N- and C-Terminal Motifs in Human αB Crystallin Play an Important Role in the Recognition, Selection, and Solubilization of Substrates

Ghosh, Jayita; Shenoy, Ananth K.; Clark, John I.

doi:10.1021/bi061471m

Cited by 51 publications

(57 citation statements)

References 40 publications

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“…3 and 4). Although an essential function for the sHSP N-terminal arm in substrate protection has been proposed (2,(20)(21)(22)(23)28), our data provide direct evidence that the N-terminal arm binds substrate. We also show that regions of the ␣-crystallin domain and C-terminal extension form substrate specific crosslinks, but at a lower intensity compared with those of the N-terminal arm (Fig.…”

Section: Discussionmentioning

confidence: 42%

“…Sequence variability and structural disorder, along with experimental evidence make the N-terminal arm a good candidate for substrate binding. Chaperone activity is altered in N-terminal chimeras, and in N-terminal point and deletion mutants, implicating the Nterminal arm in substrate protection (20)(21)(22)(23). However, these data do not distinguish between disruption of substrate interaction sites on the N-terminal arm, versus perturbation of some other sHSP property, such as oligomer integrity, which then indirectly impacts chaperone activity.…”

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confidence: 97%

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Substrate binding site flexibility of the small heat shock protein molecular chaperones

Jaya

García

Vierling

2009

Proc. Natl. Acad. Sci. U.S.A.

220

190

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Small heat shock proteins (sHSPs) serve as a first line of defense against stress-induced cell damage by binding and maintaining denaturing proteins in a folding-competent state. In contrast to the well-defined substrate binding regions of ATP-dependent chaperones, interactions between sHSPs and substrates are poorly understood. Defining substrate-binding sites of sHSPs is key to understanding their cellular functions and to harnessing their aggregation-prevention properties for controlling damage due to stress and disease. We incorporated a photoactivatable cross-linker at 32 positions throughout a well-characterized sHSP, dodecameric PsHsp18.1 from pea, and identified direct interaction sites between sHSPs and substrates. Model substrates firefly luciferase and malate dehydrogenase form strong contacts with multiple residues in the sHSP N-terminal arm, demonstrating the importance of this flexible and evolutionary variable region in substrate binding. Within the conserved ␣-crystallin domain both substrates also bind the ␤-strand (␤7) where mutations in human homologs result in inherited disease. Notably, these binding sites are poorly accessible in the sHSP atomic structure, consistent with major structural rearrangements being required for substrate binding. Detectable differences in the pattern of cross-linking intensity of the two substrates and the fact that substrates make contacts throughout the sHSP indicate that there is not a discrete substrate binding surface. Our results support a model in which the intrinsicallydisordered N-terminal arm can present diverse geometries of interaction sites, which is likely critical for the ability of sHSPs to protect efficiently many different substrates.alpha-crystallin ͉ cross-linking ͉ intrinsic disorder ͉ P-benzoylphenylalanine ͉ protein-protein interactions

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Section: Discussionmentioning

confidence: 42%

mentioning

confidence: 97%

Substrate binding site flexibility of the small heat shock protein molecular chaperones

Jaya

García

Vierling

2009

Proc. Natl. Acad. Sci. U.S.A.

220

190

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“…4A). One further hydrophobic pocket is formed by I10 and several residues within the N-terminal segment 44-55, which were suggested to be involved in binding of completely unfolded proteins (32). A third pocket is formed by the N-terminal residues 3-5 together with hydrophobic residues of the C-terminal segment encompassing residues 152-171 (Fig.…”

Section: Resultsmentioning

confidence: 99%

Multiple molecular architectures of the eye lens chaperone αB-crystallin elucidated by a triple hybrid approach

Braun

Zacharias²,

Peschek³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

150

177

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The molecular chaperone αB-crystallin, the major player in maintaining the transparency of the eye lens, prevents stress-damaged and aging lens proteins from aggregation. In nonlenticular cells, it is involved in various neurological diseases, diabetes, and cancer. Given its structural plasticity and dynamics, structure analysis of αB-crystallin presented hitherto a formidable challenge. Here we present a pseudoatomic model of a 24-meric αB-crystallin assembly obtained by a triple hybrid approach combining data from cryoelectron microscopy, NMR spectroscopy, and structural modeling. The model, confirmed by cross-linking and mass spectrometry, shows that the subunits interact within the oligomer in different, defined conformations. We further present the molecular architectures of additional well-defined αB-crystallin assemblies with larger or smaller numbers of subunits, provide the mechanism how "heterogeneity" is achieved by a small set of defined structural variations, and analyze the factors modulating the oligomer equilibrium of αB-crystallin and thus its chaperone activity.T he most prominent member of the small heat-shock protein (sHsp) family, α-crystallin, is expressed at high concentrations in the vertebrate eye lens (1) where it plays a major role in maintaining lens transparency (2). Moreover, it protects lens epithelial cells from environmental stress by preventing aggregation of stress-damaged proteins (3). In the low protein turnover milieu of the eye lens, proteins gradually deteriorate throughout the lifespan due to posttranslational modifications and become increasingly prone to aggregation leading to opacity. Thus, the chaperone action of α-crystallin is vital for maintaining the eye lens transparency. Lenticular α-crystallin is composed of two homologous polypeptides, αA-and αB-crystallin, which comprise 173 and 175 amino acid residues, respectively (1, 4). Both proteins possess a three-domain organization consisting of the α-crystallin domain (ACD), a consensus sequence of approximately 90 amino acids common to all sHsps, flanked by a diverse N-terminal region and a moderately conserved C-terminal extension (5, 6).Of the two constituents of α-crystallin, αB-crystallin is the more widespread chaperone with versatile functions: Besides the eye lens, it is abundantly expressed in other tissues (7) and upregulated by various stresses (8). There is growing evidence for its implications in several neuropathological diseases (9) including Parkinson disease, Alzheimer's disease, and multiple sclerosis (10, 11) as well as in cancer (12). In vitro, αB-crystallin prevents the stress-induced aggregation of partially folded polypeptides (3, 13).The αB-crystallin assembles into homooligomers with a variable number of subunits, primarily 24-32 (14-16), and the subunits exchange between homooligomers (17). These properties have hampered high-resolution structural studies on the functionally assembled, full-length protein. An earlier cryoelectron microscopy (cryo-EM) study (18) presented at low resolution (3...

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“…6,37,38 Deletion of the C-terminal extension in ABC leads to a reduction in chaperone activity, an increase in polydispersity, and an increase in oligomeric size. 39 The C-terminal extension functions in recognition and selection of unfolded protein substrates. 39 …”

Section: Resultsmentioning

confidence: 99%

“…39 The C-terminal extension functions in recognition and selection of unfolded protein substrates. 39 …”

Section: Resultsmentioning

confidence: 99%

Crystal structures of truncated alphaA and alphaB crystallins reveal structural mechanisms of polydispersity important for eye lens function

et al. 2010

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Small heat shock proteins alphaA and alphaB crystallin form highly polydisperse oligomers that frustrate protein aggregation, crystallization, and amyloid formation. Here, we present the crystal structures of truncated forms of bovine alphaA crystallin (AAC ) and human alphaB crystallin (ABC 68-162 ), both containing the C-terminal extension that functions in chaperone action and oligomeric assembly. In both structures, the C-terminal extensions swap into neighboring molecules, creating runaway domain swaps. This interface, termed DS, enables crystallin polydispersity because the C-terminal extension is palindromic and thereby allows the formation of equivalent residue interactions in both directions. That is, we observe that the extension binds in opposite directions at the DS interfaces of AAC 59-163 and ABC . A second dimeric interface, termed AP, also enables polydispersity by forming an antiparallel beta sheet with three distinct registration shifts. These two polymorphic interfaces enforce polydispersity of alpha crystallin. This evolved polydispersity suggests molecular mechanisms for chaperone action and for prevention of crystallization, both necessary for transparency of eye lenses.Keywords: X-ray diffraction; small heat shock protein; protein chaperone; desmin-related myopathy; cataract; eye lens transparency Abbreviations: AAC 59-163 , alphaA crystallin residues 59-163; AAC 59-163 -Zn, zinc-bound alphaA crystallin residues 59-163; ABC 68-162 , alphaB crystallin residues 68-162; ABC 68-157 , alphaB crystallin residues 68-157; ABC 67-157 , alphaB crystallin residues 67-157; ADH, alcohol dehydrogenase; AP, antiparallel beta sheet interface; AP x , antiparallel beta sheet registration state x; DS, domain-swapped interface; Hsp20 65-162 , rat heat shock protein 20 residues 65-162; MjHsp16.5, Methanococcus janaschii heat shock protein 16.5; MS, mass spectrometry; sHSP, small heat shock protein; WhHsp16.9, wheat heat shock protein 16.9.Additional Supporting Information may be found in the online version of this article.

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N- and C-Terminal Motifs in Human αB Crystallin Play an Important Role in the Recognition, Selection, and Solubilization of Substrates

Cited by 51 publications

References 40 publications

Substrate binding site flexibility of the small heat shock protein molecular chaperones

Substrate binding site flexibility of the small heat shock protein molecular chaperones

Multiple molecular architectures of the eye lens chaperone αB-crystallin elucidated by a triple hybrid approach

Crystal structures of truncated alphaA and alphaB crystallins reveal structural mechanisms of polydispersity important for eye lens function

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