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Mutational analysis of the cellular prion protein (PrPC) has revealed various regions of the protein that modulate prion propagation. However, most approaches involve deletions, insertions, or replacements in the presence of the wild-type cellular protein, which may mask the true phenotype. Here, site-directed alanine mutagenesis of PrPCwas conducted to identify sites particularly a ‘surface patch’ of the protein pertinent to prion propagation in the absence of the wild-type prion protein. Mutations were targeted to the helical, sheet and loop regions of PrPC, or a combination thereof and the mutated proteins expressed in PK1 cells in which endogenous PrPChad been silenced. PK1 cells are a clone of mouse neuroblastoma cells that are highly susceptible to Rocky Mountain Laboratory mouse prions. Using the scrapie cell assay, a highly sensitive cell culture-based bioassay for quantifying infectious titres of mouse prions, we found that all mutations within the structured 121-230 domain, irrespective of secondary structure, severely reduced prion propagation. The reduction was most pronounced for mutations within conformationally variable regions of the protein (G123A.L124A.G125A and V188A.T191A.T192A) and those neighbouring or within helix 1 (S134A.R135A.M153A and H139A.G141A.D146A). While mutations G123A and G125A would likely disrupt the structure of the prion fibril, the other mutations are unlikely to cause disruption. Our data therefore suggests that conformationally variable regions within the structured domain of PrPCare the major determinants of prion propagation efficacy.

Using a combination of NMR and fluorescence measurements we have investigated the structure and dynamics of the complexes formed between calcium loaded calmodulin (CaM) and the potent breast cancer inhibitor idoxifene, a derivative of tamoxifen. High affinity binding (Kd ~ 300 nM) saturates with a 2:1 idoxifene:CaM complex. The complex is an ensemble where each idoxifene molecule is predominantly in the vicinity of one of the two hydrophobic patches of CaM but, in contrast with the lower affinity antagonists TFP, J-8 and W-7, does not substantially occupy the hydrophobic pocket. At least four idoxifene orientations per domain of CaM are necessary to satisfy the intermolecular NOE restraints, and this requires that the idoxifene molecules switch rapidly between positions. The CaM molecule is predominantly in the form where the N and C-terminal domains are in close proximity allowing for the idoxifene molecules to contact both domains simultaneously. Hence, the 2:1 idoxifene:CaM complex illustrates how high affinity binding occurs without the loss of extensive positional dynamics.

Prion diseases, a group of incurable, lethal neurodegenerative disorders of mammals including humans, are caused by prions, assemblies of misfolded host prion protein (PrP). A single point mutation (G127V) in human PrP prevents prion disease, however the structural basis for its protective effect remains unknown. Here we show that the mutation alters and constrains the PrP backbone conformation preceding the PrP β-sheet, stabilising PrP dimer interactions by increasing intermolecular hydrogen bonding. It also markedly changes the solution dynamics of the β2-α2 loop, a region of PrP structure implicated in prion transmission and cross-species susceptibility. Both of these structural changes may affect access to protein conformers susceptible to prion formation and explain its profound effect on prion disease., Through X-ray crystallography and NMR, Hosszu et al. show that V127 mutation in the human prion protein induces structural changes, leading to conformational restrictions in key regions of the protein associated with prion disease. These may prevent prion formation and explain this mutation′s profound effect on prion disease.

Prion diseases are a group of fatal neurodegenerative diseases that can be sporadic, inherited or acquired. Inherited prion diseases are caused by mutations in the prion protein gene, PRNP , usually single nucleotide substitutions or structural variants of an octapeptide repeat encoding region. Although the classical presentation of sporadic Creutzfeldt Jakob disease (CJD) is rapidly progressive ataxia, myoclonus and cognitive decline, the presentation of genetic cases is variable and decline can be much slower. Prion diseases can mimic many neurodegenerative diseases. Genetic dementias are pleiotropic, with similar clinical syndromes being caused by mutations in different genes; additionally, mutations in a single gene may cause diverse clinical phenotypes. Due to the diagnostic difficulties arising from this clinical and genetic heterogeneity, next-generation sequencing technologies including gene panels and exome sequencing are helpful in elucidating genetic causes of dementia.1 Here we report a novel variant in PRNP in a patient diagnosed with semantic dementia, a variant of frontotemporal dementia (FTD). Patients with semantic dementia develop progressive loss of semantic knowledge resulting in significant early language impairment, with subsequent wider cognitive impairment and behavioural problems. MRI appearances are characteristic with focal asymmetric anteroinferior temporal lobe atrophy. The predominant histopathological finding is accumulation of Tar DNA-binding protein 43 (TDP-43) deposits in the temporal and frontal lobes, although other pathologies, including Alzheimer’s disease, are occasionally observed.2 A Mendelian genetic aetiology is rarely documented. A woman in her seventh decade presented with a 1-year history of rapidly progressive language problems and behavioural change. She had particular difficulty naming objects and suffered from impaired comprehension, but had preserved recognition of faces and …

Temperature-jump perturbation was used to examine the relaxation kinetics of folding of the human prion protein. Measured rates were very fast (≈3,000 s −1 ), with the extrapolated folding rate constant at ≈20 °C in physiological conditions reaching 20,000 s −1 . By a mutational analysis of core residues, we found that only 2, on the interface of helices 2 and 3, have significant ϕ-values in the transition state. Interestingly, a mutation sandwiched between the above 2 residues on the helix–helix contact interface had very little effect on the overall free energy of folding but led to the formation of a monomeric misfolded state, which had to unfold to acquire the native PrP C conformation. Another mutation that led to a marked destabilization of the native fold also formed a misfolded intermediate, but this was aggregation-prone despite the native state of this mutant being soluble. Taken together, the data imply that this fast-folding protein has a transition state that is not compact ( m value analysis gives a β t value of only 0.3) but contains a developing nucleus between helices 2 and 3. The fact that a mutation in this nucleus had a negligible effect on stability but still led to formation of aberrant conformations during folding implies an easily perturbed folding mechanism. It is notable that in inherited forms of human prion disease, where point mutations produce a lethal dominant condition, 20 of the 33 amino acid replacements occur in the helix-2/3 sequence.

Although a functional role in copper binding has been suggested for the prion protein, evidence for binding at affinities characteristic of authentic metal-binding proteins has been lacking. By presentation of copper(II) ions in the presence of the weak chelator glycine, we have now characterized two high-affinity binding sites for divalent transition metals within the human prion protein. One is in the N-terminal octapeptide-repeat segment and has a K d for copper(II) of 10 −14 M, with other metals (Ni 2+ , Zn 2+ , and Mn 2+ ) binding three or more orders of magnitude more weakly. However, NMR and fluorescence data reveal a previously unreported second site around histidines 96 and 111, a region of the molecule known to be crucial for prion propagation. The K d for copper(II) at this site is 4 × 10 −14 M, whereas nickel(II), zinc(II), and manganese(II) bind 6, 7, and 10 orders of magnitude more weakly, respectively, regardless of whether the protein is in its oxidized α-helical (α-PrP) or reduced β-sheet (β-PrP) conformation. A role for prion protein (PrP) in copper metabolism or transport seems likely and disturbance of this function may be involved in prion-related neurotoxicity.

Prions, the causative agents of Creutzfeldt-Jacob Disease (CJD) in humans and bovine spongiform encephalopathy (BSE) and scrapie in animals, are principally composed of PrPSc, a conformational isomer of cellular prion protein (PrPC). The propensity of PrPC to adopt alternative folds suggests that there may be an unusually high proportion of alternative conformations in dynamic equilibrium with the native state. However, the rates of hydrogen/deuterium exchange demonstrate that the conformation of human PrPC is not abnormally plastic. The stable core of PrPC has extensive contributions from all three alpha-helices and shows protection factors equal to the equilibrium constant for the major unfolding transition. A residual, hyper-stable region is retained upon unfolding, and exchange analysis identifies this as a small nucleus of approximately 10 residues around the disulfide bond. These results show that the most likely route for the conversion of PrPC to PrPSc is through a highly unfolded state that retains, at most, only this small nucleus of structure, rather than through a highly organized folding intermediate.

Human PrP (residues 91-231) expressed in Escherichia coli can adopt several conformations in solution depending on pH, redox conditions and denaturant concentration. Oxidised PrP at neutral pH, with the disulphide bond intact, is a soluble monomer which contains 47% alpha-helix and corresponds to PrPC. Denaturation studies show that this structure has a relatively small, solvent-excluded core and unfolds to an unstructured state in a single, co-operative transition with a DeltaG for folding of -5.6 kcal mol-1. The unfolding behaviour is sensitive to pH and at 4.0 or below the molecule unfolds via a stable folding intermediate. This equilibrium intermediate has a reduced helical content and aggregates over several hours. When the disulphide bond is reduced the protein adopts different conformations depending upon pH. At neutral pH or above, the reduced protein has an alpha-helical fold, which is identical to that observed for the oxidised protein. At pH 4 or below, the conformation rearranges to a fold that contains a high proportion of beta-sheet structure. In the reduced state the alpha- and beta-forms are slowly inter-convertible whereas when oxidised the protein can only adopt an alpha-conformation in free solution. The data we present here shows that the human prion protein can exist in multiple conformations some of which are known to be capable of forming fibrils. The precise conformation that human PrP adopts and the pathways for unfolding are dependent upon solvent conditions. The conditions we examined are within the range that a protein may encounter in sub-cellular compartments and may have implications for the mechanism of conversion of PrPC to PrPSc in vivo. Since the conversion of PrPC to PrPSc is accompanied by a switch in secondary structure from alpha to beta, this system provides a useful model for studying major structural rearrangements in the prion protein.

It is demonstrated that the identity of residues accessing excited conformational states that are of low free energy relative to the ground state in proteins can be obtained from amide proton NMR chemical shift temperature dependences displaying significant curvature. For the N-terminal domain of phosphoglycerate kinase, hen egg-white lysozyme and BPTI, conformational heterogeneity arises from a number of independent sources, including: structural instability resulting from deletion of part of the protein; a minor conformer generated through disulphide bond isomerisation; an alternative hydrogen bond network associated with buried water molecules; alternative hydrogen bonds involving backbone amides and surface-exposed side-chain hydrogen bond acceptors; and the disruption of loops, ends of secondary structural elements and chain termini. In many of these cases, the conformational heterogeneity at these sites has previously been identified by X-ray and/or NMR studies, but conformational heterogeneity of buried water molecules has hitherto received little attention. These multiple independent low free-energy excited states each involve a small number of residues and are shown to be within 2.5 kcal mol ˇ1 of the ground state. Their relationship with the partially

The structural integrity of the isolated N-domain (residues 1-174) of Bacillus stearothermophilus 3-phosphoglycerate kinase (PGK) has been investigated using heteronuclear NMR spectroscopy. Analysis of 13C chemical shifts, amide protection, and comparison of observed and expected sequential NOE intensities calculated from the crystal structure of the domain in the intact protein indicate that the secondary structure of the isolated domain is unchanged from that found in the intact molecule. Markedly shifted 1H resonances, amide protection, and long-range NOEs indicate that the tertiary structure is similarly unaffected. These results are confirmed by an excellent agreement (standard deviation 0.28 ppm) between observed H alpha chemical shifts and those calculated from the high-resolution (1.6 A) crystal structure of intact PGK [Davies et al. (1994) Acta Crystallogr. D50, 202-209]. The only region perturbed by loss of interactions with the C-domain is a small portion of the substrate-binding site (residues 148-152) whose amide protons are poorly protected from solvent. These results provide a structural basis for the analysis of the folding of the domains of PGK as isolated units and within the intact molecule [Parker et al. (1996) Biochemistry (in press)] and contrast with the notion that the native tertiary fold of the N-domain of PGK requires the whole polypeptide chain, including the entire C-domain [Mas et al. (1995) Biochemistry 34, 7931-7940]. Assignments of backbone 13C, 15N, HN, and H alpha resonances are provided.

Prion diseases are associated with a conformational switch in the prion protein (PrP) from its normal cellular form (denoted PrP(C)) to a disease-associated "scrapie" form (PrP(Sc)). A number of PrP(Sc)-like conformations can be generated by incubating recombinant PrP(C) at low pH, indicating that protonation of key residues is likely to destabilize PrP(C), facilitating its conversion to PrP(Sc). Here, we examine the stability of human PrP(C) with pH and find that PrP(C) fold stability is significantly reduced by the protonation of two histidine residues, His187 and His155. Mutation of His187 to an arginine, which imposes a permanently positively charged residue in this region of the protein, has a dramatic effect on the folding of PrP(C), resulting in a molecule that displays a markedly increased propensity to oligomerize. The oligomeric form is characterized by an increased ?-sheet content, loss of fixed side chain interactions, and partial proteinase resistance. Hence, the protonation state of H187 appears to be crucial in determining the conformation of PrP; the unprotonated form favors native PrP(C), while the protonated form favors PrP(Sc)-like conformations. These results are relevant to the pathogenic H187R mutation found in humans, which is associated with an inherited prion disease [also termed Gerstmann-Stra?ussler-Scheinker (GSS) syndrome] with unusual features such as childhood neuropsychiatric illness. Our data imply that the intrinsic instability of the PrP(C) conformation in this variant is caused by a positive charge at this site in the protein. This mutation is distinct from all those associated with GSS, which have much more subtle physical consequences. The degree of instability might be the cause of the unusually early onset of mental disturbance in affected individuals.

It has been shown previously that the unfolded N-terminal domain of the prion protein can bind up to six Cu2+ ions in vitro. This domain contains four tandem repeats of the octapeptide sequence PHGGGWGQ, which, alongside the two histidine residues at positions 96 and 111, contribute to its Cu2+ binding properties. At the maximum metal-ion occupancy each Cu2+ is co-ordinated by a single imidazole and deprotonated backbone amide groups. However two recent studies of peptides representing the octapeptide repeat region of the protein have shown, that at low Cu2+ availability, an alternative mode of co-ordination occurs where the metal ion is bound by multiple histidine imidazole groups. Both modes of binding are readily populated at pH 7.4, while mild acidification to pH 5.5 selects in favour of the low occupancy, multiple imidazole binding mode. We have used NMR to resolve how Cu2+ binds to the full-length prion protein under mildly acidic conditions where multiple histidine co-ordination is dominant. We show that at pH 5.5 the protein binds two Cu2+ ions, and that all six histidine residues of the unfolded N-terminal domain and the N-terminal amine act as ligands. These two sites are of sufficient affinity to be maintained in the presence of millimolar concentrations of competing exogenous histidine. A previously unknown interaction between the N-terminal domain and a site on the C-terminal domain becomes apparent when the protein is loaded with Cu2+. Furthermore, the data reveal that sub-stoichiometric quantities of Cu2+ will cause self-association of the prion protein in vitro, suggesting that Cu2+ may play a role in controlling oligomerization in vivo.

Although the physiological function of the prion protein remains unknown, in vitro experiments suggest that the protein may bind copper (II) ions and play a role in copper transport or homoeostasis in vivo. The unstructured N-terminal region of the prion protein has been shown to bind up to six copper (II) ions, with each of these ions co-ordinated by a single histidine imidazole and nearby backbone amide nitrogen atoms. Individually, these sites have micromolar affinities, which is weaker than would be expected of a true cuproprotein. In the present study, we show that with subsaturating levels of copper, different forms of co-ordination will occur, which have higher affinity. We have investigated the copper-binding properties of two peptides representing the known copper-binding regions of the prion protein: residues 57–91, which contains four tandem repeats of the octapeptide GGGWGQPH, and residues 91–115. Using equilibrium dialysis and spectroscopic methods, we unambiguously demonstrate that the mode of copper co-ordination in both of these peptides depends on the number of copper ions bound and that, at low copper occupancy, copper ions are co-ordinated with sub-micromolar affinity by multiple histidine imidazole groups. At pH 7.4, three different modes of copper co-ordination are accessible within the octapeptide repeats and two within the peptide comprising residues 91–115. The highest affinity copper (II)-binding modes cause self-association of both peptides, suggesting a role for copper (II) in controlling prion protein self-association in vivo.

The role of conformational intermediates in the conversion of prion protein from its normal cellular form (PrP(C)) to the disease-associated "scrapie" form (PrP(Sc)) remains unknown. To look for such intermediates in equilibrium conditions, we have examined the unfolding transitions of PrP(C), primarily using the chemical denaturant guanidine hydrochloride (GuHCl). When the protein conformation is assessed by NMR, there is a gradual shift of NMR signals in the regions between residues 125-146 and 186-196. The denaturant dependence of these shifts shows that in aqueous solution the native and locally unfolded conformations are both significantly populated. Following this shift, there is the major unfolding transition to generate a substantially unfolded population. However, analysis of NMR chemical shift and intensity changes shows that there is persistent structure in the molecule well beyond this major cooperative unfolding transition. Residual structure within this state is extensive and encompasses the majority of the secondary structure elements found in the native state of the protein.

There are two common forms of prion protein (PrP) in humans, with either methionine or valine at position 129. This polymorphism is a powerful determinant of the genetic susceptibility of humans toward both sporadic and acquired forms of prion disease and restricts propagation of particular prion strains. Despite its key role, we have no information on the effect of this mutation on the structure, stability, folding, and dynamics of the cellular form of PrP (PrP(C)). Here, we show that the mutation has no measurable effect on the folding, dynamics, and stability of PrP(C). Our data indicate that the 129M/V polymorphism does not affect prion propagation through its effect on PrP(C); rather, its influence is likely to be downstream in the disease mechanism. We infer that the M/V effect is mediated through the conformation or stability of disease-related PrP (PrP(Sc)) or intermediates or on the kinetics of their formation.

The characterization of early folding intermediates is key to understanding the protein folding process. Previous studies of the N-domain of phosphoglycerate kinase (PGK) from Bacillus stearothermophilus combined equilibrium amide exchange data with a kinetic model derived from stopped-flow kinetics. Together, these implied the rapid formation of an intermediate with extensive native-like hydrogen bonding. However, there was an absence of protection in the region proximal to the C-domain in the intact protein. We now report data for the intact PGK molecule, which at 394 residues constitutes a major extension to the protein size for which such data can be acquired. The methods utilised to achieve the backbone assignment are described in detail, including a semi-automated protocol based on a simulated annealing Monte Carlo technique. A substantial increase in the stability of the contact region is observed, allowing protection to be inferred on both faces of the beta-sheet in the intermediate. Thus, the entire N-domain acts concertedly in the formation of the kinetic refolding intermediate rather than there existing a distinct local folding nucleus.

Prion propagation involves the conversion of cellular prion protein (PrP C ) into a disease-specific isomer, PrP Sc , shifting from a predominantly α-helical to β-sheet structure. Here, conditions were established in which recombinant human PrP could switch between the native α conformation, characteristic of PrP C , and a compact, highly soluble, monomeric form rich in β structure. The soluble β form (β-PrP) exhibited partial resistance to proteinase K digestion, characteristic of PrP Sc , and was a direct precursor of fibrillar structures closely similar to those isolated from diseased brains. The conversion of PrP C to β-PrP in suitable cellular compartments, and its subsequent stabilization by intermolecular association, provide a molecular mechanism for prion propagation.

The pH-dependence of hydrogen-exchange, in native conditions, is used to probe the formation of secondary structure in the folding of an immunoglobulin domain (CD2.d1). The intermediate and transition states in the reaction are insensitive to pH, a simplification that allows us to equate structure formation and folding kinetics. The crucial residues in the folding reaction are grouped in the B, C, E and F strands which constitute a ‘crossover’ nucleus in the β-sandwich. These residues show the highest sequence conservation within the family of folds to which this domain belongs and are located in a unit of structure that is the most consistent topo-logical feature of the immunoglobulin family.