Elucidation of the structure of mammalian prions continues to be one major research challenge. The mechanism of propagation of these infectious agents will not be understood until their structure is solved. However, the polymeric and insoluble nature of prions makes this task very difficult, as high resolution techniques such as NMR or X-ray crystallography cannot be used. Thus, a number of lower resolution analytical approaches have been used and provided important structural information on PrPSc.
Among these, limited proteolysis has been successfully used to pinpoint flexible regions in the PrPSc molecule, which are more accessible and susceptible to proteolytic cleavage. In this respect, it has been known for a long time that PrPSc features two regions, one spanning from the amino terminus up to approximately position 80-90, which is extremely susceptible to proteinase K (PK), and a second one spanning from that site up to the C-terminus which is very resistant to PK. The exceedingly high sensitivity of the N-terminal sequence has been interpreted as indicating that the sequence adopts a flexible, unstructured conformation, just like in PrPC. In contrast, the high resistance to PK of the C-terminal part of of PrPSc suggests that this region is folded into a very compact, -strand rich conformation.
More recent studies have detected a number of minor PK cleavage points, which suggest the presence of short flexible turns or loops within that overall compact block. A recent Priority study (Vázquez-Fernández et al., PLoS ONE 7(11): e50111) provides a complete survey of of flexible, proteinase K (PK) susceptible regions in GPI-less PrPSc samples. PK-resistant peptides identified correspond to molecules cleaved at positions 81, 85, 89, 116, 118, 133, 134, 141, 152, 153, 162, 169 and 179. These results reinforce the hypothesis that the structure of PrPSc consists of a series of highly PK-resistant β-sheet strands connected by short flexible PK-sensitive loops and turns. Of note, the most protease resistant region of PrPSc corresponds to its C-terminus, from position 153 to the C-terminus itself. This contradicts the notion that this region consists of the preserved -helices seen in PrPC.
Proteolysis Fig. 1: A map of protease-sensitive spots within a mammalian prion. Priority researchers are probing the structure of mammalian prions by means of limited proteolysis with proteinase K (PK). PK destroys a sizeable portion of PrPSc, corresponding to its amino-terminal sequence up to position ~90, and leaving an intact C-terminal protease-resistant core. However, it also produces an array of less abundant shorter C-terminal protease-resistant cores. The N-termini of such cores should signal the frontier of PK-resistant, β-strands. Using a combination of immunoassays and mass spectrometry, they have identified these spots and constructed a map of protease-sensitive spots within the murine prion GPI-anchorless PrPSc. The map (A) suggests that PrPSc consists of a succession of β-strands connected by short loops and/or turns, very likely devoid of any a-helical secondary structure. It is hoped that it will be possible to dock these results into low-resolution structures of GPI-anchorless PrPSc obtained from cryo-EM studies, currently in course, to complete a structural model of PrPSc. (Figure adapted from Vázquez-Fernández et al., PLoS One, 2012;7(11):e50111).
For more information: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0050111
Proteolysis 2 Fig. 2: A cartoon showing the distribution of some identified flexible, PK-sensitive stretches within PrPSc. The choice of a 4-layer -solenoid architecture is arbitrary, but agrees with recent