3D structure of longer hamster prion
Off-page, on-site: Yes, we have hamster pdb coordinates!
Comments on hamster structure
Show me the pictures
Reference sequences -- color coded
Protein X: interaction with prion protein
Prion membrane interaction -- Warwicker
Macrophages transport prions to CNS -- Dealler
Immunodetection of rogue prion in spleens
Details on new fungal prion
Prion repeats comared to yeast S35 repeats
PNAS Vol. 94, pp. 10086-10091, September 1997 free fulltext Thomas L.James1, He Liu1,.,Ingrid Mehlhorn, Stanley B.Prusiner2, and Fred E.CohenHighlights:
(1: emails unknown 2:does not use email)
... Hamster Helix A is two residues longer distally than mouse, 144-156 with the last turn distored; Helix B 172-194, is two turns longer (first irregular) than mouse 179-193; Helix C runs 3 full turns longer than mouse, so 200-227 versus 200-217, with distal irregularity. The anti-parallel beta sheet is also found in hamster (129-131 and 161-163) but lacks standard geometry, with a beta-bridge only between Leu130 and Tyr162, although extensive cross-strand connectivities are found between 129-134 and 159-165.
... Residues 113-125 forming a hydrophobic cluster that packs against an irregular geometry beta-sheet whereas residues 90-112 still exhibit little defined structure. "Although identifiable secondary structure is largely lacking in the N terminus, paradoxically this N terminus increases the amount of secondary structure in the remainder of
[of the structure].".
... The surface of a long helix (residues 200-227) and a structured loop (residues 165-171) form a discontinuous epitope for binding of protein X. Polymorphisms within this epitope seem to modulate susceptibility of sheep and humans to prion disease. Conformational change at the N terminus may be the structural key to the transformation of normal to rogue, but the discontinuous epitope near the C terminus may controls it. ... During purification of the protein from E. coli, it was solubilized in 8İM guanidinium hydrochloride [denaturing] and 100 mM dithiothreitol [disulphide reducing] at pH 8.0. Its alpah-helical state was stable for at least 15İdays at temperatures from 4C to 30C as judged by circular dichroism. In contrast, 1İday at 35C led to a substantial loss of -helix and a concomitant acquisition of -sheet that was concentration-dependent.
Pdb coordinates or accession number will soon be available for the hamster structure. There is no record at Brookhaven just yet Dr. Shauna Farr-Jones is correcting this situation on 9.26.97. But we have some pdb coordinates for you! [First of 15 in ensemble]
... Antibody fragments that bind the N terminus (residues 90-112) recognize
normal but not rogue prion, whereas other epitopes in the C-terminal region bind to
both and to denatured rogue, so as rogue conformer is formed, epitopes exposed in normal conformer become buried. The pH was found to be critical: N-terminal epitopes (residues 90-112) that were observed to be partially buried at pH 5.2-8.0 by ELISA using N-terminal antibodies became completely exposed at pH 4.8İor lower.
... Spin-lattice and spin-spin relaxation time measurements indicate that the protein is undergoing rapid interconversion between a weak dimer and monomer. No specific intermolecular interactions have been identified to date.
(A) Comparison of the 15İbest-scoring structures. The color scheme is:
disulfide between Cys 179 and Cys 214, yellow;
sites of glycosidation Asn181 and Asn197, gold;
hydrophobic cluster composed of residues 113-126, red;
helices, pink; loops, gray; residues129-134, green;
encompassing strand S1 and residues 159-165, encompassing strand S2 blue
(B) Residues 113-132 illustrating the interaction of the hydrophobic cluster with the first -strand.
(C) Van der Waals surface of rPrP turned approximately 180 from A, illustrating the interaction of helix A with helix C.İHelices A, B, and C are colored magenta, cyan, and gold, respectively.
(D) Proximity of helix C to the 165-171 loop and the end of helix B, where residues Gln168 and Gln172 are depicted with a low-density van der Waals rendering and helix C residues Thr215 and Gln219 are depicted with a high-density.
(E) Highlights in white the residues corresponding to point mutations that lead to human prion diseases.
9.26.97 webmasterIt is especially helpful in reading Cohen-Prusiner to view the hamster sequence colored for helices, sheets, and numbering. The pdb coordinates aren't posted like they said, but FC told me this morning that the situation will be corrected shortly.
Don't make the mistake of thinking this is a me-too prion structure. This is a larger fragment, the helices are significcantly longer and possibly more physiological, it's a different species, the resolution on the "beta-sheet" is phenomenal, they have the non-contiguous residues identified for protein X binding, plus they are making the data available to the research community without an 18 month artificial hold. These are block-buster articles, fabulous graphics, you will be studying them for months.
Longer helices really changes the interpretation of certain human mutations and sheep alleles. I am up at DSC right now looking to see where the helices should be in these other species. The other cool thing to do is color your sequences with a list of exterior and interior sidechains, plus the non-contiguous interacting pairs. This allows a hunt for the much predicted but seldom found concerted residue substitutions; basically, toss the invariants and singlets, intersect with synapomorphies: these are the candidates.
I should have the structural coordinates for about 70 species, plus all the mutants, alleles, and key phylogenetic nodes posted by noon on Monday. This is done by threading changes in amino acid sequence onto the known hamster structure and adjusting the energies. This can be done with Swiss Prot Modeller (since the homologies are close to 90%) or Threader for the more ancient nodes or the bird sequences. I will extend the Swiss long incubation mouse to hamster length and do short incubation mouse as well. Just like having a whole bunch of aligned sequences helps in predicting secondary structure accurately, two independent high quality structures can help with the threading exercises.
One thing I hadn't realized earlier is that the long incubation mouse is actually an unnatural inbred strain. It is the short incubation mouse that represents the wild type. This is obvious upon aligning all 10 rodent sequences plus primates and artiodactyls. I predict that the two bizarre substitutions in long mouse will substantially degrade normal mouse prion functions [which don't include incubation time]. The phenylalanine substitution (from leucine) is within the hinge region studied by Cohen-Prusiner and the valine (from threonine) is in Helix B.
In other words, the Swiss structure was done on the wrong mouse. Still very useful, particarly if they later do normal mouse. However, the point of reference has to be a normal phylogenetically evolved structure.
Neither group has a good handle on the pre-repeat, repeat, or core invariant regions, which make up a third of the protein. The new structure includes the final pseudo-repeat and the core; 113 on has some structure that reminded me of what Inouye found earlier in the rogue fiber and clearly is associating in the right place with the beta sheets.
Warwicker's new paper addresses these issues more insightfully, if speculatively. He considers membrane attachment or interaction with another protein as integral to getting these regions to adopt stable conformations. Interestingly, Cohen-Prusiner found some evidence for a weak dimer in solution (but they couldn't nail the dimer interface). Georg Schulz kindly sent me the unpublished QW pdb coordinates, I will process them shortly.
The xray people snort at very mention of nmr, say you'd get the dimer (and maybe the fiber interface) for free in the unit cell.
>hamster_Syrian MANLSYWLLALFVAMWTDVGLC KKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGTWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGW 89 GQGGGTHNQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPMMHFGNDWEDRYYRENMNRYPNQVYYR 163 PVDQYNNQNNFVHDCVNITIKQHTVTTTTKGENFTETDIKIMERVVEQMCTTQYQKESQAYYDGRRSS 231 .AVLFSSPPVILLISFLIFLMVG >Mus_mouse_long MANLGYWLLALFVTMWTDVGLC KKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGG.TWGQPHGGGWGQPHGGSWGQPHGGSWGQPHGGGW GQGGGTHNQWNKPSKPKTNFKHVAGAAAAGAVVGGLGGYMLGSAMSRPMIHFGNDWEDRYYRENMYRYPNQVYYR PVDQYSNQNNFVHDCVNITIKQHTVVTTTKGENFTETDVKMMERVVEQMCVTQYQKESQAYYDGRRSSSTVLFSS PPVILLISFLIFLIVG >Mus_mouse_short MANLGYWLLALFVTMWTDVGLC KKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGG.TWGQPHGGGWGQPHGGSWGQPHGGSWGQPHGGGW GQGGGTHNQWNKPSKPKTNLKHVAGAAAAGAVVGGLGGYMLGSAMSRPMIHFGNDWEDRYYRENMYRYPNQVYYR PVDQYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCVTQYQKESQAYYDGRRSSSTVLFSS PPVILLISFLIFLIVG >homo_sap_human MANLGCWMLVLFVATWSDLGLC KKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGW GQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYR PMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQ.RGSS.MVLFSS PPVILLISFLIFLIVG >Ovis_aries_sheep MVKSHIGSWILVLFVAMWSDVGLC KKRPKPGGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGG WGQGGSHSQWNKPSKPKTNMKHVAGAAAAGAVVGGLGGYMLGSAMSRPLIHFGNDYEDRYYRENMYRYPNQVYYR PVDQYSNQNNFVHDCVNITVKQHTVTTTTKGENFTETDIKIMERVVEQMCITQYQRESQAYYQRGASVILFSS PPVILLISFLIFLIVG >Bovine_short MVKSHIGSWILVLFVAMWSDVGLC KKRPKPGGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGG WGQGGTHGQWNKPSKPKTNMKHVAGAAAAGAVVGGLGGYMLGSAMSRPLIHFGSDYEDRYYRENMHRYPNQVYYR PVDQYSNQNNFVHDCVNITVKEHTVTTTTKGENFTETDIKMMKRVVEQMCITQYQRESQAYYQRGASVILFSS PPVILLISFLIFLIVG
PNAS 1997 Sep 16;94(19):10069-10074 free fulltext Kaneko K, Zulianello L, Scott M, Cooper CM, Wallace AC, James TL, Cohen FE, Prusiner SBStudies on the transmission of human prions to transgenic mice suggested that another molecule provisionally designated protein X participates in the formation of nascent scrapie isoform of prion protein. We report the identification of the site at which protein X binds to the cellular isoform using scrapie-infected mouse neuroblastoma cells transfected with chimeric genes even though protein X has not yet been isolated. Substitution of a human residue at position 214 or 218 prevented rogue prion formation. The side chains of these residues protrude from the same surface of the C-terminal alpha-helix and form a discontinuous epitope with residues 167 and 171 in an adjacent loop.
Substitution of a basic residue at positions 167, 171, or 218 also prevented rogue prion formation: at a mechanistic level, these mutant PrPs appear to act as "dominant negatives" by binding protein X and rendering it unavailable for prion propagation. Our findings seem to explain the protective effects of basic polymorphic residues in PrP of humans and sheep and suggest therapeutic and prophylactic approaches to prion diseases.
Figure 3 explains he role of protein X in rogue prion formation and the influence of mutations on the prion replication cycle. Part (E) shows the dominant-negative effect of tight binding mutants. K218 successfully competes with wild type for binding to protein X.İThe complex is formed but conversion is inhibited. Part (F) shows Type 3İinhibition: normal prion from a distinct species is able to bind mouse protein X, but the complex is not competent for conversion. The result is that protein X is sequestered and normal prions are not recruited.
Biochem Biophys Res Commun 1997 Sep 8;238(1):185-190 Warwicker JIn considering a protein-only model for prion pathogenesis in TSEs, one key challenge is to explain the existence of strains. These have traditionally been characterised by neuropathology and incubation times and more recently through biochemical analysis of prion protein, which shows differences in protease-resistant fragment size and glycoform ratios. It is now suggested that the prion protein possesses two faces which on the basis of conservation and non-polar nature could each (physiologically) interact either with membrane or with neighbouring protein. This model leads to the construction of two clearly different membrane-attached prion orientations, with consequences for protease resistance and glycoform incorporation that qualitatively match to experiment.
J Gen Virol 1997 Sep;78( Pt 9):2389-2396 Somerville RA, Birkett CR, Farquhar CF, Hunter N, Goldmann W, Doman J, Grover D, Hennion RM, Percy C, Foster J, Jeffrey MThe development of diagnostic tools for transmissible spongiform encephalopathies (TSEs) would greatly assist their study and may provide assistance in controlling the disease. The detection of an abnormal form of the host protein PrP in noncentral nervous system tissues may form the basis for diagnosis of TSEs. Using a new antibody reagent to PrP produced in chickens, PrP can be readily detected in crude tissue extracts. PrP from uninfected spleen had a lower molecular mass range than PrP from brain, suggesting a lower degree of glycosylation. A simple method for detecting the abnormal form of the protein, PrPSc, in ruminant brain and spleen has been developed. PrPSc was detected in sheep spleen extracts from a flock affected by natural scrapie and was also found in spleens from some, but not all, experimental TSE cases. In spleens from cattle with bovine spongiform encephalopathy (BSE) no PrPSc was detected. It is therefore suggested that there is differential targeting of PrPSc deposition between organs in these different types of TSE infection which, with other factors, depends on strain of infecting agent.
Med Hypotheses 1997 Sep;49(3):213-220 Dealler S Burnley General Hospital, UK.It is suggested that the agent for transmissible spongiform encephalopathies is trasferred from an original peripheral site of infection into the brain by recruited and selected circulating macrophages/monocytes. It is because of this selection that strains of disease appear to be different when infecting separate species, but retain characteristics when infecting a single species.
PNAS Vol. 94, pp. 10012-10014, September 1997 free fulltext Commentary by Read WicknerA new prion controls fungal cell fusionİincompatibility: Het-s is like scrapie, and unlike URE3 and PSI, in that the prion form produces a phenotype by doing mischief, not by simply causing the absence of the active normal form of the het-s protein. The normal form of the protein is dispensable for growth, mating, and heterokaryon formation.
Unlike all the other putative prions, the prion form of the het-s protein, is carrying out a normal fungal cell function. Heterokaryon incompatibility systems are widespread among filamentous fungi and usually are controlled by genetic loci showing none of the characteristics suggestive of prions. Is there an advantage to Podospora in using a prion to signal heterokaryon incompatibility? Because this is a purposeful cell death, and many viruses produce apoptosis in their host cells, could this heterokaryon incompatibility reaction be a form of fungal apoptosis?
The het-s protein has no evident similarity to other putative prion proteins. The prion domains of Ure2p and Sup35p are rich in asparagine and glutamine residues, but this is not true of either the prion or the het-s protein. Sup35p and PrP have similar octapeptide repeats, but these appear to be outside the prion domain and are not found in Ure2p or the het-s protein. Whether structural similarities will be found among the normal or prion forms of these proteins remains to be determined.
MSDSNQGNNQQNYQQYSQNGNQQQGNNRYQGYQAYNAQAQPAGGYYQNYQGYSGYQQGGY QQYNPDAGYQQQYNPQGGYQQYNPQGGYQQQFNPQGGRGNYKNFNYNNNLQGYQAGFQPQ SQGMSLNDFQKQQKQ....Alignment of a mammalian prion repeat region with yeast S35:
mink ---------------------MVKSHIGSWLLVLFVATWSDIGFCKKRPKPGGGWNTGGS 39 yeast MSDSNQGNNQQNYQQYSQNGNQQQGNNRYQGYQAYNAQAQPAGGYYQNYQGYSGYQQGGY 60 :.: : * . * :. : .*:: ** mink RYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHG-GGWGQPHGGGWG---QPHGGGGWGQ 95 yeast QQYNPDAGYQQQYNPQGGYQQYNPQGGYQQQFNPQGGRGNYKNFNYNNNLQGYQAGFQPQ 120 : . .: ::* **** :*:** * : ** *: :. .:. * : .* *