Copper and Zinc Ions Specifically Promote Nonamyloid Aggregation of the Highly Stable Human γ‑D Crystallin Liliana Quintanar,* ,† Jose ́ A. Domínguez-Calva, † Eugene Serebryany, ‡ Lina Rivillas-Acevedo, § Cameron Haase-Pettingell, ‡ Carlos Amero, § and Jonathan A. King* ,‡ † Departamento de Química, Centro de Investigació n y de Estudios Avanzados (Cinvestav), 07360 Mexico City, Mé xico ‡ Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States § Centro de Investigaciones Químicas, Instituto de Investigació n en Ciencias Ba ́ sicas y Aplicadas, Universidad Autó noma del Estado de Morelos, 62209 Cuernavaca, Me ́ xico *S Supporting Information ABSTRACT: Cataract is the leading cause of blindness in the world. It results from aggregation of eye lens proteins into high-molecular-weight complexes, causing light scattering and lens opacity. Copper and zinc concentrations in cataractous lens are increased significantly relative to a healthy lens, and a variety of experimental and epidemiological studies implicate metals as potential etiological agents for cataract. The natively monomeric, β-sheet rich human γD (HγD) crystallin is one of the more abundant proteins in the core of the lens. It is also one of the most thermodynamically stable proteins in the human body. Surprisingly, we found that both Cu(II) and Zn(II) ions induced rapid, nonamyloid aggregation of HγD, forming high-molecular-weight light-scattering aggregates. Unlike Zn(II), Cu(II) also substantially decreased the thermal stability of HγD and promoted the formation of disulfide-bridged dimers, suggesting distinct aggregation mechanisms. In both cases, however, metal-induced aggregation depended strongly on temperature and was suppressed by the human lens chaperone αB-crystallin (HαB), implicating partially folded intermediates in the aggregation process. Consistently, distinct site-specific interactions of Cu(II) and Zn(II) ions with the protein and conformational changes in specific hinge regions were identified by nuclear magnetic resonance. This study provides insights into the mechanisms of metal-induced aggregation of one of the more stable proteins in the human body, and it reveals a novel and unexplored bioinorganic facet of cataract disease. C ataract is the leading cause of blindness in the world, and it is projected to affect 50 million people by 2050 in the U.S. alone. 1 Cataracts are formed upon aggregation of lens proteins into high-molecular-weight complexes, causing light scattering and lens opacity. 2,3 The currently available treatment for cataract is eye surgery, which though effective, is costly and not risk-free. Developed countries like the United States spend billions of dollars per year in cataract surgery, while in developing countries, cataract has become the major cause of visual disability. The eye lens is responsible for transparency and focusing of light onto the retina, essential for normal vision. The lens is formed of elongated fiber cells with high protein content, depleted of nuclei and organelles; this unique differentiation occurs in the embryonic stage. 4 Fully differentiated fiber cells have a very low metabolism, and they are void of protein synthesis and degradation machineries. Crystallins constitute the most abundant proteins in the lens, and their solubility and stability are essential to maintain its transparency throughout the lifetime of an individual. 2,3 Crystallins are classified as α-, β-, and γ- crystallins, and they constitute more than 90% of the protein content in the human lens. 5 α-crystallins belong to the family of small heat shock proteins, and they function as molecular chaperones by recognizing exposed hydrophobic patches in partially folded β- and γ-crystallins and complexing them to prevent their aggregation. 2,6,7 The β- and γ-crystallins are composed of duplicated domains that share double Greek key β-sheet folds. Although the β-crystallin family members form dimers and oligomers, the γ-crystallins are monomeric. Human γD (HγD) crystallin is one of the more abundant crystallins in the core of the lens, and its nonamyloid aggregation is associated with cataracts. 5 HγD crystallin is a highly stable protein, resisting denaturation by heat (80 °C) and chemical agents (8 M urea or 2−3 M guanidinium chloride). 8 However, when partially folded molecules of HγD crystallin are formed, these are prone to aggregation in the absence of chaperones. 9,10 Aggregation of partially folded proteins into high-molecular- weight aggregates has emerged as a major hallmark of degenerative diseases. 11−14 Particular attention has been focused on cases in which the aggregated state is an amyloid Received: July 16, 2015 Accepted: November 18, 2015 Published: November 18, 2015 Articles pubs.acs.org/acschemicalbiology © 2015 American Chemical Society 263 DOI: 10.1021/acschembio.5b00919 ACS Chem. Biol. 2016, 11, 263−272