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Hamster coordinate hang-up
Structure of full-length hamster prion 29-231

Hamster coordinates held back at James lab

11 Dec 97 -- webmaster
Coordinates for hamster prion -- even the short version -- are still not available from the Protein Data Base, even though two papers have appeared that depend on these coordinates. Brookhaven said on 10 Dec 97 that "a problem was discovered" and the coordinates sent back to the authors to resolve but that there has been no response. The possibilities are:

  • they don't want to make the data available to other researchers, especially to threaders,
  • the coordinates contain an internal inconsistency or steric conflict that cannot be resolved,
  • it is some minor thing, they are simply too busy to deal with it right now.

    Structure of full-length hamster prion 29-231: N terminus is highlyİflexible

    PNAS Vol. 94, pp. 13452-13457, 9 Dec 1997
    D G Donne, JH Viles, D Groth, I Mehlhorn, TL James, FE Cohen, SB Prusiner, PE Wright, and HJ Dyson 
    Best snippets:
    The secondary structure of the recombinant Syrian hamster PrP of residues 29-231 is investigated...Most striking is the random-coil nature of chemical shifts for residues 30-124 in the full-length PrP. There may be transient interactions between the additional residues and the structured core. Backbone dynamics studies indicate that residues 29-124 are highly flexible. This plastic region could feature in the conversion of PrPC to PrPSc by template-assisted formation of beta-structure.

    Fragments lack the biologically important N-terminal octarepeat region ([P(Q/H)GGG(G/-)WGQ]x5) and do not contain all of the sites of pathologic point mutations (9)... The disordered loop between the last two helices in mouse121-231 shows a greater degree of order in hamster 90-231, and residues 113-125 show some interactions with the folded core of the protein. Because PrP can adopt two or more conformations, it is unclear whether fragments will display context-dependent structural behavior in the full protein.

    The largely conserved octarepeat region may play an important role in the conversion of PrPC to PrPSc, either by participation in the profound conformational change, or in nonspecific binding to membranes (14) in the subcellular compartment where the conversion takes place (15). An earlier study of an octarepeat peptide by CD spectroscopy suggested that the repeat region adopts a nonrandom, extended conformation with the properties of a poly-L-proline left-handed helix (14). Here, the secondary structure of hamster 29-231 is determined, and the dynamic properties of the protein backbone measured. We postulate a plausible role for the octarepeat region in prion disease pathogenesis. Similar results were obtained earlier with full length mouse prion(16).

    NMR samples were prepared in 20İmM sodium acetate-d5 buffer at a final pH of 5.2İin 90% 1H2O/10% 2H2O. All solutions contained 0.005% sodium azide to inhibit bacterial growth [ie, no EDTA mentioned, no copper added --webmaster]. The protein was found to be monomeric and stable for up to 3İmonths at pH 5.2İand 30ƒC

    Resonances of corresponding residues in four of five octarepeats (PHGGGWGQ) essentially were unresolved. The first octarepeat has a slightly different sequence, PQGGGTWGQ [rodents are anomalous in having a threonine in this position, all other species have glycine or a deletion. -- webmaster] from the other four, and the GTW portion could be assigned sequence-specifically. Residues in the same position in the remaining four octarepeats were degenerate in all frequencies.

    [Reference repeats -- webmaster]
    human        pqggggwgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq
    sheep        pqggggwgq.phgggwgq.phgggwgq.phgggwgq.phggggwgq
    hamster      pqgggtwgq.phgggwgq.phgggwgq.phgggwgq.phgggwgq
    mice 	     pqgg-twgq.phgggwgq.phggswgq.phggswgq.phgggwgq
    rats         pgsggtwgq.phgggwgq.phgggwgq.phgggwgq.phgggwsq
    Secondary structure: the most striking feature is that almost half of the protein is indistinguishable in chemical shift from random coil. Helix A spans residues 144-156, helix B contains residues 172-193, and helix C includes residues 200-227. Differences in buffer conditions and the more acidic pH may contribute to variations in secondary structure. Comparing full length and fragmentary hamster, residues 187-193 display consistent and significant differences, with amide protons and nitrogens giving the most significant changes. Backbone flexibility data is summarized in Fig. 4.

    Most of the residues between 130-227 give large positive NOEs, indicating these residues are within the structured core. Residues 190-197, corresponding to the loop between helix B and helix C, have smaller values, indicating these residues are relatively more flexible than those in regular secondary structure. Five residues at the C terminus are flexible, a property common to the termini of many proteins. The region from 90-124 is clearly flexible, with local correlation times less than 1ns, indicating that these residues do not form part of any stable secondary or tertiary fold and are highly flexible (43). The flexibility of the various parts of the polypeptide chain is shown schematically in Fig. 5. Residues 23-89 were hand-built for illustration purposes only [ie, are not experimentally determined --webmaster]

    The presence of the 61 additional residues here does not have a significant effect on the secondary structure elements. In particular, residues 90-124 remain highly flexible in the full-length protein. This is consistent with recent studies of the antigenic structures of PrPC and PrPSc, which document that the structure of the region between 90-124 varies between the cellular and scrapie isoforms whereas the C-terminal region is unaltered (46).

    The only major difference between full length and fragmentary hamster is an apparent consistent difference in 13C chemical shifts for residues 187-193 (Fig. 3), suggesting that stabilization of helical structure has occurred in helix B in 29-231. This effect may well be caused by transient tertiary interactions between the N-terminal residues containing the octarepeats and helix B in the longer fragment. This region is important in PrPC/PrPSc recognition but also must serve a normal physiologic function.

    Although the function of the octarepeat region remains uncertain, several possibilities can be re-evaluated in light of our structural observations. First, the octarepeats may participate in binding to the membrane or another protein. In either case, the flexibility of this region, as documented by heteronuclear NOE data, would facilitate binding to a partner macromolecule. Alternatively, the octarepeats may be in an apoprotein conformation that will adopt a more organized structure in the presence of a cofactor. Candidates for this cofactor would include Cu(II) (J Stoeckel, F.E.C., and S.B.P., unpublished observations). [As first suggested years ago in already-published and also newer studies of copper binding to the repeat region --webmaster]

    A small amount of context-dependent beta-structure may exist in PrPC, but its relevance is uncertain. The flexible regions are clearly biologically important, if only in the process of conversion from PrPC to PrPSc. It is not clear whether they participate in the normal cellular function of the PrP in the same unstructured state.

    PrPSc recognizes a surface that includes residues from the regions 90-144 and 180-205, whereas protein X recognizes an antipodal surface that includes residues from the regions 160-180 and 205-231 (48). The energy barrier to the formation of the beta-sheet containing PrPSc will be much lower from an initial "random coil" than from a structure that already contains stable secondary structure.

    Overlooked earlier work on copper binding to repeat region.

     Miura T, et al. 
           Metal-dependent alpha-helix formation promoted by the glycine-rich octapeptide
           region of prion protein. 
           FEBS Lett. 1996 Nov 4; 396(2-3): 248-252. 
    Hornshaw MP, et al.  
           Copper binding to the N-terminal tandem repeat region of mammalian and avian prion
           protein: structural studies using synthetic peptides. 
           Biochem Biophys Res Commun. 1995 Sep 25; 214(3): 993-999. 
    Hornshaw MP, et al. 
           Copper binding to the N-terminal tandem repeat regions of mammalian and avian
           prion protein. 
           Biochem Biophys Res Commun. 1995 Feb 15; 207(2): 621-629. 





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