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 ...

Protein-protein interactions are essential in every aspect of cellular activity. Multiprotein complexes form and dissociate constantly in a specifically tuned manner, often by conserved mechanisms. Protein domains that bind proline-rich motifs (PRMs) are frequently involved in signaling events. The unique properties of proline provide a mechanism for highly discriminatory recognition without requiring high affinities. We present herein a detailed, quantitative assessment of the structural features that define the interfaces between PRM-binding domains and their target PRMs, and investigate the specificity of PRM recognition. Together with the analysis of peptide-library screens, this approach has allowed the identification of several highly conserved key interactions found in all complexes of PRM-binding domains. The inhibition of protein-protein interactions by using small-molecule agents is very challenging. Therefore, it is important to first pinpoint the critical interactions that must be considered in the design of inhibitors of PRM-binding domains.

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The Ena-VASP family of proteins act as molecular adaptors linking the cytoskeletal system to signal transduction pathways. Their N-terminal EVH1 domains use groups of exposed aromatic residues to speci®cally recognize`FPPPP' motifs found in the mammalian zyxin and vinculin proteins, and ActA protein of the intracellular bacterium Listeria monocytogenes. Here, evidence is provided that the af®nities of these EVH1±peptide interactions are strongly dependent on the recognition of residues¯anking the core FPPPP motifs. Determination of the VASP EVH1 domain solution structure, together with peptide library screening, measurement of individual K d s bȳ uorescence titration, and NMR chemical shift mapping, revealed a second af®nity-determining epitope present in all four ActA EVH1-binding motifs. The epitope was shown to interact with a complementary hydrophobic site on the EVH1 surface and to increase strongly the af®nity of ActA for EVH1 domains. We propose that this epitope, which is absent in the sequences of the native EVH1-interaction partners zyxin and vinculin, may provide the pathogen with an advantage when competing for the recruitment of the host VASP and Mena proteins in the infected cell.

Since the initial discovery of the role of histamine in allergic conditions (1) serious efforts have been made to develop drugs that inhibit the actions of histamine. Already in 1933, Fourneau and Bovet (2) reported the first "antihistamine" piperoxan. Following this finding many potent H 1 antagonists that can be considered as variations of diaryl-substituted ethylamines (e.g. diphenhydramine and mepyramine) have been developed (for review see Ref.3). These "first generation" H 1 antagonists are quite effective in humans in allergic rhinitis and urticaria, but because of central nervous system penetration and central H 1 receptor blockade their clinical use is hampered by sedative side effects (3-5). A "second generation" of nonsedative H 1 antagonists (e.g. astemizole, acrivastine, cetirizine, loratidine, and terfenadine) has recently been developed (for review see Ref.3). Their altered pharmacokinetics result in good clinical effectiveness combined with a strongly reduced sedative potential (3-5).The development of H 1 antagonists has so far been directed by traditional medicinal chemistry (3). With the availability of the genetic information of the histamine H 1 receptor (6), the rationalization of drug-protein interaction has become a major challenge for this therapeutically important class of drugs. Like all aminergic G-protein coupled receptors (GPCR), 1 the H 1 receptor contains an aspartate residue (Asp 116 ) in transmembrane domain (TM) III (6), that is involved in the binding of the protonated amine function found in both agonists and antagonists structures (7,8). Mutagenesis studies have furthermore shown that the imidazole ring of histamine is accommodated by Lys 200 and Asn 207 in TM V (9, 10). In view of the low sequence similarity between GPCRs and bacteriorhodopsin (BR) much controversy exists on the validity of models derived for GPCRs based on the homology with BR (11-13). Nevertheless, despite the speculative nature of BRderived GPCR models they have been quite helpful in understanding and predicting drug-receptor interactions for a variety of receptors (see e.g. Refs. 14 -16). Previously, we also developed a three-dimensional computer model of the histamine H 1 receptor based on the homology with BR, incorporating the results obtained from mutagenesis studies on the agonist binding site (17). In the present study this computer model of the H 1 receptor was combined with a pharmacophoric model of the H 1 antagonistic binding site (18). This ligand-based model for the H 1 antagonistic binding site is based upon an interaction of the protonated amine function of various first generation, semi-rigid H 1 antagonists with an aspartate residue (Asp 116 in the guinea pig H 1 receptor) (18) and precisely positions the cis-and trans-aromatic rings of the H 1 antagonists relative to the C ␣ and C ␤ carbon atoms of this aspartate residue. Combining the three-dimensional receptor model and the ligand-based pharmacophoric model of the H 1 antagonist binding site resulted in the prediction of interactions...

Intracellular protein interaction domains are essential for eukaryotic signaling. In T cells, the CD2BP2 adaptor binds two membrane‐proximal proline‐rich motifs in the CD2 cytoplasmic tail via its GYF domain, thereby regulating interleukin‐2 production. Here we present the structure of the GYF domain in complex with a CD2 tail peptide. Unlike SH3 domains, which use two surface pockets to accommodate proline residues of ligands, the GYF domain employs phylogenetically conserved hydrophobic residues to create a single interaction surface. NMR analysis shows that the Fyn but not the Lck tyrosine kinase SH3 domain competes with CD2BP2 GYF‐domain binding to the same CD2 proline‐rich sequence in vitro. To test the in vivo significance of this competition, we used co‐immunoprecipitation experiments and found that CD2BP2 is the ligand of the membrane‐proximal proline‐rich tandem repeat of CD2 in detergent‐ soluble membrane compartments, but is replaced by Fyn SH3 after CD2 is translocated into lipid rafts upon CD2 ectodomain clustering. This unveils the mechanism of a switch of CD2 function due to an extracellular mitogenic signal.

Major histocompatibility complex (MHC) molecules are a key element of the cellular immune response. Encoded by the MHC they are a family of highly polymorphic peptide receptors presenting peptide antigens for the surveillance by T cells. We have shown that certain organic compounds can amplify immune responses by catalyzing the peptide loading of human class II MHC molecules HLA-DR. Here we show now that they achieve this by interacting with a defined binding site of the HLA-DR peptide receptor. Screening of a compound library revealed a set of adamantane derivatives that strongly accelerated the peptide loading rate. The effect was evident only for an allelic subset and strictly correlated with the presence of glycine at the dimorphic position ␤86 of the HLA-DR molecule. The residue forms the floor of the conserved pocket P1, located in the peptide binding site of MHC molecule. Apparently, transient occupation of this pocket by the organic compound stabilizes the peptide-receptive conformation permitting rapid antigen loading. This interaction appeared restricted to the larger Gly ␤86 pocket and allowed striking enhancements of T cell responses for antigens presented by these "adamantyl-susceptible" MHC molecules. As catalysts of antigen loading, compounds targeting P1 may be useful molecular tools to amplify the immune response. The observation, however, that the ligand repertoire can be affected through polymorphic sites form the outside may also imply that environmental factors could induce allergic or autoimmune reactions in an allele-selective manner.

Double-stranded RNA deaminase I (ADAR1) contains the Z-DNA binding domain Z␣. Here we report the solution structure of free Z␣ and map the interaction surface with Z-DNA, confirming roles previously assigned to residues by mutagenesis. Comparison with the crystal structure of the (Z␣) 2͞Z-DNA complex shows that most Z-DNA contacting residues in free Z␣ are prepositioned to bind Z-DNA, thus minimizing the entropic cost of binding. Comparison with homologous (␣؉␤)helix-turn-helix͞B-DNA complexes suggests that binding of Z␣ to B-DNA is disfavored by steric hindrance, but does not eliminate the possibility that related domains may bind to both B-and Z-DNA. RNA editing in mammals alters codons in mRNA through site-specific deamination of adenosines and cytosines, leading to proteins with modified function. Adenosine to inosine (A 3 I) editing modulates the calcium permeability of neural glutamate receptors (1) and reduces the G-protein coupling efficacy of serotonin 2C receptors (2). Double-stranded RNA deaminases I and II (ADAR1͞2) catalyze these A 3 I conversions, but unknown auxiliary factors are thought to be involved in the control of editing efficiency in vivo (3). ADAR1, but not ADAR2, has two left-handed Z-DNA binding domains, Z␣ and Z␤, at its N terminus. These domains may contribute to the control of ADAR1-mediated editing in vivo (4). Z-DNA formation in vivo has been shown to be transcription dependent in prokaryotes and eukaryotes (5). Z-DNA can be generated transiently 5Ј to a moving RNA polymerase in alternating purine͞pyrimidine sequences (5), thereby providing a transient binding site for Z␣ and Z␤. Thus, Z-DNA binding may ensure that the catalytic activity of ADAR1 is targeted to sites where nascent pre-mRNA substrates emerge (5).Here we have determined the solution structure of free Z␣ and mapped the interaction surface between Z␣ and a 6-bp d(CG) substrate DNA by two-dimensional (2D) 15 N-heteronuclear single quantum correlation (HSQC) NMR spectroscopy. Z␣ binds this substrate with high affinity (K d ϭ 30 nM) and a stoichiometry of 2:1 (protein͞DNA) (6-10). The map of the interaction surface in solution agrees well with the crystal structure of Z␣ complexed with Z-DNA (7). Further the structure of Z␣ free in solution demonstrates that there are only minor conformational changes upon binding Z-DNA. Not only is the overall structure the same, but unexpectedly, most Z-DNA contacting residues are prepositioned in free Z␣ to fit Z-DNA. This study also examines why Z␣ preferentially binds Z-DNA rather than B-DNA despite its high structural homology to (␣ϩ␤) helix-turn-helix (␣ϩ␤HTH) B-DNA binding proteins. Materials and MethodsProtein Preparation. The Z␣ domain, comprising residues 119-200 of human ADAR1 (GenBank accession no. U10439), has been described (8). Z␣ was expressed as a fusion protein with a N-terminal (His) 6 -tag from a pET-21a vector (Novagen) in Escherichia coli strain HM174(DE3). For isotope labeling, bacteria were grown in M9 medium containing 1 g͞liter 15 NH 4 Cl and 1.5 g͞liter 13 C-gl...