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Prion Protein-Detergent Micelle Interactions Studied by NMR in Solution

2009; Elsevier BV; Volume: 284; Issue: 34 Linguagem: Inglês

10.1074/jbc.m109.000430

1083-351X

Simone Hornemann, Christine von Schroetter, Fred F. Damberger, Kurt Wüthrich,

Infectious Encephalopathies and Encephalitis

Cellular prion proteins, PrPC, carrying the amino acid substitutions P102L, P105L, or A117V, which confer increased susceptibility to human transmissible spongiform encephalopathies, are known to form structures that include transmembrane polypeptide segments. Herein, we investigated the interactions between dodecylphosphocholine micelles and the polypeptide fragments 90–231 of the recombinant mouse PrP variants carrying the amino acid replacements P102L, P105L, A117V, A113V/A115V/A118V, K110I/H111I, M129V, P105L/M129V, and A117V/M129V. Wild-type mPrP-(90–231) and mPrP[M129V]-(91–231) showed only weak interactions with dodecylphosphocholine micelles in aqueous solution at pH 7.0, whereas discrete interaction sites within the polypeptide segment 102–127 were identified for all other aforementioned mPrP variants by NMR chemical shift mapping. These model studies thus provide evidence that amino acid substitutions within the polypeptide segment 102–127 affect the interactions of PrPC with membranous structures, which might in turn modulate the physiological function of the protein in health and disease. Cellular prion proteins, PrPC, carrying the amino acid substitutions P102L, P105L, or A117V, which confer increased susceptibility to human transmissible spongiform encephalopathies, are known to form structures that include transmembrane polypeptide segments. Herein, we investigated the interactions between dodecylphosphocholine micelles and the polypeptide fragments 90–231 of the recombinant mouse PrP variants carrying the amino acid replacements P102L, P105L, A117V, A113V/A115V/A118V, K110I/H111I, M129V, P105L/M129V, and A117V/M129V. Wild-type mPrP-(90–231) and mPrP[M129V]-(91–231) showed only weak interactions with dodecylphosphocholine micelles in aqueous solution at pH 7.0, whereas discrete interaction sites within the polypeptide segment 102–127 were identified for all other aforementioned mPrP variants by NMR chemical shift mapping. These model studies thus provide evidence that amino acid substitutions within the polypeptide segment 102–127 affect the interactions of PrPC with membranous structures, which might in turn modulate the physiological function of the protein in health and disease. Transmissible spongiform encephalopathies (TSEs), 2The abbreviations used are: TSEtransmissible spongiform encephalopathyDPCdodecylphosphocholineGPIglycosylphosphatidylinositolHSQCheteronuclear single quantum coherencePrPprion proteinmPrPmouse prion proteinAPPamyloid precursor protein. such as Creutzfeldt-Jakob disease and the Gerstmann-Sträussler-Scheinker syndrome in humans, are accompanied by the appearance in the brain of an aggregated "scrapie" isoform of the host-encoded prion protein, PrPSc (1Prusiner S.B. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 13363-13383Crossref PubMed Scopus (5167) Google Scholar, 2Collinge J. Annu. Rev. Neurosci. 2001; 24: 519-550Crossref PubMed Scopus (1113) Google Scholar, 3Weissmann C. Enari M. Klöhn P.C. Rossi D. Flechsig E. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 16378-16383Crossref PubMed Scopus (102) Google Scholar). The cellular form, PrPC, consists of an unstructured N-terminal "tail" of residues 23–125 and a globular domain of residues 126–231, and is attached by a C-terminal glycosylphosphatidylinositol (GPI) anchor to the outer plasma membrane. This structure ensures a role of membrane interactions in the physiological function of PrPC and probably also in the disease-related events leading to TSEs. For example, transgenic mice expressing a prion protein variant lacking the GPI membrane anchor did not develop the typical clinical signs of TSE after inoculation with infectious brain homogenate, although significant amounts of PrPSc accumulated in the brain (4Chesebro B. Trifilo M. Race R. Meade-White K. Teng C. LaCasse R. Raymond L. Favara C. Baron G. Priola S. Caughey B. Masliah E. Oldstone M. Science. 2005; 308: 1435-1439Crossref PubMed Scopus (536) Google Scholar). This finding led to the conclusion that membrane-association of PrPC is necessary for the development of a TSE. Independent evidence for the importance of membrane interactions for the onset of prion diseases was derived from cell-free conversion assays and cell culture experiments (5Baron G.S. Wehrly K. Dorward D.W. Chesebro B. Caughey B. EMBO J. 2002; 21: 1031-1040Crossref PubMed Scopus (238) Google Scholar, 6Baron G.S. Caughey B. J. Biol. Chem. 2003; 278: 14883-14892Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). transmissible spongiform encephalopathy dodecylphosphocholine glycosylphosphatidylinositol heteronuclear single quantum coherence prion protein mouse prion protein amyloid precursor protein. Data have also been presented that indicate that in addition to the normal form with the C terminus linked to a GPI anchor and the C-terminal domain located on the cell surface, PrPC can adopt two different transmembrane topologies, CtmPrP and NtmPrP, which have the C-terminal polypeptide segment located in the lumen of the endoplasmic reticulum (CtmPrP) or in the cytoplasm (NtmPrP) (7Harris D.A. Br. Med. Bull. 2003; 66: 71-85Crossref PubMed Scopus (142) Google Scholar, 8Harrison C.F. Barnham K.J. Hill A.F. J. Neurochem. 2007; 103: 1709-1720Crossref PubMed Scopus (13) Google Scholar, 9Hegde R.S. Voigt S. Lingappa V.R. Mol. Cell. 1998; 2: 85-91Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The population of the CtmPrP variant is mPrP[A117V]-(90–231) > mPrP[P102L]-(91–231). Only weak interactions were observed for mPrP-(90–231) and mPrP[M129V]-(91–231). Within the aforementioned binding regions, the largest chemical shift changes were observed for the residues Val113, Val118, and Gly123 in mPrP[A113V,A115V,A118V]-(90–231), and for the residues Val112, Ala113, and Gly123 in mPrP[K110I,H111I]-(90–231). The resonances that show the largest 15N chemical shift changes upon addition of DPC also exhibit the greatest degree of line-broadening. Comparison of pairs of proteins carrying either methionine or valine at position 129, with otherwise identical amino acid sequences, showed that they have nearly identical 15N chemical shift changes upon addition of DPC (in Fig. 3, compare A and B, D and E, and F and G, respectively), indicating that the M129V polymorphism does not noticeably influence the interactions of the proteins with DPC. To further investigate the nature of the detergent interactions causing the 15N chemical shift changes in Fig. 3, two-dimensional 15N,1H-correlation NMR spectra of mPrP[A113V,A115V,A118V]-(90–231) were measured during a titration with small DPC concentrations. Below the critical micelle concentration of 1.5 mm, no changes in the 15N chemical shifts were observed, indicating that the protein does not interact with DPC monomers, but is rather interacting with DPC micelles (Fig. 4). To obtain an estimate of the stability of the PrP-detergent micelle complexes, we measured 15N chemical shift changes for selected well resolved resonances upon stepwise addition of DPC in the range above the cmc up to a concentration of 40 mm. Fig. 5 shows for all the proteins studied that over this range of concentrations the upfield chemical shift changes increase monotonously with increasing DPC concentrations. The slope of the 15N chemical shift changes versus the DPC concentration over the near-linear part of the curves in Fig. 5 was highest for mPrP[A113V,A115V,A118V]-(90–231) and mPrP[K110I,H111I]-(90–231), and for the other proteins it decreased in the order mPrP[P105L]-(91–231), mPrP[P105L,M129V]-(91–231), mPrP[A117V]-(90–231), mPrP[A117V,M129V]-(90–231), mPrP[P102L]-(91–231), mPrP[M129V]-(90–231), and mPrP-(90–231) (Fig. 5). The addition of DPC micelles also caused line broadening and precipitation of variable fractions of the individual proteins. For mPrP[A113V,A115V,A118V]-(90–231) and mPrP[K110I,H111I]-(90–231), these side effects limited the data analysis to DPC concentrations below 18.6 and 6.8 mm, respectively (Fig. 5, H and I). Although the 15N chemical shifts show a linear dependence on the detergent concentration over most of the DPC concentration range covered in Fig. 5, saturation at the higher DPC concentrations could be measured and fitted with Equation 1 (see "Experimental Procedures") for mPrP[P105L]-(91–231), which shows the strongest binding among the proteins for which data could be obtained up to 40 mm DPC concentration (Fig. 5). This approach yielded a dissociation constant, Kd, of 0.9 ± 0.2 mm, which can be considered to represent an upper limit of the affinity of DPC micelles for the PrP species of Fig. 1C with the exception of the last two species listed. These may bind more tightly. However, the limited solubility of these two species at elevated DPC concentrations prevents a quantitative statement regarding their affinity for DPC micelles. The proteins mPrP[P105L]-(91–231) and mPrP[A117V]-(90–231) were selected for further studies, based on the fact that they show large chemical shift changes upon interaction with DPC and are associated with TSEs in humans, to quantify the population and determine the residue boundaries of helical conformation induced by the interaction with the micelles. The 13Cα downfield chemical shifts induced by the addition of DPC in these two proteins (Fig. 6, B and C) indeed manifest increased α-helical character of the polypeptide segments 102–124 and 110–122, respectively, which had independently been identified as the DPC binding regions (Fig. 3). Wild-type mPrP-(90–231) was used as a control, and it showed no specific DPC effects on 13Cα shifts (Fig. 6A). The striking result of this in vitro study is that amino acid exchanges in PrP, which are ot