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The small heat shock protein (sHSP) αB-crystallin (αB) plays a key role in the cellular protection system against stress. For decades, high-resolution structural studies on heterogeneous sHSPs have been confounded by the polydisperse nature of αB oligomers. We present an atomic-level model of full-length αB as a symmetric 24-subunit multimer based on solid-state NMR, small-angle X-ray scattering (SAXS), and EM data. The model builds on our recently reported structure of the homodimeric a-crystallin domain (ACD) and C-terminal IXI motif in the context of the multimer. A hierarchy of interactions contributes to build multimers of varying sizes: Interactions between two ACDs define a dimer, three dimers connected by their C-terminal regions define a hexameric unit, and variable interactions involving the N-terminal region define higher-order multimers. Within a multimer, N-terminal regions exist in multiple environments, contributing to the heterogeneity observed by NMR. Analysis of SAXS data allows determination of a heterogeneity parameter for this type of system. A mechanism of multimerization into higher-order asymmetric oligomers via the addition of up to six dimeric units to a 24-mer is proposed. The proposed asymmetric multimers explain the homogeneous appearance of αB in negative-stain EM images and the known dynamic exchange of αB subunits. The model of αB provides a structural basis for understanding known disease-associated missense mutations and makes predictions concerning substrate binding and the reported fibrilogenesis of αB.

Small heat shock proteins (sHSPs) are essential ‘holdase’ chaperones that form large assemblies and respond dynamically to pH and temperature stresses to protect client proteins from aggregation. While the alpha-crystallin domain (ACD) dimer of sHSPs is the universal building block, how the ACD transmits structural changes in response to stress to promote holdase activity is unknown. We found that the dimer interface of HSPB5 is destabilized over physiological pHs and a conserved histidine (His-104) controls interface stability and oligomer structure in response to acidosis. Destabilization by pH or His-104 mutation shifts the ACD from dimer to monomer but also results in a large expansion of HSPB5 oligomer states. Remarkably, His-104 mutant-destabilized oligomers are efficient holdases that reorganize into structurally distinct client–bound complexes. Our data support a model for sHSP function wherein cell stress triggers small perturbations that alter the ACD building blocks to unleash a cryptic mode of chaperone action. Proteins are composed of one or more long chain-like molecules that must fold into complex three-dimensional shapes in order to work properly. Incorrectly folded proteins cannot function and often aggregate into toxic states that are associated with a number of neurological diseases including Alzheimer's, Huntington's, and Parkinson's. Elevated temperatures, increased acidity, and other stressful conditions in the cell can hinder the folding process and may cause existing proteins to unfold and aggregate. However, when cells experience these stresses, certain proteins—known as small heat shock proteins (or sHSPs for short)—act as ‘holdase chaperones’ to protect cells from protein misfolding. HSPB5 is one such chaperone that binds to and stabilizes other proteins (called ‘clients’) to prevent their aggregation. The core structure of HSPB5 and other similar chaperone proteins is well known. But, it is not clear how chaperones sense stressful conditions and respond to increase their activity to help stabilize client proteins. Now, Rajagopal et al. have identified a single amino acid in HSPB5 that is sensitive to pH changes. When the environment inside a cell becomes more acidic, this amino acid (a histidine) triggers changes in HSPB5's structure that enhance the chaperone's activity. This histidine was then replaced with another amino acid in an attempt to lock HSPB5 into a low-pH state that mimics an active HSPB5 chaperone inside a stressed cell. Further experiments revealed that this mutant HSPB5 is a super-active holdase chaperone, and that it dramatically changes its structure to bind to a client protein in the holdase state. From this, Rajagopal et al. propose a model to explain how cellular stress triggers small changes in HSPB5 that propagate through the chaperone in a response mechanism that increases its activity. Future studies will investigate whether inherited mutations in HSPB5 and other similar chaperones—which are associated with cardiac, muscle, and nerve disorders—exert their effect by disrupting this response mechanism.

The RING domain of the breast and ovarian cancer tumor suppressor BRCA1 interacts with multiple cognate proteins, including the RING protein BARD1. Proper function of the BRCA1 RING domain is critical, as evidenced by the many cancer-predisposing mutations found within this domain. We present the solution structure of the heterodimer formed between the RING domains of BRCA1 and BARD1. Comparison with the RING homodimer of the V(D)J recombination-activating protein RAG1 reveals the structural diversity of complexes formed by interactions between different RING domains. The BRCA1–BARD1 structure provides a model for its ubiquitin ligase activity, illustrates how the BRCA1 RING domain can be involved in associations with multiple protein partners and provides a framework for understanding cancer-causing mutations at the molecular level.

Cataracts reduce vision in 50% of individuals over 70 years of age and are a common form of blindness worldwide. Cataracts are caused when damage to the major lens crystallin proteins causes their misfolding and aggregation into insoluble amyloids. Using a thermal stability assay, we identified a class of molecules that bind α-crystallins (cryAA and cryAB) and reversed their aggregation in vitro. The most promising compound improved lens transparency in the R49C cryAA and R120G cryAB mouse models of hereditary cataract. It also partially restored protein solubility in the lenses of aged mice in vivo and in human lenses ex vivo. These findings suggest an approach to treating cataracts by stabilizing α-crystallins.

The structure of the small heat shock protein αB-crystallin, associated with multiple sclerosis and Alzheimer's disease, has eluded biologists for years. Small angle X-ray scattering and solid-state NMR reveal a curved dimer that modulates substrate interactions upon a change in pH. The small heat shock protein αB-crystallin (αB) contributes to cellular protection against stress. For decades, high-resolution structural studies on oligomeric αB have been confounded by its polydisperse nature. Here, we present a structural basis of oligomer assembly and activation of the chaperone using solid-state NMR and small-angle X-ray scattering (SAXS). The basic building block is a curved dimer, with an angle of ∼121° between the planes of the β-sandwich formed by α-crystallin domains. The highly conserved IXI motif covers a substrate binding site at pH 7.5. We observe a pH-dependent modulation of the interaction of the IXI motif with β4 and β8, consistent with a pH-dependent regulation of the chaperone function. N-terminal region residues Ser59-Trp60-Phe61 are involved in intermolecular interaction with β3. Intermolecular restraints from NMR and volumetric restraints from SAXS were combined to calculate a model of a 24-subunit αB oligomer with tetrahedral symmetry.

Small heat shock proteins (sHSP) are a class of ATP-independent protein chaperones found throughout nature. They share a common ability to maintain partly unfolded proteins in soluble states under cellular stress conditions. All sHSPs contain a central domain called the α-crystallin domain (ACD); the domain is found in all sHSPs and in no other proteins and therefore defines the family. Though most sHSPs form large, often polydisperse oligomers from varying numbers of subunits, the ACD is both necessary and sufficient for formation of a dimer, the fundamental building block for oligomers. HSPB1 (also known as Hsp27) is unique among the ten human sHSPs because it contains a Cys residue in its dimer interface. HSPB1 is highly expressed under conditions of oxidative stress and is proposed to serve as a redox-sensitive chaperone. HSPB1 residue Cys137 has been proposed to modulate function by existing in either its oxidized (disulfide) or reduced (thiol) form (Chalova et al 2014). Here we report the solution-state NMR structure of oxidized HSPB1-ACD and compare it to a previously determined crystal structure of the reduced state. Formation of the disulfide-bond across the dimer interface yields a locked dimer structure with increased accessible hydrophobic surface. In the context of full-length HSPB1 oligomers, oxidation of Cys137 is associated with enhanced ability to bind the hydrophobic dye, 8-Anilinonapthalene-1-sulfonic-acid, implying an increased ability to interact with client proteins under oxidative stress.

INTEREST in triple and quadruple DNA helices built from homopurine and homopyrimidine strands has recently intensified principally because such structures may occur in vivo 1 but also because of the potential use of triplexes both in forming highly sequence-specific complexes for use in chromosome mapping 2,3 and in repressing transcription 4 . From fibre diffraction data, models for triplex structures with poly(U)·poly(A)·poly(U) and poly(dT)·poly(dA)·poly(dT) have been proposed 5 , in which the purine and one pyrimidine strand are Watson–Crick paired in an A' helix, and the other pyrimidine strand is Hoogsteen base-paired parallel to the purine strand along the major groove. A similar base-pairing scheme involving G and C would require protonation of C for Hoogsteen base-pair formation, and models for such triplexes have been proposed 1,6 by analogy to the single-sequence fibre diffraction data. To date, however, there have been no single crystal or NMR structural data on DNA triplexes, and no direct observation of the protonated C in such a context. We present here the first NMR evidence for triplex formation in DNA from the homopurine d(G-A) 4 and homopyrimidine d(T-C) 4 oligonucleotides, and report direct observation of imino protons from protonated cytosines in the triplex.

Cataracts are the most common cause of vision loss, especially in our ever-increasing elderly population. Cataracts arise when crystallin, a major protein component of the eye lens, begins to aggregate, which causes the lens to become cloudy. Makley et al. explored whether small molecules that reverse this aggregation might have therapeutic potential for treating cataracts, which normally require surgery (see the Perspective by Quinlan). They used a screening method that monitors the effect of ligands on temperature-dependent protein unfolding and identified several compounds that bind and stabilize the soluble form of crystallin. In proof-of-concept studies, one of these compounds improved lens transparency in mice. Science, this issue p. 674; see also p. 636 Cataracts reduce vision in 50% of individuals over 70 years of age and are a common form of blindness worldwide. Cataracts are caused when damage to the major lens crystallin proteins causes their misfolding and aggregation into insoluble amyloids. Using a thermal stability assay, we identified a class of molecules that bind α-crystallins (cryAA and cryAB) and reversed their aggregation in vitro. The most promising compound improved lens transparency in the R49C cryAA and R120G cryAB mouse models of hereditary cataract. It also partially restored protein solubility in the lenses of aged mice in vivo and in human lenses ex vivo. These findings suggest an approach to treating cataracts by stabilizing α-crystallins.

The small heat shock protein (sHSP) αB-crystallin (αB) plays a key role in the cellular protection system against stress. For decades, high-resolution structural studies on heterogeneous sHSPs have been confounded by the polydisperse nature of αB oligomers. We present an atomic-level model of full-length αB as a symmetric 24-subunit multimer based on solid-state NMR, small-angle X-ray scattering (SAXS), and EM data. The model builds on our recently reported structure of the homodimeric α-crystallin domain (ACD) and C-terminal IXI motif in the context of the multimer. A hierarchy of interactions contributes to build multimers of varying sizes: Interactions between two ACDs define a dimer, three dimers connected by their C-terminal regions define a hexameric unit, and variable interactions involving the N-terminal region define higher-order multimers. Within a multimer, N-terminal regions exist in multiple environments, contributing to the heterogeneity observed by NMR. Analysis of SAXS data allows determination of a heterogeneity parameter for this type of system. A mechanism of multimerization into higher-order asymmetric oligomers via the addition of up to six dimeric units to a 24-mer is proposed. The proposed asymmetric multimers explain the homogeneous appearance of αB in negative-stain EM images and the known dynamic exchange of αB subunits. The model of αB provides a structural basis for understanding known disease-associated missense mutations and makes predictions concerning substrate binding and the reported fibrilogenesis of αB. [PUBLICATION ABSTRACT]