Proc. Natd. Acad. Sci. USA Vol. 91, pp. 7139-7143, July 1994 Biophysics Proposed three-dimensional structure for the cellular prion protein (protein conformation/secondary structure prediction/tertiary structure predictIon/computer modelng/prion diseases) ZIWEI HUANG*t, JEAN-MARC GABRIELtI, MICHAEL A. BALDWINt, ROBERT J. FLETTERICK*§, STANLEY B. PRUSINERt§, AND FRED E. COHEN*§1¶I Departments of *Pharmaceutical Chemistry, tNeurology, §Biochemistry and Biophysics, and NMedicine, University of California, San Francisco, CA 94143 Contributed by Stanley B. Prusiner, February 14, 1994 ABSTRACT Prion diseases are a group of neurodegenera- tive disorders in human and animals that seem to result from a conformatlonal change in the prion protein (PrP). Utilizing data obtained by circular dichroism and in spectroscopy, computational studies prdIe the tedi nal s ture of the cellular form of PrP (PrPC). A heuristic approach consistng of the prediction of so structures and of an evalnation of the pig of s y elements was used to search for plausible teriay strucures. After a series of exper- imental and theoretical ants were applied, four structural models of four-helix bundles emerged. A group of amino acids within the four predicted helices were Identified as important for tertiary interactions between helices. These amino acids could be essential for ma ining a stable tertiary suture of PrPC. Among four plausible s al models for PrPC, the X-bundle model seemed to correlate best with 5 of 11 known point mutations that segregate with the inherited prion diseases. These 5 mutions cluster around a central hydrophobic core in the X-bundle structure. Furthermore, these mutations occur at or near those amino acds which are predicted to e important for helix-helix interactions. The three-dimensonal structure of PrPc proposed here may not only provide a basis for rational- izing mutations of the PrP gene in the inherited prion diseases but also guide design of genticafly engineered NP molecules for further experimental studies. Prions are a novel class of "infectious" pathogens distinct from viroids and viruses with respect to both their structure and the neurodegenerative diseases that they -cause (1). Prion diseases are manifest as sporadic, inherited, and infectious disorders including scrapie, mink encephalopathy, chronic wasting disease, bovine spongiform encephalopathy, feline spongiform encephalopathy, and exotic ungulate encepha- lopathy of animals (2-4) as well as kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Striussler-Scheinker syndrome, and fatal familial insomnia of humans (5-7). The prion protein (PrP) is the major, if not the only, component of prions (1). PrP exists in two isoforms: the normal cellular form (PrP9 and the abnormal disease (scrapie)-related form (PrPSc) (8, 9). The entire open reading frame of PrP genes is contained in a single exon, eliminating the possibility that PrPC and PrPSc arise from alternative RNA splicing (10). Attempts to identify a posttranslational chemical modification that features in the conversion of PrPC to PrPSc have been unsuccessful (11). Structural studies of PrPC and PrPSC using Fourier-transform infrared and circular dichroism spectroscopy indicated that PrPc and PrPSC differ from each other in their conformations (12-17). These findings suggest that prion diseases are dis- orders of protein conformation and result from a change in the structure of PrPC when it is converted into PrPsc. Elucidating the three-dimensional structures of PrPC and PrPSC and the conformational changes that occur during the production of PrPsc is central to understanding the molecular mechanisms of prion diseases. Because of the low level of expression of PrPC and insolubility of PrPSc, efforts to obtain crystals for x-ray structure determinations have been unsuc- cessful. Exploiting recent advances in protein structure pre- diction algorithms, we carried out computational studies to predict the three-dimensional structure of PrPC based on a family of homologous amino acid sequences. Our predictive studies were facilitated by spectroscopic findings showing that PrPC has a secondary structure which contains ==43% a-helix and is virtually devoid of 3-sheet (15). Although the problem of predicting protein structure from the amino acid sequence information alone remains unsolved, recent advances in secondary and tertiary structure predic- tion of proteins have shown that reasonable structures can often be proposed by using a heuristic approach in conjunc- tion with experimental data (18). For all-a-helical proteins, this approach has been applied to generate low-resolution structural models for a number of proteins (19-21). Some of the structural features proposed by these methods have been verified by subsequent x-ray or NMR experiments (22). In the study reported here, three-dimensional structures of PrPc were generated by applying the heuristic approach coupled with experimental data. In the absence of a three- dimensional structure of PrPC from either x-ray crystallog- raphy or NMR spectroscopy, these models provide a vehicle to rationalize much of the available data and help design further experimental studies. METHODS The computational procedures used for the prediction of the three-dimensional structures of PrPC involved four major steps: (i) alignment of a family of homologous sequences, (ii) prediction of secondary structures, (iii) packing of secondary elements to generate all plausible tertiary structures, and (iv) selection and refinement of final structural models. PrP amino acid sequences from 1 avian and 11 mammalian sources including chicken, cow, sheep, rat, mouse, hamster, mink, and human were used. The alignment of these se- quences was reported previously (23), using the Feng and Doolittle algorithm (24). Methods for secondary and tertiary structure prediction were applied independently to all 12 PrP sequences. As suggested by Benner and Gerloff (25), the use of a broad family of homologous sequences improves the accuracy of structure prediction. Secondary Structure Prediction. An initial attempt to pre- dict the secondary structure of PrPC was made by several different methods, including the Chou-Fasman method (26), GOR algorithm (27), and PHD program (28). The GOR algorithm Abbreviations: PrP, prion protein; PrPC, cellular isoform of PrP; PrPs, scrapie isoform of PrP; CJD, Creutzfeldt-Jakob disease. ITo whom reprint requests should be addressed at: Department of Pharmaceutical Chemistry, University of California, San Fran- cisco, CA 94143-0446. tPresent address: Department of Research, Neurobiology Labora- tory, Kantonsspital, CH-4031 Basel, Switzerland. 7139 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.