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A histidine switch for Zn-induced aggregation of #-crystallins reveals a metal-bridging mechanism relevant to cataract disease José A. Domínguez-Calva, Cameron Haase-Pettingell, Eugene Serebryany, Jonathan A. King, and Liliana Quintanar Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00436 • Publication Date (Web): 31 Jul 2018 Downloaded from http://pubs.acs.org on August 4, 2018
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Biochemistry
A histidine switch for Zn-induced aggregation of γ−crystallins reγ− veals a metal-bridging mechanism relevant to cataract disease. José Antonio Domínguez-Calva,1 Cameron Haase-Pettingell,2 Eugene Serebryany,2 Jonathan Alan King,2, Liliana Quintanar1,* 1
Departamento de Química, Centro de Investigación y de Estudios Avanzados (Cinvestav), Mexico City, México
2
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA
Supporting Information Placeholder ABSTRACT: Cataract disease results from non-
amyloid aggregation of eye lens proteins and it is the leading cause of blindness in the world. Zinc concentrations in cataractous lenses are increased significantly relative to healthy lens. It was recently reported that Zn(II) ions induce the aggregation of one of the more abundant proteins in the core of the lens: human γD-crystallin. Here, the mechanism of Zninduced aggregation has been revealed through a comparative study of three homologous human lens γ-crystallins, and a combination of spectroscopic, electron microscopy and site-directed mutagenesis studies. This work reveals that a single His residue acts as a “switch” for the Zn-induced non-amyloid aggregation of human γ-crystallins. Aggregation can be reversed by a chelating agent, revealing a metalbridging mechanism. This study sheds light into an aberrant Zn-crystallin interaction that promotes aggregation, a process that is relevant to cataracts disease.
Cataract disease is the leading cause of blindness in the world, affecting over 60 million people worldwide.1 Cataracts are formed upon aggregation of lens proteins into high molecular weight complexes, causing light scattering and lens opacity. Crystallins constitute the most abundant proteins in the lens, and their solubility and stability are essential to maintain its transparency.2, 3 Crystallins are classified as: αcrystallins, which are multimeric, belong to the family of small heat shock proteins, and function as molecular chaperones; the dimeric β-crystallins, and the monomeric γ-crystallins.3-5 Upon aging, the lens crystallins suffer oxidative damage that cause the formation of partially folded intermediates prone to
non-amyloid aggregation. α-crystallins are able to recognize partially folded β- and γ-crystallins and bind them to prevent their aggregation.2, 6 Although the cellular environment of fiber cells in the human lens has a very low metabolism, metal ions are required for enzymatic activity.7 Thus, a healthy human lens contains copper, zinc, and iron at concentrations that range between 0.4 and 30 µg of metal per gram of dry lens tissue.7-10 However, copper and zinc concentrations in the lens are significantly increased during cataract disease: cataractous lenses have approximately twice as much Zn and ten times more Cu, as compared to normal lenses.10 Moreover, several studies implicate metals as a potential etiological agent for cataract.11 A recent study demonstrated that Cu(II) and Zn(II) ions can induce the non-amyloid aggregation of human γD-crystallin (HγD), one of the more abundant γ-crystallins in the core of the lens, involving site-specific interactions with this lens protein.12 In this study, we have evaluated the effect of Zn ions in the aggregation of the homologous human γC- and γS- crystallins (HγC and HγS, respectively), and have investigated the mechanism of Zn-induced aggregation of HγD crystallin. HγC and HγS crystallins are the most abundant γcrystallins in the nucleus and cortex of the human lens, respectively, underscoring the importance to determine if they are also sensitive to Zn-induced aggregation. Turbidity assays with HγD, HγC and HγS crystallins at 37 °C in the presence of different concentrations of Zn(II) ions clearly show that only HγD is sensitive to Zn-induced aggregation (Figure 1A), while Zn exerts no effect on HγC and HγS crystallins (Figures 1B and 1C, respectively). Although, HγD, HγC and HγS share a common Greek key fold struc-
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ture and 80% similarity in their sequences (Scheme 1), the striking difference in their sensitivity to Zninduced aggregation may be due to a different content of His and Cys residues, which may act as Znanchoring sites. In order to identify the amino acid residues in HγD crystallin that might be involved in Zn-induced aggregation, the effect of this metal ion in the aggregation of its separate N- and C-terminal domains was evaluated (Figures S1A and S1B, respectively). Interestingly, only the N-terminal domain of HγD is sensitive to Zn-induced aggregation (Figure S1A). By inspection of the structures and sequence alignment of the three γ-crystallins, His22 and Cys18 stand out as putative residues in the N-terminal domain that are not conserved, yet possibly important in Zn-induced aggregation of HγD crystallin (Scheme1). Indeed, the H22Q mutation in HγD crystallin abolishes Zninduced aggregation (Figure 1D), but the C18S mutation does not (Figure S1D). The H22Q/C18S double mutation also abolishes Zn-induced aggregation (Figure S1F), underscoring the important role of His22. Overall, these results clearly indicate that Zninduced aggregation of HγD crystallin must be due to a specific interaction of this metal ion with His22. A previous study had identified His83 and 87 as potential Zn binding residues in HγD crystallin;12 while Zn ions might bind to those residues, such interaction is not involved in Zn-induced aggregation.
C22H HγC (H), and C26H HγS (I) crystallins, at the end point of the turbidity assays with 10 equiv of Zn(II).
Interestingly, His22 is not conserved in HγC and HγS crystallins, which contain a Cys residue at their homologous positions: 22 and 26, respectively (Scheme 1). Thus, we next evaluated if placing a His residue at such positions would make HγC and HγS sensitive to Zn-induced aggregation. Turbidity assays of C22H HγC and C26H HγS crystallins clearly show that these mutations “switch on” the Zn-induced aggregation of these proteins (Figures 1E and 1F, respectively), and in fact the effect is more drastic than for HγD. Electron microscopy analysis of the Zninduced aggregates of wt HγD, C22H HγC and C26H HγS shows that the aggregation is non-amyloid in nature (Figures 1G-I). It is important to note that C22H HγC and C26H HγS crystallins are not prone to aggregation in the absence of Zn ions (grey traces in Figures 1E and 1F), indicating that these mutations do not compromise the stability of these proteins; and thus, their increased sensitivity towards Zn ions is not due to a loss of protein stability. Overall, these results underscore the important role that this His plays in the Zn-induced aggregation of γ-crystallins.
Scheme 1. Sequence alignment of HγD (PDB:1HK0), HγC (PDB:2NBR) and HγS (PDB:2M3T) crystallin proteins at the first 30 residues; full sequence alignment is presented in Figure S2. Structural alignment of HγD (dark blue), HγC (green) and HγS (gray) displaying the positions of His22 and Cys18 in HγD and the corresponding residues in HγC and HγS crystallins (His: blue, Cys: yellow and Thr: red) (A). Zoom view of the site (B).
Figure 1. Turbidity assays of HγD (A), HγC (B), HγS (C), H22Q HγD (D), C22H HγC (E), and C26H HγS (F) crystallins (50 µM) in the absence (gray) or presence of 2, 4, 6, 8 and 10 equiv of Zn(II) (yellow to red traces). All assays were performed at 37 °C, and turbidity at 400 nm was followed after the addition of Zn. Electron microscopy images for Zn-induced aggregates of HγD (G),
In order to gain further insight into the mechanism of Zn-induced aggregation of HγD, the effect of Zn(II) ions in the folding of HγD wt, N-terminal domain of HγD, C22H HγC and C26H HγS crystallins was evaluated. The proteins were titrated with increasing amounts of this metal ion at 37 °C, and circular dichroism (CD) data were collected in the UV region. γ-Crystallins contain four Greek key motifs, and their CD spectra display an intense negative
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Biochemistry
transition at 218 nm, indicative of β-sheet structure. Upon addition of Zn(II) ions, no changes are observed in the CD spectra of these proteins (data for HγD wt are shown in Figure 2A, and for all proteins in Figures S3 A-D), even after 60 min after adding the metal ion (Figure S3E), or when incubating at 42 °C (Figure S3F). Consistently, Trp fluorescence of HγD is not impacted by the addition of Zn(II) ions (Figure 2B). These results indicate that Zn(II) ions do not cause unfolding of these proteins, and thus, the mechanism of Zn-induced aggregation does not involve the formation of partially folded intermediates. Strikingly, the lens chaperone human αB-crystallin (HαB) prevents Zn induced aggregation of HγD (Figure 2C), C26H HγS and C22H HγC (Figure S4). Thus, it is quite puzzling that HαB crystallin prevents Zn-induced aggregation, when CD spectroscopy shows no evidence for Zn-induced protein unfolding. However, HαB crystallin can bind at least two Zn(II) ions, involving His residues from a His-rich region at its C-terminal; while a dissociation constant in the sub-millimolar range has been reported.13 The crystal structure of a truncated Zn-bound form of bovine αB crystallin reveals that the His ligands are provided by three different molecules. 14, 15 Thus, the observed protective effect of HαB in Zn-induced aggregation of γ-crystallins is likely due to its ability to bind Zn(II) ions, and not to its capacity to recognize partially folded intermediates; although a detailed study of the relative binding affinities for Zn(II) of these crystallins would be needed to confirm this proposal.
Figure 2. Elucidating the mechanism of Zn-induced aggregation of HγD crystallin. Titrations of wt HγD crystallin (2 µM) with 0 (black spectra), 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 equiv of Zn(II) (yellow to brown traces), as followed by CD (A) and Trp fluorescence (B) at 37 °C. Turbidity assays of HγD crystallin after the addition of 2 or 10 equiv of Zn(II) in the presence of 4 equiv of HαB crystallin (purple traces) (C); for reference, turbidity assays in the absence of the chaperone (orange and red traces) are also shown. Turbidity assays of HγD crystallin in the presence of 20 (D) equiv of Zn(II); 100 equiv of EDTA were added at the time indicated by the arrow. All assays were performed at 50 µM protein concentration, 37 °C, and the turbidity at 400 nm is reported as a function of time after the addition of the metal ion. Electron microscopy images for the Zn-induced aggregates of HγD crystallin obtained at the endpoint (200 min) of a turbidity assay with 10 equiv of Zn(II), before (E) and after (F) the addition of 10 equiv of EDTA.
Since the Zn-induced aggregation of HγD crystallin does not involve partial unfolding of the protein, another possibility is that the mechanism might involve the formation of metal-bridged species. Thus, the effect of a chelating agent, ethylenediaminetetraacetic acid (EDTA), in the Zn-induced aggregation of γ-crystallins was studied. Turbidity assays clearly show that EDTA can revert completely the Zn-induced aggregation of HγD WT (Figure 2D), C26H HγS and C22H HγC crystallins (Figure S4). Even when the chelating agent is added at an advanced stage of the Zn-induced aggregation, the addition of EDTA completely removes solution turbidity. In fact, the Zn-induced aggregates can be almost fully dissociated upon addition of EDTA, as demonstrated by electron microscopy (Figures 2E&F HγD, Figure S5 for all other proteins). Generally, these results support the notion that the mechanism of Zn-induced aggregation of HγD crystallin must involve the formation of metal-bridged species, which can be reversed by a chelating agent. Overall, our results point to a metal-bridging mechanism for the Zn-induced aggregation of HγD crystallin, involving a specific interaction with His22 at the N-terminal domain (Scheme 2). The fact that a metal-His interaction drives non-amyloid aggregation of HγD crystallin begs the question if other metal ions with preference to coordinate imidazole groups might also induce protein aggregation via a metal-bridging mechanism. Thus, the effect of Ni(II) and Co(II) ions in the aggregation of HγD WT was evaluated, finding that none of them induces protein aggregation (Figure S6), while it is known that Cu(II) induces drastic aggregation.12 The metal ion specificity for the Zn-induced aggregation of HγD crystallin
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cannot be fully explained by the geometric preferences of each metal ion: while Zn(II) prefers to adopt a tetrahedral geometry and Co(II) is used as surrogate for Zn(II) in biological systems, Ni(II) and Cu(II) can adopt different geometries including square planar and octahedral arrangements, which might be more challenging to achieve in a metalbridging mechanism. Thus, the fact that Zn(II) and Cu(II) are the only two ions in this series to induce aggregation of HγD crystallin might be due to their higher affinity for His residues. We hypothesize that Zn(II) could be anchoring to His22 and the nearby Cys18, while recruiting two more ligands from another protein monomer to adopt a tetrahedral geometry. Indeed, Cys18 and His22 are contained in a CXXXH motif, which resembles a typical binding sequence in Zn finger proteins.16, 17 In such proteins, Zn(II) adopts a tetrahedral geometry by completing its coordination sphere with two ligands from a separate secondary structure element. In the case of HγD crystallin, there are no other regions of the protein within the same monomer that could provide ligands to complete a tetrahedral geometry for Zn. Thus, the metal ion likely recruits ligands from a separate monomer (Scheme 2A). This might also explain the fact that the chaperone HαB crystallin rescues HγD from Zn-induced aggregation, likely forming soluble complexes (Scheme 2B). However, if His22 residues from two monomers completed its coordination sphere, a non-aggregating head-to-head dimer would be formed. Alternatively, given that Zninduced aggregation increases with temperature,12 the possibilities that a transient local unfolding event could lead to aggregation of such head-to-head dimer and/or that a second Zn-binding site might be exposed by transient unfolding cannot be discarded. Further spectroscopic and mutational studies should help elucidate the nature of the coordination sphere of Zn(II) and the ligands involved in the metalbridging mechanism that induces non-amyloid aggregation of HγD crystallin. In summary, our study demonstrates that Zninduced aggregation of HγD crystallin is due to a specific interaction of this metal ion with His22. HγC and HγS crystallins are not sensitive to Zn-induced aggregation because His22 is not conserved in these γ-crystallins; however, Zn-induced aggregation is a phenomenon that can be “switched on” by placing a His at this position. The mechanism of Zn-induced aggregation involves the formation of metal-bridged species, which can be reversed by a chelating agent. This study demonstrates that chelating the metal ion can prevent aberrant metal-protein interactions in
vitro that induce non-amyloid aggregation of lens γcrystallins, a process involved in cataract disease.
Scheme 2. Proposed mechanism of Zn-induced aggregation of HγD crystallin. ASSOCIATED CONTENT Supporting Information. Materials and Methods. Turbidity assays and CD spectroscopic analysis of several variants of γ-crystallins. Evaluation of the effects of: chaperone, EDTA, Ni(II) or Co(II) in γ-crystallin ag-
gregation by turbidity assays and/or electron microscopy. The Supporting Information is available free of charge on the ACS Publications website.
AUTHOR INFORMATION Corresponding Authors
[email protected] ACKNOWLEDGMENTS This research has been supported by the National Council for Science and Technology in Mexico (CONACYT grants # 221134 and PN2076 to L.Q. and fellowships to J.A.D.C.), MIT-Seed Funds, NIH EY015834 grant to J.A.K., and Fulbright-García Robles fellowship and Cátedra Marcos Moshinsky to L.Q. Authors would like to thank Dr. Claudia Andreini and Dr. Acely Garza-García for useful discussions, and Lourdes Rojas at the Unit of Microscopy of Cinvestav.
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Nonamyloid Aggregation of the Highly Stable Human gamma-D Crystallin, ACS Chem. Biol. 11, 263-272. [13] Biswas, A., and Das, K. P. (2008) Zn2+ enhances the molecular chaperon function and stability of alpha-crystallin, Biochemistry 47, 804-816. [14] Laganowsky, A. B., L. P.; Landau, M.; Ding, L.; Sawaya, M. R.; Cascio, D.; Huang, Q.; Robinson C. V.; Horwitz, J.; Eisenberg, D. (2010) Crystal structures of truncated alphaA and alphaB crystallins reveal structural mechanisms of polydispersity important for eye lens function, Protein Sci. 19, 1031-1043. [15] Karmakar, S. D., K. P. . (2012) Identification of Histidine Residues Involved in Zn2+ Binding to aA- and aB-Crystallin by Chemical Modification and MALDI TOF Mass Spectrometry, Protein J. 31, 623-640. [16] Andreini, C. B., I.; Cavallaro, G. (2011) Minimal Functional Sites Allow a Classification of Zinc Sites in Proteins, PLoS ONE. 6, 26325-26337. [17] Valasatava, Y. R. A. C. G. A., C. (2014) MetalS3, a databasemining tool for the identification of structurally similar metal sites, JBIC 19, 937-945.
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