Molecular mechanisms for protein-encoded inheritance

Abstract

In prion inheritance and transmission, strains are phenotypic variants encoded by protein 'conformations'. However, it is unclear how a protein conformation can be stable enough to endure transmission between cells or organisms. Here we describe new polymorphic crystal structures of segments of prion and other amyloid proteins, which offer two structural mechanisms for the encoding of prion strains. In packing polymorphism, prion strains are encoded by alternative packing arrangements (polymorphs) of beta-sheets formed by the same segment of a protein; in segmental polymorphism, prion strains are encoded by distinct beta-sheets built from different segments of a protein. Both forms of polymorphism can produce enduring conformations capable of encoding strains. These molecular mechanisms for transfer of protein-encoded information into prion strains share features with the familiar mechanism for transfer of nucleic acid-encoded information into microbial strains, including sequence specificity and recognition by noncovalent bonds.

Figure 1

Packing polymorphism of steric zippers, determined by X-ray microcrystallography. A steric zipper is a pair of interdigitated β-sheets, generally with a dry interface between them. The views here look down the fibril axes, showing three layers of the zipper. In actual fibrils and microcrystals, there are tens of thousands of layers. Each strand forms backbone hydrogen bonds to strands above and below it. Water molecules are shown as aqua spheres a. Registration polymorphism of SSTNVG from islet amyloid polypeptide (IAPP). The left steric zipper (PDB code 3DG1) can be transformed to the right steric zipper by moving the top sheet to the left, and flipping side chains S2 and N4. b. Registration polymorphism of VQIVYK from tau protein. The left zipper (PDB code 2ON9) can be transformed to the right zipper by moving the top sheet to the right. c. Facial polymorphism of NNQQ from yeast prion Sup35. The left NNQQ steric (PDB codes 2ONX) zipper displays ‘face-to-back’ packing with N1 and Q3 amino acid side chains (yellow) of the top sheet interdigitated with Q4 and N2 (white) of the bottom sheet. In contrast, the right NNQQ steric zipper (PDB codes 2OLX ) displays ‘face-to-face’ packing, with N1 and Q3 side chains (yellow) of both sheets forming the interdigitated interface. d. Facial polymorphism of NNQNTF from elk prion protein. Both NNQNTF setric zippers are found in the same crystal structure, one face-to-face (right), with N1, Q3 and T5 (yellow) of both sheets forming the interdigitated interface; the other back-to-back, with sidechains N2, N4, and F6 interdigitated (white).

Figure 2. Segmental polymorphism in islet amyloid polypeptide (IAPP)

a. IAPP sequences and segment propensities for fibril formation. The sequence of human IAPP (hIAPP) is shown at bottom, with residue replacements in mouse IAPP (mIAPP) below. The histogram at top shows the estimated energies of steric zippers formed by six-residue segments (starting at the listed residue) of IAPP. Segments having energies of −23kcal/mol or lower are predicted to form fibrils⁴². b. The six IAPP segments (highlighted with horizontal bars in a) were synthesized and found to form fibrils, as shown in the electron micrographs, top (scale bars are 100 nm). Each of the segments also forms microcrystals, shown in the light micrographs, upper middle (scale bars 50 μm). The structures of the six segments were determined, lower middle, and each revealed a steric zipper. Resolutions and R-factors are given at the bottom; details are described in Supplementary Information. The electron micrographs of segments HSSNNF and NNFGAIL seem to show both microcrystals and fibrils.

Figure 3. Evidence for at least two steric zipper polymorphs in full length IAPP

a. Mouse IAPP (mIAPP) does not form fibrils, in contrast to human IAPP (hIAPP) which rapidly forms fibrils. The mutation of R18 to H in mIAPP now permits mIAPP R18H to form fibrils. b. The pH dependence of mIAPP R18H fibrillization supports the involvement of His18. The error bars are the standard deviation of six replicates. c. hIAPP fibrils are commonly twisted and ~8 nm in width; mIAPP R18H fibrils are uniformly wider (~9–10 nm) and untwisted (scale bars are 50 nm), suggesting a different underlying structure.

Figure 4. Schematic summary of steric-zipper mechanisms for amyloid and prion polymorphism

On the left, an amyloid-forming protein is depicted with two segments (blue and yellow) each capable of forming a self-complementary steric zipper. Below the linear sequence is shown a steric zipper formed by the yellow segment with two β-sheets face-to-face. a. Packing polymorphism, in which the yellow segment has a sequence capable of forming a second steric zipper with the two β-sheets packing face-to-back as well as face-to-face. b. Segmental polymorphism, in which both the yellow and blue segments have sequences capable of forming self-complementary steric zippers. c. Combinatorial polymorphism, in which the blue and yellow segments have sequences capable of engaging in a steric zipper. d. Single-chain registration polymorphism, in which two segments of the same chain form two steric zippers with different registrations of their sidechains. Compare this to Figures 1a and 1b where the registration polymorphs are formed from identical segments of different chains. Neither combinatorial nor single-chain-registration polymorphisms have yet been observed at atomic resolution.