RESUMO
Proteins are the workhorses of biology, orchestrating a myriad of cellular functions through intricate conformational changes. Protein allostery, the phenomenon where binding of ligands or environmental changes induce conformational rearrangements in the protein, is fundamental to these processes. We have previously shown that transition metal Förster resonance energy transfer (tmFRET) can be used to interrogate the conformational rearrangements associated with protein allostery and have recently introduced novel FRET acceptors utilizing metal-bipyridyl derivatives to measure long (>20 Å) intramolecular distances in proteins. Here, we combine our tmFRET system with fluorescence lifetime measurements to measure the distances, conformational heterogeneity, and energetics of maltose-binding protein, a model allosteric protein. Time-resolved tmFRET captures near-instantaneous snapshots of distance distributions, offering insights into protein dynamics. We show that time-resolved tmFRET can accurately determine distance distributions and conformational heterogeneity of proteins. Our results demonstrate the sensitivity of time-resolved tmFRET in detecting subtle conformational or energetic changes in protein conformations, which are crucial for understanding allostery. In addition, we extend the use of metal-bipyridyl compounds, showing that Cu(phen)2+ can serve as a spin label for pulse dipolar electron paramagnetic resonance (EPR) spectroscopy, a method that also reveals distance distributions and conformational heterogeneity. The EPR studies both establish Cu(phen)2+ as a useful spin label for pulse dipolar EPR and validate our time-resolved tmFRET measurements. Our approach offers a versatile tool for deciphering conformational landscapes and understanding the regulatory mechanisms governing biological processes.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Proteínas Ligantes de Maltose , Conformação Proteica , Regulação Alostérica , Proteínas Ligantes de Maltose/química , Proteínas Ligantes de Maltose/metabolismo , Fatores de TempoRESUMO
With the great progress on determining protein structures over the last decade comes a renewed appreciation that structures must be combined with dynamics and energetics to understand function. Fluorescence spectroscopy, specifically Förster resonance energy transfer (FRET), provides a great window into dynamics and energetics due to its application at physiological temperatures and ability to measure dynamics on the ångström scale. We have recently advanced transition metal FRET (tmFRET) to study allosteric regulation of maltose binding protein and have reported measurements of maltose-dependent distance changes with an accuracy of â¼1.5 Å. When paired with the noncanonical amino acid Acd as a donor, our previous tmFRET acceptors were useful over a working distance of 10 to 20 Å. Here, we use cysteine-reactive bipyridyl and phenanthroline compounds as chelators for Fe2+ and Ru2+ to produce novel tmFRET acceptors to expand the working distance to as long as 50 Å, while preserving our ability to resolve even small maltose-dependent changes in distance. We compare our measured FRET efficiencies to predictions based on models using rotameric ensembles of the donors and acceptors to demonstrate that steady-state measurements of tmFRET with our new probes have unprecedented ability to measure conformational rearrangements under physiological conditions.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Fenantrolinas , Fenantrolinas/química , Ligantes , 2,2'-Dipiridil/química , 2,2'-Dipiridil/análogos & derivados , Maltose/química , Maltose/metabolismo , Maltose/análogos & derivados , Proteínas Ligantes de Maltose/química , Proteínas Ligantes de Maltose/metabolismoRESUMO
Proteins are the workhorses of biology, orchestrating a myriad of cellular functions through intricate conformational changes. Protein allostery, the phenomenon where binding of ligands or environmental changes induce conformational rearrangements in the protein, is fundamental to these processes. We have previously shown that transition metal Förster resonance energy transfer (tmFRET) can be used to interrogate the conformational rearrangements associated with protein allostery and have recently introduced novel FRET acceptors utilizing metal-bipyridyl derivatives to measure long (>20 Å) intramolecular distances in proteins. Here, we combine our tmFRET system with fluorescence lifetime measurements to measure the distances, conformational heterogeneity, and energetics of maltose binding protein (MBP), a model allosteric protein. Time-resolved tmFRET captures near-instantaneous snapshots of distance distributions, offering insights into protein dynamics. We show that time-resolved tmFRET can accurately determine distance distributions and conformational heterogeneity of proteins. Our results demonstrate the sensitivity of time-resolved tmFRET in detecting subtle conformational or energetic changes in protein conformations, which are crucial for understanding allostery. In addition, we extend the use of metal-bipyridyl compounds, showing Cu(phen)2+ can serve as a spin label for pulse dipolar electron paramagnetic resonance (EPR) spectroscopy, a method which also reveals distance distributions and conformational heterogeneity. The EPR studies both establish Cu(phen)2+ as a useful spin label for pulse dipolar EPR and validate our time-resolved tmFRET measurements. Our approach offers a versatile tool for deciphering conformational landscapes and understanding the regulatory mechanisms governing biological processes.
RESUMO
With the great progress on determining protein structures over the last decade comes a renewed appreciation that structures must be combined with dynamics and energetics to understand function. Fluorescence spectroscopy, specifically Förster resonance energy transfer (FRET), provides a great window into dynamics and energetics due to its application at physiological temperatures and ability to measure dynamics on the ångström scale. We have recently advanced transition metal FRET (tmFRET) to study allosteric regulation of maltose binding protein and have reported measurements of maltose-dependent distance changes with an accuracy of ~1.5 Å. When paired with the noncanonical amino acid Acd as a donor, our previous tmFRET acceptors were useful over a working distance of 10 Å to 20 Å. Here, we use cysteine-reactive bipyridyl and phenanthroline compounds as chelators for Fe2+ and Ru2+ to produce novel tmFRET acceptors to expand the working distance to as long as 50 Å, while preserving our ability to resolve even small maltose-dependent changes in distance. We compare our measured FRET efficiencies to predictions based on models using rotameric ensembles of the donors and acceptors to demonstrate that steady-state measurements of tmFRET with our new probes have unprecedented ability to measure conformational rearrangements under physiological conditions.
RESUMO
Site-directed spin-labeling (SDSL)âin combination with double electron-electron resonance (DEER) spectroscopyâhas emerged as a powerful technique for determining both the structural states and the conformational equilibria of biomacromolecules. DEER combined with in situ SDSL in live cells is challenging since current bioorthogonal labeling approaches are too slow to allow for complete labeling with low concentrations of spin label prior to loss of signal from cellular reduction. Here, we overcome this limitation by genetically encoding a novel family of small, tetrazine-bearing noncanonical amino acids (Tet-v4.0) at multiple sites in proteins expressed in Escherichia coli and in human HEK293T cells. We achieved specific and quantitative spin-labeling of Tet-v4.0-containing proteins by developing a series of strained trans-cyclooctene (sTCO)-functionalized nitroxidesâincluding a gem-diethyl-substituted nitroxide with enhanced stability in cellsâwith rate constants that can exceed 106 M-1 s-1. The remarkable speed of the Tet-v4.0/sTCO reaction allowed efficient spin-labeling of proteins in live cells within minutes, requiring only sub-micromolar concentrations of sTCO-nitroxide. DEER recorded from intact cells revealed distance distributions in good agreement with those measured from proteins purified and labeled in vitro. Furthermore, DEER was able to resolve the maltose-dependent conformational change of Tet-v4.0-incorporated and spin-labeled MBP in vitro and support assignment of the conformational state of an MBP mutant within HEK293T cells. We anticipate the exceptional reaction rates of this system, combined with the relatively short and rigid side chains of the resulting spin labels, will enable structure/function studies of proteins directly in cells, without any requirements for protein purification.
Assuntos
Aminoácidos , Compostos Heterocíclicos , Animais , Humanos , Aminoácidos/química , Marcadores de Spin , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Células HEK293 , Proteínas/química , Mamíferos/metabolismoRESUMO
Studying protein structures and dynamics directly in the cellular environments in which they function is essential to fully understand the molecular mechanisms underlying cellular processes. Site-directed spin-labeling (SDSL)-in combination with double electron-electron resonance (DEER) spectroscopy-has emerged as a powerful technique for determining both the structural states and the conformational equilibria of biomacromolecules. In-cell DEER spectroscopy on proteins in mammalian cells has thus far not been possible due to the notable challenges of spin-labeling in live cells. In-cell SDSL requires exquisite biorthogonality, high labeling reaction rates and low background signal from unreacted residual spin label. While the bioorthogonal reaction must be highly specific and proceed under physiological conditions, many spin labels display time-dependent instability in the reducing cellular environment. Additionally, high concentrations of spin label can be toxic. Thus, an exceptionally fast bioorthogonal reaction is required that can allow for complete labeling with low concentrations of spin-label prior to loss of signal. Here we utilized genetic code expansion to site-specifically encode a novel family of small, tetrazine-bearing non-canonical amino acids (Tet-v4.0) at multiple sites in green fluorescent protein (GFP) and maltose binding protein (MBP) expressed both in E. coli and in human HEK293T cells. We achieved specific and quantitative spin-labeling of Tet-v4.0-containing proteins by developing a series of strained trans -cyclooctene (sTCO)-functionalized nitroxides-including a gem -diethyl-substituted nitroxide with enhanced stability in cells-with rate constants that can exceed 10 6 M -1 s -1 . The remarkable speed of the Tet-v4.0/sTCO reaction allowed efficient spin-labeling of proteins in live HEK293T cells within minutes, requiring only sub-micromolar concentrations of sTCO-nitroxide added directly to the culture medium. DEER recorded from intact cells revealed distance distributions in good agreement with those measured from proteins purified and labeled in vitro . Furthermore, DEER was able to resolve the maltose-dependent conformational change of Tet-v4.0-incorporated and spin-labeled MBP in vitro and successfully discerned the conformational state of MBP within HEK293T cells. We anticipate the exceptional reaction rates of this system, combined with the relatively short and rigid side chains of the resulting spin labels, will enable structure/function studies of proteins directly in cells, without any requirements for protein purification.
RESUMO
In this issue of Neuron, Xue et al. report high-resolution structures of the human cGMP-activated ion channel CNGA1 from rod photoreceptors. These structures provide valuable insights into the processes of cGMP-dependent activation and Ca2+ block and permeation.
Assuntos
GMP Cíclico , Canais de Cátion Regulados por Nucleotídeos Cíclicos , HumanosRESUMO
Cyclic nucleotide-gated (CNG) ion channels are essential components of mammalian visual and olfactory signal transduction. CNG channels open upon direct binding of cyclic nucleotides (cAMP and/or cGMP), but the allosteric mechanism by which this occurs is incompletely understood. Here, we employed double electron-electron resonance (DEER) spectroscopy to measure intersubunit distance distributions in SthK, a bacterial CNG channel from Spirochaeta thermophila Spin labels were introduced into the SthK C-linker, a domain that is essential for coupling cyclic nucleotide binding to channel opening. DEER revealed an agonist-dependent conformational change in which residues of the B'-helix displayed outward movement with respect to the symmetry axis of the channel in the presence of the full agonist cAMP, but not with the partial agonist cGMP. This conformational rearrangement was observed both in detergent-solubilized SthK and in channels reconstituted into lipid nanodiscs. In addition to outward movement of the B'-helix, DEER-constrained Rosetta structural models suggest that channel activation involves upward translation of the cytoplasmic domain and formation of state-dependent interactions between the C-linker and the transmembrane domain. Our results demonstrate a previously unrecognized structural transition in a CNG channel and suggest key interactions that may be responsible for allosteric gating in these channels.
Assuntos
Sítio Alostérico/fisiologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/química , Canais de Cátion Regulados por Nucleotídeos Cíclicos/fisiologia , Spirochaeta/metabolismo , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Escherichia coli/metabolismo , Ativação do Canal Iônico/fisiologia , Modelos Moleculares , Nucleotídeos Cíclicos , Conformação ProteicaRESUMO
Cyclic nucleotide-gated (CNG) channels produce the initial electrical signal in mammalian vision and olfaction. They open in response to direct binding of cyclic nucleotide (cAMP or cGMP) to a cytoplasmic region of the channel. However, the conformational rearrangements occurring upon binding to produce pore opening (i.e. gating) are not well understood. SthK is a bacterial CNG channel that has the potential to serve as an ideal model for structure-function studies of gating but is currently limited by its toxicity, native cysteines, and low open probability (Po). Here, we expressed SthK in giant Escherichia coli spheroplasts and performed patch-clamp recordings to characterize SthK gating in a bacterial membrane. We demonstrated that the Po in cAMP is higher than has been previously published and that cGMP acts as a weak partial SthK agonist. Additionally, we determined that SthK expression is toxic to E. coli because of gating by cytoplasmic cAMP. We overcame this toxicity by developing an adenylate cyclase-knockout E. coli cell line. Finally, we generated a cysteine-free SthK construct and introduced mutations that further increase the Po in cAMP. We propose that this SthK model will help elucidate the gating mechanism of CNG channels.
Assuntos
Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/química , Ativação do Canal Iônico , Técnicas de Patch-Clamp , Conformação Proteica , Esferoplastos/metabolismoRESUMO
The function of the cellular prion protein (PrPC), while still poorly understood, is increasingly linked to its ability to bind physiological metal ions at the cell surface. PrPC binds divalent forms of both copper and zinc through its unstructured N-terminal domain, modulating interactions between PrPC and various receptors at the cell surface and ultimately tuning downstream cellular processes. In this chapter, we briefly discuss the molecular features of copper and zinc uptake by PrPC and summarize evidence implicating these metal ions in PrP-mediated physiology. We then focus our review on recent biophysical evidence revealing a physical interaction between the flexible N-terminal and globular C-terminal domains of PrPC. This interdomain cis interaction is electrostatic in nature and is promoted by the binding of Cu2+ and Zn2+ to the N-terminal octarepeat domain. These findings, along with recent cellular studies, suggest a mechanism whereby NC interactions serve to regulate the activity and/or toxicity of the PrPC N-terminus. We discuss this potential mechanism in relation to familial prion disease mutations, lethal deletions of the PrPC central region, and neurotoxicity induced by certain globular domain ligands, including bona fide prions and toxic amyloid-ß oligomers.
Assuntos
Cobre/farmacologia , Neurotoxinas/antagonistas & inibidores , Neurotoxinas/química , Proteínas Priônicas/antagonistas & inibidores , Proteínas Priônicas/química , Zinco/farmacologia , Animais , Humanos , Modelos Moleculares , Domínios ProteicosRESUMO
Copper plays a critical role in prion protein (PrP) physiology. Cu(2+) binds with high affinity to the PrP N-terminal octarepeat (OR) domain, and intracellular copper promotes PrP expression. The molecular details of copper coordination within the OR are now well characterized. Here we examine how Cu(2+) influences the interaction between the PrP N-terminal domain and the C-terminal globular domain. Using nuclear magnetic resonance and copper-nitroxide pulsed double electron-electron resonance, with molecular dynamics refinement, we localize the position of Cu(2+) in its high-affinity OR-bound state. Our results reveal an interdomain cis interaction that is stabilized by a conserved, negatively charged pocket of the globular domain. Interestingly, this interaction surface overlaps an epitope recognized by the POM1 antibody, the binding of which drives rapid cerebellar degeneration mediated by the PrP N terminus. The resulting structure suggests that the globular domain regulates the N-terminal domain by binding the Cu(2+)-occupied OR within a complementary pocket.
Assuntos
Cobre/metabolismo , Proteínas Priônicas/química , Animais , Sítios de Ligação , Camundongos , Simulação de Acoplamento Molecular , Proteínas Priônicas/metabolismo , Ligação ProteicaRESUMO
Site-directed spin labeling (SDSL) is a powerful tool for the characterization of protein structure and dynamics; however, its application in many systems is hampered by the reliance on unique and benign cysteine substitutions for the site-specific attachment of the spin label. An elegant solution to this problem involves the use of genetically encoded unnatural amino acids (UAAs) containing reactive functional groups that are chemically orthogonal to those of the 20 amino acids found naturally in proteins. These unique functional groups can then be selectively reacted with an appropriately functionalized spin probe. In this chapter, we detail the genetic incorporation of the ketone-bearing amino acid p-acetyl phenylalanine (pAcPhe) into recombinant proteins expressed in E. coli. Incorporation of pAcPhe is followed by chemoselective reaction of the ketone side chain with a hydroxylamine-functionalized nitroxide to afford the spin-labeled side chain "K1," and we present two protocols for successful K1 labeling of proteins bearing site-specific pAcPhe. We outline the basic requirements for pAcPhe incorporation and labeling, with an emphasis on practical aspects that must be considered by the researcher if high yields of UAA incorporation and efficient labeling reactions are to be achieved. To this end, we highlight recent advances that have led to increased yields of pAcPhe incorporation, and discuss the use of aniline-based catalysts allowing for facile conjugation of the hydroxylamine spin label under mild reaction conditions. To illustrate the utility of K1 labeling in proteins where traditional cysteine-based SDSL methods are problematic, we site-specifically K1 label the cellular prion protein at two positions in the C-terminal domain and determine the interspin distance using double electron-electron resonance EPR. Recent advances in UAA incorporation and ketone-based bioconjugation, in combination with the commercial availability of all requisite reagents, should make K1 labeling an increasingly viable alternative to cysteine-based methods for SDSL in proteins.
Assuntos
Espectroscopia de Ressonância de Spin Eletrônica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Príons/química , Marcadores de Spin , Aminoácidos/química , Cisteína/química , Mutagênese Sítio-Dirigida , Fenilalanina/análogos & derivados , Fenilalanina/química , Estrutura Secundária de ProteínaRESUMO
Dimers of 2-substituted N,N'-dimethylbenzimidazoline radicals, (2-Y-DMBI)2 (Y=cyclohexyl (Cyc), ferrocenyl (Fc), ruthenocenyl (Rc)), have recently been reported as n-dopants for organic semiconductors. Here their structural and energetic characteristics are reported, along with the mechanisms by which they react with acceptors, A (PCBM, TIPS-pentacene), in solution. X-ray data and DFT calculations both indicate a longer C-C bond for (2-Cyc-DMBI)2 than (2-Fc-DMBI)2 , yet DFT and ESR data show that the latter dissociates more readily due to stabilization of the radical by Fc. Depending on the energetics of dimer (D2 ) dissociation and of D2 -to-A electron transfer, D2 reacts with A to form D(+) and A(-) by either of two mechanisms, differing in whether the first step is endergonic dissociation or endergonic electron transfer. However, the D(+) /0.5 D2 redox potentials-the effective reducing strengths of the dimers-vary little within the series (ca. -1.9â V vs. FeCp2 (+/0) ) (Cp=cyclopentadienyl) due to cancelation of trends in the D(+/0) potential and D2 dissociation energy. The implications of these findings for use of these dimers as n-dopants, and for future dopant design, are discussed.
Assuntos
Radicais Livres/química , Imidazolinas/química , Semicondutores , Derivados de Benzeno/química , Cristalografia por Raios X , Dimerização , Transporte de Elétrons , Modelos Moleculares , Naftacenos/química , Oxirredução , Teoria Quântica , TermodinâmicaRESUMO
Chemoselective protein labeling remains a significant challenge in chemical biology. Although many selective labeling chemistries have been reported, the practicalities of matching the reaction with appropriately functionalized proteins and labeling reagents is often a challenge. For example, we encountered the challenge of site specifically labeling the cellular form of the murine Prion protein with a fluorescent dye. To facilitate this labeling, a protein was expressed with site specific p-acetylphenylalanine. However, the utility of this acetophenone reactive group is hampered by the severe lack of commercially available aminooxy fluorophores. Here we outline a general strategy for the efficient solid phase synthesis of adapter reagents capable of converting maleimido-labels into aminooxy or azide functional groups that can be further tuned for desired length or solubility properties. The utility of the adapter strategy is demonstrated in the context of fluorescent labeling of the murine Prion protein through an adapted aminooxy-Alexa dye.
Assuntos
Corantes/metabolismo , Príons/metabolismo , Coloração e Rotulagem , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Eletroforese em Gel de Poliacrilamida , Indicadores e Reagentes , Camundongos , Dados de Sequência Molecular , Peptídeos/químicaRESUMO
The cellular form of the prion protein (PrP(C)) is found in both full-length and several different cleaved forms in vivo. Although the precise functions of the PrP(C) proteolytic products are not known, cleavage between the unstructured N-terminal domain and the structured C-terminal domain at Lys-109↓His-110 (mouse sequence), termed α-cleavage, has been shown to produce the anti-apoptotic N1 and the scrapie-resistant C1 peptide fragments. ß-Cleavage, residing adjacent to the octarepeat domain and N-terminal to the α-cleavage site, is thought to arise from the action of reactive oxygen species produced from redox cycling of coordinated copper. We sought to elucidate the role of key members of the ADAM (a disintegrin and metalloproteinase) enzyme family, as well as Cu(2+) redox cycling, in recombinant mouse PrP (MoPrP) cleavage through LC/MS analysis. Our findings show that although Cu(2+) redox-generated reactive oxygen species do produce fragmentation corresponding to ß-cleavage, ADAM8 also cleaves MoPrP in the octarepeat domain in a Cu(2+)- and Zn(2+)-dependent manner. Additional cleavage by ADAM8 was observed at the previously proposed location of α-cleavage, Lys-109↓His-110 (MoPrP sequencing); however, upon addition of Cu(2+), the location of α-cleavage shifted by several amino acids toward the C terminus. ADAM10 and ADAM17 have also been implicated in α-cleavage at Lys-109↓His-110; however, we observed that they instead cleaved MoPrP at a novel location, Ala-119↓Val-120, with additional cleavage by ADAM10 at Gly-227↓Arg-228 near the C terminus. Together, our results show that MoPrP cleavage is far more complex than previously thought and suggest a mechanism by which PrP(C) fragmentation responds to Cu(2+) and Zn(2+).
Assuntos
Proteínas ADAM/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Proteínas de Membrana/metabolismo , Proteínas PrPC/metabolismo , Proteínas ADAM/genética , Proteína ADAM10 , Proteína ADAM17 , Secretases da Proteína Precursora do Amiloide/genética , Animais , Cromatografia Líquida , Cobre/química , Cobre/metabolismo , Humanos , Espectrometria de Massas , Proteínas de Membrana/genética , Camundongos , Modelos Moleculares , Oxirredução , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteínas PrPC/química , Proteínas PrPC/genética , Estrutura Terciária de Proteína , Proteólise , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/metabolismo , Zinco/química , Zinco/metabolismoRESUMO
The discovery of air-stable n-dopants for organic semiconductor materials has been hindered by the necessity of high-energy HOMOs and the air sensitivity of compounds that satisfy this requirement. One strategy for circumventing this problem is to utilize stable precursor molecules that form the active doping complex in situ during the doping process or in a postdeposition thermal- or photo-activation step. Some of us have reported on the use of 1H-benzimidazole (DMBI) and benzimidazolium (DMBI-I) salts as solution- and vacuum-processable n-type dopant precursors, respectively. It was initially suggested that DMBI dopants function as single-electron radical donors wherein the active doping species, the imidazoline radical, is generated in a postdeposition thermal annealing step. Herein we report the results of extensive mechanistic studies on DMBI-doped fullerenes, the results of which suggest a more complicated doping mechanism is operative. Specifically, a reaction between the dopant and host that begins with either hydride or hydrogen atom transfer and which ultimately leads to the formation of host radical anions is responsible for the doping effect. The results of this research will be useful for identifying applications of current organic n-doping technology and will drive the design of next-generation n-type dopants that are air stable and capable of doping low-electron-affinity host materials in organic devices.
Assuntos
Imidazóis/química , Transporte de Elétrons , Cinética , Soluções , TermodinâmicaRESUMO
The cellular prion protein PrP(C) consists of two domains--a flexible N-terminal domain, which participates in copper and zinc regulation, and a largely helical C-terminal domain that converts to ß sheet in the course of prion disease. These two domains are thought to be fully independent and noninteracting. Compelling cellular and biophysical studies, however, suggest a higher order structure that is relevant to both PrP(C) function and misfolding in disease. Here, we identify a Zn²âº-driven N-terminal to C-terminal tertiary interaction in PrP(C). The C-terminal surface participating in this interaction carries the majority of the point mutations that confer familial prion disease. Investigation of mutant PrPs finds a systematic relationship between the type of mutation and the apparent strength of this domain structure. The structural features identified here suggest mechanisms by which physiologic metal ions trigger PrP(C) trafficking and control prion disease.