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Understanding Prion Structure and Stability

Wuthrich et al. made an important observation (Nature 382: 180 1996): mouse prion fragment 121-231 is markedly bipolar: one side of the disks is negative, the other positive. They interpret this latter feature as electrostatic binding to a negatively-charged outer phospholipid plasma membrane surface which then orients the negative face of prion protein outwards.

The dimensional scale is missing from the article but fortunately the axis of helix H1 is parallel to the plane of the diagram, so an axial translation of 1.5 Angstrom per residue is appropriate. Overall the fragment of prion protein studied would fit into a 35 x 35 x10 angstrom box. It thus has a high surface area to volume ratio compared to a globular protein of the same size.

Prion protein has a GPI anchor already so it doesn't need electrostatic binding to anchor itself to the membrane. Major plasma membrane components like sphingomyelin, cerebrosides, phosphatidyl ethanolamine, phosphatidyl choline, sterols, and integral membrane proteins can provide neutral membrane amphipathicity; neuronal membranes are highly specialized mosaics; and prion protein is reported localized within calveolar patches. Bipolarity is not consistently reported from other GPI caveolar proteins. The undetermined amino terminus of mature prion could mask the fragment's positive surface. In short, the proferred explanation for bipolarity is problematic.

The authors do not calculate the electric dipole moment and dipole axis corresponding to this surface, nor did they measure it by dielectric constant relaxation frequency. An accurate calculation is easy given refined coordinates [Biophys J 94:1550 1993]; as a point of departure, the Glockshuber-Wuthrich structure can be modelled as a oblate ellisoid and the dipole moment estimated (with an unscreened uniform dielectric coulombic model) at some 225 debyes orthogonal to the plane of the disc with only the 3 histidines presenting a computational issue at the isoelectric point. (Coordinates can be reverse-engineered from published rendered projections using "thin-sectioning" techniques in IHS color space.) Charge separation is thus a force holding the protein together and could contribute to the exceptional stability of prion protein to heat denaturation or to kinetics of renaturation.

Now the three alpha helices have significant dipole moments of their own (from aligned hydrogen bonds and peptide bonds) not related to that from surface charge, unlike the beta sheet. These align constructively along the helical axis to form macrodipoles (positive axis N-terminal) of 39, 43, and 59 debyes. The pattern is out-of-sequence anti-parallel, ie, there are constructive dipole-dipole interactions between pairs of helices and also with the bipolar dipole, as the dipoles are oriented nose to tail and a few angstroms apart in the 3D picture. Normally,1H NMR directly correlates helix dipole strength and amide proton chemical shifts (J Mol Biol 222: 311 1991).

The accompaning graphic shows the relationship between these features -- it is consistent with the idea of an important dipole contribution to structural stability of prion protein. A previously predicted helix H0 at 109-122 (PNAS 91: 7139 1996) is shown in faint colors in a speculative alignment that matches its dipole with the unpaired end of helix H2. This stretch is the core invariant region of prion protein, hardly changing from chicken to marsupial to mammal since the Devonion, and a likely site for ligand binding. The figure also shows (with a red X) a proposed binding site for a small ligand or catalytic binding site based on dipole distribution. (Helix HO should folded over this but is not, for clarity.) These roles for the observed surface charge distribution could either supplement the membrane binding proposal, or displace it.

A very important further consideration for an alpha helix is its cap, commonly a macrodipole-compensatory charged residues facing the end of the helix. These need to be considered in prion structure and disease, as do the effects on helix stability of various internal changes (e.g. alanine can improve stability). The leading and trailing four residues of alpha-helices also differ from the others by a partial inability to make intra-helical hydrogen bonds. There is a statistical preference for certain cap residues at the C and N termini that fulfill these special hydrogen bonding requirements (Nature 342: 296 1989).

Five CJD-causing mutations are helix-proximal, attributable perhaps to cap effects. Internal helical changes associated with disease may follow inverse propensity to form alpha helix [given in Science 250: 646 1990], to the extent that a weakened helix is conducive to formation of the scrapie isoform.

If prion protein forms a oligomer, we might expect the dipole moment of each subunit to be antiparallel, causing partial cancellation of the net dipole moment, as Takashima observed for tetramers such as normal hemoglobin (Biophys J 64: 1550 1993). It is worth noting that sickle cell tetramers have a signficantly smaller dipole because of two glutamate-to-valine changes. With prion strains, scrapie isoform seems to induce symmetric changes in cellular isoform [Science 20 Dec 1996], consistent with Monod's theory of homodimer Z(2) symmetry and thus reinforcing anti-parallel subunit dipoles.

The final observation pertinent to electrostatics and prion protein conformational change is that of Przybycien and Bailey (BBA 1076: 103-11 1991): a chaotropic salt, KSCN, caused formation of beta sheet at the expense of alpha helix in proportion to the dipole moment, fraction of charged amino acids in the primary sequence, surface area, and initial helical content, in 12 proteins. Precipatates could not be redissolved without a solubilizing agent.

In other words, conversion of cellular to scrapie isoforms could essentially be salting out nucleated by a slightly abnormal isoform. Beta sheet is commonly seen at a quaternary subunit interface.

The roles of global electrostatic interaction in proteins are reviewed in Quart. Rev Biophys 29: 1-90 1996. Horse cytochrome c is strongly bipolar: a 300 debye dipole contributes signficantly to interaction with substrate (JBC 257: 4426 1982). Human neuropeptide Y has a charge clustering giving rise to a dipole moment of 325-450 debye directed toward the receptor-binding region, a dipole moment antiparallel to that of the alpha-helix (Biochemistry 32: 2954 1993). Acetylcholinesterase exhibits charge steering (Biopolymers 39: 85-94 1996). The overall dipole moment of red algal flavodoxin is said to be important in its interactions with other proteins (J Biol Chem 265: 15804 1990).

There are many examples in protein chemistry where helix dipoles have been implicated in ligand binding and catalysis, reviewed in Adv Biophys 19: 133 1985, where nine examples are given of alpha helix N-termini near an active site. For papain and cathepsin (Biochemistry 35: 12495 1996), tryptophan synthase (Proteins 21: 130 1995), and thioredoxin (J Mol Biol 253: 799 1995), a helix macrodipole has a binding role. Cohen observed twelve proteins with anti-parallel orientation of neighboring helices and concluded that "this distribution is in accordance with structure types expected if the helix macro dipole effect makes a substantial contribution to the stability of the native structure (PNAS 86: 6592 1989).

Alpha helical caps have been found significant in many proteins: for lysozyme, introduced acidic groups designed to interact with dipole partial positive charge at the N-termini of alpha-helices increased stability (Ciba Found Symp 161: 52 1991) as did internal alanine; similar results were found for malate dehydrogenase (Biochemistry 32: 39131993); Tyr to Asp in cro protein was a stabilizing capping interaction N-terminal to an alpha helix (Proteins 19: 310 1994); capping improved heat stability in ribonuclease (Nature 342: 296 1989); histidine adversely affects alpha-helix stability by interacting with the backbone charges (PNAS 90: 11337 1993); and ribonuclease T1was destabilized by acidic to amide dipole cap changes (J Mol Biol 252: 133 1995).

Lys to Ser or Asp at a helix N-terminus improved neutral protease thermostability (Protein Eng 5: 165 1992); ribonuclease A also exhibited a strong charged cap effect (Nature 326: 5631987); barnase exhibited stabilization of protein structure by interaction of alpha-helix dipole with a charged side chain and a markedly increased pKa for a histidine (Nature 335: 740 1988); in glutaredoxin, the electrostatic field associated with an alpha-helix N terminal to a cysteine residue lowered the thiol pKa value by 5 units (J Mol Biol 253: 799 1995); and a simple set of rules accounted for the interaction of charged groups with the alpha helix dipole in myoglobin from 62 different species (J Biomol Struct Dyn 7: 973 1990).

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