prion diseases seems to be a conformational change in cellular prion protein (PrP C ) whereby it is converted into the pathologic isoform PrP Sc . In fatal familial insomnia (FFI), the protease-resistant fragment of PrP Sc after deglycosylation has a size of 19 kilodaltons, whereas that from other inherited and sporadic prion diseases is 21 kilodaltons. Extracts from the brains of FFI patients transmitted disease to transgenic mice expressing a chimeric human-mouse PrP gene about 200 days after inoculation and induced formation of the 19-kilodalton PrP Sc fragment, whereas extracts from the brains of familial and sporadic Creutzfeldt-Jakob disease patients produced the 21-kilodalton PrP Sc fragment in these mice. The results presented indicate that the conformation of PrP Sc functions as a template in directing the formation of nascent PrP Sc and suggest a mechanism to explain strains of prions where diversity is encrypted in the conformation of PrP Sc .
N-terminus (Y145STOP) fragment of the prion protein plays a role in prion misfolding
2012
Prion diseases are transmissible protein misfolding disorders in which misfolding of a host-encoded prion protein (PrP) occurs. They comprise of a group of distinct diseases in animals and humans, which show similar clinical and neuropathological changes. Human prion diseases can arise sporadically, be hereditary or be acquired. Sporadic human prion diseases include Cruetzfeldt-Jacob disease (CJD) and fatal insomnia. Genetic or familial prion diseases are caused by autosomal dominantly inherited mutations in the gene encoding for PrP C and include familial or genetic CJD, fatal familial insomnia and Gerstmann-Sträussler-Scheinker syndrome. Acquired human prion diseases account for only 5% of cases of human prion disease. They include Kuru, iatrogenic CJD and a new variant form of CJD that is transmitted to humans from affected cattle via meat consumption. Despite considerable effort in understanding the structure of the prion protein, the precise role of different prion protein domains that may be important in basic misfolding is poorly understood. The linkage between Prnp mutations and hereditary prion disease provide support for the central role of PrP in pathogenesis, because the genetic disease can be propagated in an infectious way. One of the most intriguing disease-related mutations is the tyrosine to stop codon substitution at position 145; Y145Stop variant of prion protein. Y145Stop variant; PrP23-144, is the only truncated prion molecule that is linked to an autosomal dominant inherited genetic TSE, Gerstmann-Sträussler-Scheinker syndrome. This disease related mutation indicates an essential role of the N-terminus fragment of the prion protein in seeding and misfolding of the mammalian prions. Therefore, it was hypothesized that the N-terminus fragment of the prion protein plays a central role in the mechanism of prion conversion. v Studies were designed to test the hypothesis using a Protein Misfolding Cyclic Amplification (PMCA) assay in the presence or absence of preexisting prions. It was identified that recombinant Y145Stop had the propensity for spontaneous conversion to protease resistant isoforms. Systematic molecular investigations established that Y145Stop was able to induce PrP Sc formation in cell culture in comparison to other recombinant PrP fragments and prions extracted from infected brain tissues. Misfolded Y145Stop showed similar kinetics as naturally occurring PrP Sc in cell culture infectivity. Lastly, it was demonstrated that the toxicity of Y145Stop in the cell culture was correlated with apoptotic cell death. The toxic activity of the peptide was dependent on the activation of caspase and p38 MAP kinase pathway. These experiments established a critical role for Y145Stop molecule in prion conversion of recombinant and mammalian prions. Experimental evidence findings lead to propose a two-step phenomenon in prion misfolding whereby, the N-terminus first interacts with metals or polyanions leading to its misfolding that then catalyzes the conformational conversion of the structured Cterminus of the molecule. Taken together, data generated from these studies will provide better understanding of the prion conversion mechanism. Elucidating the role of the Nterminus in seeding and misfolding of mammalian prions has important implications for designing diagnostics and therapeutics of prion diseases, as well as for understanding pathogenic mechanisms operative in interspecies transmission. vi
Continuum of prion protein structures enciphers a multitude of prion isolate-specified phenotypes
Proceedings of the National Academy of Sciences, 2006
On passaging synthetic prions, two isolates emerged with incubation times differing by nearly 100 days. Using conformational-stability assays, we determined the guanidine hydrochloride (Gdn⅐HCl) concentration required to denature 50% of disease-causing prion protein (PrP Sc ) molecules, denoted as the [Gdn⅐HCl]1/2 value. For the two prion isolates enciphering shorter and longer incubation times, [Gdn⅐HCl]1/2 values of 2.9 and 3.7 M, respectively, were found. Intrigued by this result, we measured the conformational stabilities of 30 prion isolates from synthetic and naturally occurring sources that had been passaged in mice. When the incubation times were plotted as a function of the [Gdn⅐HCl] 1/2 values, a linear relationship was found with a correlation coefficient of 0.93. These findings demonstrate that (i) less stable prions replicate more rapidly than do stable prions, and (ii) a continuum of PrP Sc structural states enciphers a multitude of incubation-time phenotypes. Our data argue that cellular machinery must exist for propagating a large number of different PrP Sc conformers, each of which enciphers a distinct biological phenotype as reflected by a specific incubation time. The biophysical explanation for the unprecedented plasticity of PrP Sc remains to be determined.
Hot spots in prion protein for pathogenic conversion
Proceedings of the National Academy of Sciences, 2007
Prion proteins are key molecules in transmissible spongiform encephalopathies (TSEs), but the precise mechanism of the conversion from the cellular form (PrP C ) to the scrapie form (PrP Sc ) is still unknown. Here we discovered a chemical chaperone to stabilize the PrP C conformation and identified the hot spots to stop the pathogenic conversion. We conducted in silico screening to find compounds that fitted into a ''pocket'' created by residues undergoing the conformational rearrangements between the native and the sparsely populated high-energy states (PrP*) and that directly bind to those residues. Forty-four selected compounds were tested in a TSE-infected cell culture model, among which one, 2-pyrrolidin-1-