Apolipoprotein E4 Forms a Molten Globule A POTENTIAL BASIS FOR ITS ASSOCIATION WITH DISEASE* Received for publication, May 17, 2002, and in revised form, October 21, 2002 Published, JBC Papers in Press, October 21, 2002, DOI 10.1074/jbc.M204898200 Julie A. Morrow‡§¶, Danny M. Hatters‡§¶, Bin Lu‡§, Peter Ho ¨ chtl, Keith A. Oberg**, Bernhard Rupp, and Karl H. Weisgraber‡§‡‡§§ From the ‡Gladstone Institutes of Cardiovascular Disease and Neurological Disease, San Francisco, California 94141- 9100, the §Cardiovascular Research Institute and the ‡‡Department of Pathology, University of California, San Francisco, California 94143, Lawrence Livermore National Laboratory, Livermore, California 94551, and **Allecure, Inc., Los Angeles, California 91355 The amino-terminal domain of apolipoprotein (apo) E4 is less susceptible to chemical and thermal denatur- ation than the apoE3 and apoE2 domains. We compared the urea denaturation curves of the 22-kDa amino-ter- minal domains of the apoE isoforms at pH 7.4 and 4.0. At pH 7.4, apoE3 and apoE4 reflected an apparent two-state denaturation. The midpoints of denaturation were 5.2 and 4.3 M urea, respectively. At pH 4.0, a pH value known to stabilize folding intermediates, apoE4 and apoE3 dis- played the same order of denaturation but with distinct plateaus, suggesting the presence of a stable folding intermediate. In contrast, apoE2 proved the most stable and lacked the distinct plateau observed with the other two isoforms and could be fitted to a two-state unfolding model. Analysis of the curves with a three-state unfold- ing model (native, intermediate, and unfolded) showed that the apoE4 folding intermediate reached its maxi- mal concentration (90% of the mixture) at 3.75 M, whereas the apoE3 intermediate was maximal at 4.75 M (80%). These results are consistent with apoE4 being more susceptible to unfolding than apoE3 and apoE2 and more prone to form a stable folding intermediate. The structure of the apoE4 folding intermediate at pH 4.0 in 3.75 M urea was characterized using pepsin prote- olysis, Fourier transform infrared spectroscopy, and dy- namic light scattering. From these studies, we conclude that the apoE4 folding intermediate is a single molecule with the characteristics of a molten globule. We propose a model of the apoE4 molten globule in which the four- helix bundle of the amino-terminal domain is partially opened, generating a slightly elongated structure and exposing the hydrophobic core. Since molten globules have been implicated in both normal and abnormal physiological function, the differential abilities of the apoE isoforms to form a molten globule may contribute to the isoform-specific effects of apoE in disease. Apolipoprotein (apo) 1 E plays a key role in lipid transport throughout the body including the nervous system and is in- volved in the maintenance and repair of neurons (1, 2). One of the common human apoE isoforms, apoE4, is a major risk factor for Alzheimer’s disease (3–5) and atherosclerosis (6 – 8). ApoE4 is also associated with poor recovery from head injury and stroke (9 –11), cognitive decline associated with coronary bypass surgery (12), increased severity of tissue damage in multiple sclerosis (13), shortening of survival after the onset of amyotrophic lateral sclerosis (14), and a poor response to other forms of central nervous system stress (15). The three common isoforms of apoE (apoE2, apoE3, and apoE4) are genetically determined and differ in cysteine and arginine content at positions 112 and 158: apoE2 (Cys 112 , Cys 158 ), apoE3 (Cys 112 , Arg 158 ), and apoE4 (Arg 112 , Arg 158 ) (16, 17). The protein contains two distinct structural domains: a 22-kDa amino-terminal domain and a 10-kDa carboxyl-termi- nal domain (18, 19). In apoE4 and not the other isoforms, the two domains interact in a unique manner. In apoE4, Arg 112 causes Arg 61 to assume a unique conformation and interact with Glu 255 in the carboxyl-terminal domain. This novel prop- erty of apoE4 is referred to as apoE4 domain interaction (20, 21) and was suggested to contribute to the association of apoE4 with disease (2, 21). Previously, we demonstrated that the two domains of apoE unfold independently for all three isoforms (19, 22) and that the 22-kDa fragments, which contain the amino acid interchanges, differ in their susceptibility to thermal and chemical denatur- ation (apoE4  apoE3  apoE2) (22, 23). Denaturation of apoE2 with guanidine at neutral pH displayed two-stage coop- erative unfolding, whereas apoE3 and apoE4 displayed nonco- operative unfolding that was much more prominent with apoE4. This noncooperative unfolding of apoE4 suggested the presence of a stable folding intermediate (22). Folding intermediates that are both stable under certain conditions and have nearly native structural features are re- ferred to as molten globules (24). Three structural features characterize the molten globule state. First, a significant amount of secondary structure of the native state is retained. Second, although there is considerable loss of tertiary struc- ture, the molten globule is structurally compact. Third, there is internal mobility with exposure of the hydrophobic core. Until recently, it was assumed that the molten globule was a rela- tively rare state for a protein. However, there is increasing evidence that molten globules are common and that they play a key role in a wide variety of physiological processes, including translocation across membranes, increased affinity for mem- branes, binding to liposomes and phospholipids, protein traf- * This work was supported in part by grant NS35939 from the Na- tional Institutes of Health (to K. H. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ¶ Both authors contributed equally to this work. §§ To whom correspondence should be addressed: Gladstone Inst. of Cardiovascular Disease, P. O. Box 419100, San Francisco, CA 94141- 9100. Tel.: 415-826-7500; Fax: 415-285-5632; E-mail: kweisgraber@ gladstone.ucsf.edu. 1 The abbreviations used are: apo, apolipoprotein; FTIR, Fourier transform infrared spectroscopy; DLS, dynamic light scattering; DMPC, dimyristoylphosphatidylcholine; R h , hydrodynamic radius; M r , molecular mass. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 52, Issue of December 27, pp. 50380 –50385, 2002 Printed in U.S.A. This paper is available on line at http://www.jbc.org 50380 by guest on May 30, 2020 http://www.jbc.org/ Downloaded from