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 pHdependent 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 (sHSPs) help to maintain protein homeostasis by interacting with unfolded, aggregated or misfolded proteins to prevent cell damage [1][2][3] . The ATP-independent chaperone αB-crystallin (αB, 20 kDa, 175 residues) is a paradigm example 4 . αB was originally found in the eye-lens as the B-subunit of α-crystallin, a protein essential for maintaining eyelens transparency. In recent years, the list of biological roles for αB has grown substantially, including involvement in the regulation of the ubiquitin-proteasome pathway as well as AUTHOR CONTRIBUTIONSS.J. contributed to all aspects of the manuscript; P.R. performed solution NMR experiments and helped to write the manuscript; B.B. performed structure calculations; S.M. did solid-state NMR and SAXS measurements as well as data analysis; R.K. contributed to modeling of C-terminal intermolecular interactions; J.R.S. prepared samples; V.A.H. contributed to assignment strategies, was involved in structure calculations and helped write the manuscript; R.E.K. contributed to the interpretation of results and wrote the manuscript; B.J.v.R. contributed to solid-state NMR measurements, discussed the results and helped to write the manuscript; H.O. designed experimental strategies, contributed to the interpretation of results and wrote the manuscript. COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/. [6][7][8][9][10][11] . In the brain of patients with Alexander's disease, the insoluble cell fraction contains protein fibers (Rosenthal fibers) coprecipitated with αB phosphorylated at Ser59, whereas unphosphorylated αB remains in the soluble fraction 7 . A missense mutation, R120G, in αB is associated with desmin-related cardiomyopathy 8,9 . The mutations D140N and Q151X are associated with congenital cataracts and myopathy, respectively 10,11 . A decreased concentration of αB in the cerebrospinal fluid was found to be associated with ...

SummaryAtomic level structural information on αB-Crystallin (αB), a prominent member of the small Heat Shock Protein (sHSP) family has been a challenge to obtain due its polydisperse, oligomeric nature. We show that magic-angle spinning solid-state NMR can be used to obtain high-resolution information on ∼ 580 kDa human αB assembled from 175-residue, 20 kDa subunits. An ∼100-residue α-crystallin domain is common to all sHSPs and solution-state NMR was performed on two different α-crystallin domain constructs isolated from αB. In vitro, the chaperone-like activities of full-length αB and the isolated α-crystallin domain are identical. Chemical shifts of the backbone and the C β resonances have been obtained for residues 64-162 (α-crystallin domain plus part of the C-terminus) in αB and the isolated α-crystallin domain by solid-and solution-state NMR, respectively. Both sets of data strongly predict six β-strands in the α-crystallin domain. A majority of residues in the α-crystallin domain have similar chemical shifts in both solid-and solution-state indicating a similar structure for the domain in its isolated and oligomeric forms. Sites of inter-subunit interaction are identified from chemical shift differences that cluster to specific regions of the α-crystallin domain. Multiple signals are observed for the resonances of M68 in the oligomer, identifying the region containing this residue as existing in heterogeneous environments within αB. Evidence for a novel dimerization motif in the human α-crystallin domain is obtained by a comparison of (i) solid-and solution-state chemical shift data and (ii) 1 H-15 N HSQC spectra as a function of pH. The isolated α-crystallin domain undergoes a dimer-monomer transition over the pH range of 7.5 to 6.8. This steep pH-dependent switch may be important for αB to function optimally, e.g., to preserve the filament integrity of cardiac muscle proteins such as actin and desmin during cardiac ischemia which is accompanied by acidosis.

Phosphodiesterase 5 (PDE5) controls intracellular levels of cGMP through its regulation of cGMP hydrolysis. Hydrolytic activity of the C-terminal catalytic domain is increased by cGMP binding to the N-terminal GAF A domain. We present the NMR solution structure of the cGMP-bound PDE5A GAF A domain. The cGMP orientation in the buried binding pocket was defined through 37 intermolecular nuclear Overhauser effects. Comparison with GAF domains from PDE2A and adenylyl cyclase cyaB2 reveals a conserved overall domain fold of a six-stranded ␤-sheet and four ␣-helices that form a well defined cGMP binding pocket. However, the nucleotide coordination is distinct with a series of altered binding contacts. The structure suggests that nucleotide binding specificity is provided by Asp-196, which is positioned to form two hydrogen bonds to the guanine ring of cGMP. An alanine mutation of Asp-196 disrupts cGMP binding and increases cAMP affinity in constructs containing only GAF A causing an altered cAMP-bound structural conformation. NMR studies on the tandem GAF domains reveal a flexible GAF A domain in the absence of cGMP, and indicate a large conformational change upon ligand binding. Furthermore, we identify a region of ϳ20 residues directly N-terminal of GAF A as critical for tight dimerization of the tandem GAF domains. The features of the PDE5 regulatory domain revealed here provide an initial structural basis for future investigations of the regulatory mechanism of PDE5 and the design of GAF-specific regulators of PDE5 function.Intracellular concentrations of the second messengers cAMP and cGMP are tightly regulated by the rate of synthesis through cyclases and hydrolysis through cyclic nucleotide phosphodiesterases (PDEs) 3 (1). The cGMP-specific, cGMP-binding phosphodiesterase, PDE5, is one of eleven identified PDE families. It has been characterized as the major cGMP-hydrolyzing PDE in numerous tissues such as lung, platelets, pulmonary artery smooth muscle cells, and the penile corpus cavernosum (2, 3). The abundance of PDE5 in smooth muscles and its role in regulating their contractile tone has made PDE5 an important drug target for the treatment of erectile dysfunction and pulmonary hypertension (2), leading to the development of potent PDE5 inhibitors, such as tadalafil (Cialis TM

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