Elucidating the Folding Problem of Helical Peptides using Empirical Parameters. II†. Helix Macrodipole Effects and Rational Modification of the Helical Content of Natural Peptides

The conversion of prion helix 1 from an ␣-helical into an extended conformation is generally assumed to be an essential step in the conversion of the cellular isoform PrP C of the prion protein to the pathogenic isoform PrP Sc . Peptides encompassing helix 1 and flanking sequences were analyzed by nuclear magnetic resonance and circular dichroism. Our results indicate a remarkably high instrinsic helix propensity of the helix 1 region. In particular, these peptides retain significant helicity under a wide range of conditions, such as high salt, pH variation, and presence of organic co-solvents. As evidenced by a data base search, the pattern of charged residues present in helix 1 generally favors helical structures over alternative conformations. Because of its high stability against environmental changes, helix 1 is unlikely to be involved in the initial steps of the pathogenic conformational change. Our results implicate that interconversion of helix 1 is rather representing a barrier than a nucleus for the PrP C 3 PrP Sc conversion.Prion protein, PrP, 1 is probably the disease-causing agent of transmissible spongiform encephalopathies such as bovine spongiform encephalopathy in cattle or Creutzfeldt-Jakob disease in man (1). Its cellular form, PrP C , is a highly conserved cell surface glycoprotein of 230 amino acids expressed in all of the mammals studied so far as well as in several species of fish and birds (2, 3). The physiological function of PrP C is not yet fully understood. PrP C seems to be involved in the maintenance of proper presynaptic copper levels as well as in protecting neurons from oxidative stress (4, 5). In addition, the physiological function of PrP C could be associated with higher neurological functions such as learning and memory (5). According to the protein-only hypothesis, disease is caused by accumulation of a misfolded pathogenic isoform, PrP Sc , which is the result of an irreversible large scale conformational change of PrP C . Although PrP C is largely ␣-helical and soluble in polar solvents and sensitive to protease K digestion, PrP Sc consists mostly of ␤-sheets, is soluble only in nonpolar, denaturing solvents, and is resistant to digestion with protease K (6). PrP Sc forms fibrillar aggregates similar to other amyloid fibrils (7). Accumulation of PrP Sc aggregates is accompanied by astrocytosis and gliosis in central nervous tissue, which in turn result in vacuoles in the brains of patients.The solution structures of human PrP-(23-230), huPrP (8), mouse PrP-(121-231) (9), bovine PrP-(23-230) (10), and Syrian hamster PrP-(29 -231) (11) have been determined by NMR spectroscopy. They possess a high degree of structural conservation consistent with the high sequence identity of these proteins. Prion proteins consist of a flexible NH 2 -terminal domain spanning residues 23-124 (huPrP C -numbering scheme), which is largely disordered. This region includes an octapeptide sequence that is repeated four times from residues 60 to 92 and that is likely to bind copper (4). This part al...

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“…2C). Helix content and relative secondary shifts for both mutations are consistent with predictions made by AGADIR (Table I) (40,41,42).…”

Section: Resultssupporting

confidence: 85%

CD and NMR Studies of Prion Protein (PrP) Helix 1

Ziegler

Sticht

Marx

et al. 2003

Journal of Biological Chemistry

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“…Not surprisingly, these electrostatically complementary residues are essentially conserved in all known c-Myb and CBP/p300 sequences (28). Table S1 reports the top six point mutant candidates that were predicted from AGADIR (68)(69)(70)(71)(72) to increase the helical propensity of the α 3 helix and therefore can potentially affect cooperative binding (SI Text). Out of all of the C-terminal residues located in the α 3 helix that make no intermolecular native contacts with either c-Myb or MLL, only six point mutations at either Lys667 or Ser670 caused a significant increase in the predicted helicity of α 3 (∼3.5-12.7%) relative to the wild-type sequence.…”

Section: Discussionmentioning

confidence: 99%

Prepaying the entropic cost for allosteric regulation in KIX

Law¹,

Gagnon²,

Mapp

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The kinase-inducible domain interacting (KIX) domain of the CREB binding protein (CBP) is capable of simultaneously binding two intrinsically disordered transcription factors, such as the mixedlineage leukemia (MLL) and c-Myb peptides, at isolated interaction sites. In vitro, the affinity for binding c-Myb is approximately doubled when KIX is in complex with MLL, which suggests a positive cooperative binding mechanism, and the affinity for MLL is also slightly increased when KIX is first bound by c-Myb. Expanding the scope of recent NMR and computational studies, we explore the allosteric mechanism at a detailed molecular level that directly connects the microscopic structural dynamics to the macroscopic shift in binding affinities. To this end, we have performed molecular dynamics simulations of free KIX, KIX-c-Myb, MLL-KIX, and MLL-KIX-c-Myb using a topology-based Go-like model. Our results capture an increase in affinity for the peptide in the allosteric site when KIX is prebound by a complementary effector and both peptides follow an effector-independent folding-and-binding mechanism. More importantly, we discover that MLL binding lowers the entropic cost for c-Myb binding, and vice versa, by stabilizing the L 12 -G 2 loop and the C-terminal region of the α 3 helix on KIX. This work demonstrates the importance of entropy in allosteric signaling between promiscuous molecular recognition sites and can inform the rational design of small molecule stabilizers to target important regions of conformationally dynamic proteins.

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“…4): A mutation destabilizes one end of the molecule, causing folding flux through the other end of the molecule. The initiation site for folding of wild-type myotrophin does not correlate with intrinsic helical propensity [as predicted by the program AGADIR (24,25)], which is below 5% for all residues except those in the second helix of ANK II for which the propensity rises to Ϸ15%. Interestingly, folding appears to be initiated preferentially at the terminal rather than internal repeats of myotrophin.…”

Section: Discussionmentioning

confidence: 99%

Rational redesign of the folding pathway of a modular protein

Lowe

Itzhaki

2007

Proc. Natl. Acad. Sci. U.S.A.

100

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The modular structures of repeat proteins afford them distinct properties compared with globular proteins, enabling them to function in a large and diverse range of cellular processes. Here, we show that they can also have different folding mechanisms. Myotrophin comprises four ankyrin repeats stacked linearly to form an elongated structure. Using site-directed mutagenesis, we find that folding of wild-type myotrophin is initiated at the C-terminal repeats. However, close examination of the mutant chevron plots reveals that simple models are insufficient to describe all of the data, and double mutant analysis subsequently confirms that there are parallel folding pathways. Destabilizing mutations in the C-terminal repeats reduce flux through the wild-type pathway, making a new route accessible in which folding is initiated at the N-terminal repeats. Thus, the folding mechanism of the repeat protein is poised on a fulcrum: When one end of the molecule is perturbed, the balance shifts between the different nucleation sites. The vast majority of studies on small globular proteins indicate a single, well defined route between the denatured and native states. By contrast, the potential to initiate folding at more than one site may be a general feature of repeat proteins arising from the symmetry inherent in their structures. We show that this simple architecture makes it straightforward to direct the folding pathway of a repeat protein by design.ankyrin repeat ͉ myotrophin ͉ ⌽ value ͉ protein engineering ͉ parallel pathways R epeat proteins, such as ankyrin repeats, leucine-rich repeats, and tetratricopeptide repeats, consist of tandem arrays of a structural motif of 20-40 aa that stack in a roughly linear fashion, creating elongated and superhelical architectures (1-3). Repeat protein structures are composed entirely of short-range interactions, either within a repeat or in adjacent repeats, in striking contrast to the more complex topologies of globular proteins that are stabilized by contacts between residues distant in sequence (4). They are ubiquitous, representing Ͼ5% of eukaryotic proteins in the Swiss-Prot database, and highly versatile, mediating molecular recognition in many different biological processes, but little is known about their folding mechanisms. The simple, modular nature of these structures has made them uniquely successful as scaffolds for engineering novel binding specificities (5-7). Does the repeat architecture likewise give rise to distinct folding properties, and are their folding pathways also amenable to design?In this study, we have addressed this question using the small, well behaved ankyrin (ANK) repeat protein myotrophin. The folding and unfolding properties of ANK repeats are of particular interest because these motifs are thought to play a role in mechanical signal transduction by virtue of their putative elastic properties (8, 9). The ANK repeat is a 33-residue motif consisting of a pair of antiparallel ␣-helices linked to the neighboring repeat via an antiparallel ␤-loop. The 11...

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Elucidating the Folding Problem of Helical Peptides using Empirical Parameters. II†. Helix Macrodipole Effects and Rational Modification of the Helical Content of Natural Peptides

Cited by 365 publications

References 81 publications

CD and NMR Studies of Prion Protein (PrP) Helix 1

CD and NMR Studies of Prion Protein (PrP) Helix 1

Prepaying the entropic cost for allosteric regulation in KIX

Rational redesign of the folding pathway of a modular protein

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