Janice Bramham

Structural diversity in p160/CREB-binding protein coactivator complexes.

Ligand-induced transcription by nuclear receptors involves the recruitment of p160 coactivators such as steroid receptor coactivator 1 (SRC1), in complex with histone acetyltransferases such as CREB-binding protein (CBP) and p300. Here we describe the solution structure of a complex formed by the SRC1 interaction domain (SID) of CBP and the activation domain (AD1) of SRC1, both of which contain four helical regions (Cα1, Cα2, Cα3, and Cα3′ in CBP and Sα1, Sα2′, Sα2, and Sα3 in SRC1). A tight four-helix bundle is formed between Sα1, Cα1, Cα2, and Cα3 that is capped by Sα3. In contrast to the structure of the AD1 domain of the related p160 protein ACTR in complex with CBP SID, the sequences forming Sα2′ and Sα2 in SRC1 AD1 are not involved in the interface between the two domains but rather serve to position Sα3. Thus, although the CBP SID domain adopts a similar fold in complex with different p160 proteins, the topologies of the AD1 domains are strikingly different, a feature that is likely to contribute to functional specificity of these coactivator complexes.

A t(1;11) translocation linked to schizophrenia and affective disorders gives rise to aberrant chimeric DISC1 transcripts that encode structurally altered, deleterious mitochondrial proteins

Disrupted-In-Schizophrenia 1 (DISC1) was identified as a risk factor for psychiatric illness through its disruption by a balanced chromosomal translocation, t(1;11)(q42.1;q14.3), that co-segregates with schizophrenia, bipolar disorder and depression. We previously reported that the translocation reduces DISC1 expression, consistent with a haploinsufficiency disease model. Here we report that, in lymphoblastoid cell lines, the translocation additionally results in the production of abnormal transcripts due to the fusion of DISC1 with a disrupted gene on chromosome 11 (DISC1FP1/Boymaw). These chimeric transcripts encode abnormal proteins, designated CP1, CP60 and CP69, consisting of DISC1 amino acids 1–597 plus 1, 60 or 69 amino acids, respectively. The novel 69 amino acids in CP69 induce increased α-helical content and formation of large stable protein assemblies. The same is predicted for CP60. Both CP60 and CP69 exhibit profoundly altered functional properties within cell lines and neurons. Both are predominantly targeted to mitochondria, where they induce clustering and loss of membrane potential, indicative of severe mitochondrial dysfunction. There is currently no access to neural material from translocation carriers to confirm these findings, but there is no reason to suppose that these chimeric transcripts will not also be expressed in the brain. There is thus potential for the production of abnormal chimeric proteins in the brains of translocation carriers, although at substantially lower levels than for native DISC1. The mechanism by which inheritance of the translocation increases risk of psychiatric illness may therefore involve both DISC1 haploinsufficiency and mitochondrial deficiency due to the effects of abnormal chimeric protein expression. GenBank accession numbers: DISC1FP1 (EU302123), Boymaw (GU134617), der 11 chimeric transcript DISC1FP1 exon 2 to DISC1 exon 9 (JQ650115), der 1 chimeric transcript DISC1 exon 4 to DISC1FP1 exon 4 (JQ650116), der 1 chimeric transcript DISC1 exon 6 to DISC1FP1 exon 3a (JQ650117).

Solution Structure of the Calponin Ch Domain and Fitting to the 3D-Helical Reconstruction of F-Actin:Calponin.

Calponin is involved in the regulation of contractility and organization of the actin cytoskeleton in smooth muscle cells. It is the archetypal member of the calponin homology (CH) domain family of actin binding proteins that includes cytoskeletal linkers such as alpha-actinin, spectrin, and dystrophin, and regulatory proteins including VAV, IQGAP, and calponin. We have determined the first structure of a CH domain from a single CH domain-containing protein, that of calponin, and have fitted the NMR-derived coordinates to the 3D-helical reconstruction of the F-actin:calponin complex using cryo-electron microscopy. The tertiary fold of this single CH domain is typical of, yet significantly different from, those of the CH domains that occur in tandem pairs to form high-affinity ABDs in other proteins. We thus provide a structural insight into the mode of interaction between F-actin and CH domain-containing proteins.

ZZ domain of dystrophin and utrophin: topology and mapping of a beta-dystroglycan interaction site.

Dystrophin forms part of a vital link between actin cytoskeleton and extracellular matrix via the transmembrane adhesion receptor dystroglycan. Dystrophin and its autosomal homologue utrophin interact with beta-dystroglycan via their highly conserved C-terminal cysteine-rich regions, comprising the WW domain (protein-protein interaction domain containing two conserved tryptophan residues), EF hand and ZZ domains. The EF hand region stabilizes the WW domain providing the main interaction site between dystrophin or utrophin and dystroglycan. The ZZ domain, containing a predicted zinc finger motif, stabilizes the WW and EF hand domains and strengthens the overall interaction between dystrophin or utrophin and beta-dystroglycan. Using bacterially expressed ZZ domain, we demonstrate a conformational effect of zinc binding to the ZZ domain, and identify two zinc-binding regions within the ZZ domain by SPOTs overlay assays. Epitope mapping of the dystrophin ZZ domain was carried out with new monoclonal antibodies by ELISA, overlay assay and immunohistochemistry. One monoclonal antibody defined a discrete region of the ZZ domain that interacts with beta-dystroglycan. The epitope was localized to the conformationally sensitive second zinc-binding site in the ZZ domain. Our results suggest that residues 3326-3332 of dystrophin form a crucial part of the contact region between dystrophin and beta-dystroglycan and provide new insight into ZZ domain organization and function.

A Function for the RING Finger Domain in the Allosteric Control of MDM2 Conformation and Activity

The MDM2 oncoprotein plays multiple regulatory roles in the control of p53-dependent gene expression. A picture of MDM2 is emerging where structurally discrete but interdependent functional domains are linked through changes in conformation. The domain structure includes: (i) a hydrophobic pocket at the N terminus of MDM2 that is involved in both its transrepressor and E3-ubiqutin ligase functions, (ii) a central acid domain that recognizes a ubiquitination signal in the core DNA binding domain of p53, and (iii) a C-terminal C2H2C4 RING finger domain that is required for E2 enzyme-binding and ATP-dependent molecular chaperone activity. Here we show that the binding affinity of MDM2s hydrophobic pocket can be regulated through the RING finger domain and that increases in pocket affinity are reflected by a gain in MDM2 transrepressor activity. Thus, mutations within the RING domain that affect zinc coordination, but not one that inhibits ATP binding, produce MDM2 proteins that have a higher affinity for the BOX-I transactivation domain of p53 and a reduced I0.5 for p53 transrepression. An allosteric model for regulation of the hydrophobic pocket is supported by differences in protein conformation and pocket accessibility between wild-type and the RING domain mutant MDM2 proteins. Additionally the data demonstrate that the complex relationship between different domains of MDM2 can impact on the efficacy of anticancer drugs directed toward its hydrophobic pocket.

Functional Insights from the Structure of the Multifunctional C345C Domain of C5 of Complement

The complement protein C5 initiates assembly of the membrane attack complex. This remarkable process results in lysis of target cells and is fundamental to mammalian defense against infection. The 150-amino acid residue domain at the C terminus of C5 (C5-C345C) is pivotal to C5 function. It interacts with enzymes that convert C5 to C5b, the first step in the assembly of the membrane attack complex; it also binds to the membrane attack complex components C6 and C7 with high affinity. Here a recombinant version of this C5-C345C domain is shown to adopt the oligosaccharide/oligonucleotide binding fold, with two helices packed against a five-stranded β-barrel. The structure is compared with those from the netrin-like module family that have a similar fold. Residues critical to the interaction with C5-convertase cluster on a mobile, hydrophobic inter-strand loop that protrudes from the open face of the β-barrel. The opposite, helix-dominated face of C5-C345C carries a pair of exposed hydrophobic side chains adjacent to a striking negatively charged patch, consistent with affinity for positively charged factor I modules in C6 and C7. Modeling of homologous domains from complement proteins C3 and C4, which do not participate in membrane attack complex assembly, suggests that this provisionally identified C6/C7-interacting face is indeed specific to C5.

A Novel p53 Phosphorylation Site within the MDM2 Ubiquitination Signal: II. A MODEL IN WHICH PHOSPHORYLATION AT SER269 INDUCES A MUTANT CONFORMATION TO p53*

The p53 DNA-binding domain harbors a conformationally flexible multiprotein binding site that regulates p53 ubiquitination. A novel phosphorylation site exists within this region at Ser269, whose phosphomimetic mutation inactivates p53. The phosphomimetic p53 (S269D) exhibits characteristics of mutant p53: stable binding to Hsp70 in vivo, elevated ubiquitination in vivo, inactivity in DNA binding and transcription, increased thermoinstability using thermal shift assays, and λmax of intrinsic tryptophan fluorescence at 403 nm rather than 346 nm, characteristic of wild type p53. These data indicate that p53 conformational stability is regulated by a phosphoacceptor site within an exposed flexible surface loop and that this can be destabilized by phosphorylation. To test whether other motifs within p53 have similarly evolved, we analyzed the effect of Ser215 mutation on p53 function because Ser215 is another inactivating phosphorylation site in the conformationally flexible PAb240 epitope. The p53S215D protein is inactive like p53S269D, whereas p53S215A is as active as p53S269A. However, the double mutant p53S215A/S269A was transcriptionally inactive and more thermally unstable than either individual Ser-Ala loop mutant. Molecular dynamics simulations suggest that (i) solvation of phospho-Ser215 and phospho-Ser269 by positive charged residues or solvent water leads to local unfolding, which is accompanied by local destabilization of the N-terminal loop and global destabilization of p53, and (ii) the double alanine 215/269 mutation disrupts hydrogen bonding normally stabilized by both Ser215 and Ser269. These data indicate that p53 has evolved two serine phosphoacceptor residues within conformationally flexible epitopes that normally stabilize the p53 DNA-binding domain but whose phosphorylation induces a mutant conformation to wild type p53.

The Mitosis and Neurodevelopment Proteins NDE1 and NDEL1 Form Dimers, Tetramers, and Polymers with a Folded Back Structure in Solution

Background: NDE1 and NDEL1 are neurodevelopmental and mitotic proteins with extended coiled-coil N termini, but unknown C-terminal structure. Results: Recombinant NDE1/NDEL1 form dimers and tetramers in which their C termini interact with their N-terminal domains. Conclusion: NDE1/NDEL1 each adopt a sharply bent back structure. Significance: This explains the existence of two distinct dynein-binding domains on NDE1/NDEL1 and instability of disease-associated mutants lacking C termini. Paralogs NDE1 (nuclear distribution element 1) and NDEL1 (NDE-like 1) are essential for mitosis and neurodevelopment. Both proteins are predicted to have similar structures, based upon high sequence similarity, and they co-complex in mammalian cells. X-ray diffraction studies and homology modeling suggest that their N-terminal regions (residues 8–167) adopt continuous, extended α-helical coiled-coil structures, but no experimentally derived information on the structure of their C-terminal regions or the architecture of the full-length proteins is available. In the case of NDE1, no biophysical data exists. Here we characterize the structural architecture of both full-length proteins utilizing negative stain electron microscopy along with our established paradigm of chemical cross-linking followed by tryptic digestion, mass spectrometry, and database searching, which we enhance using isotope labeling for mixed NDE1-NDEL1. We determined that full-length NDE1 forms needle-like dimers and tetramers in solution, similar to crystal structures of NDEL1, as well as chain-like end-to-end polymers. The C-terminal domain of each protein, required for interaction with key protein partners dynein and DISC1 (disrupted-in-schizophrenia 1), includes a predicted disordered region that allows a bent back structure. This facilitates interaction of the C-terminal region with the N-terminal coiled-coil domain and is in agreement with previous results showing N- and C-terminal regions of NDEL1 and NDE1 cooperating in dynein interaction. It sheds light on recently identified mutations in the NDE1 gene that cause truncation of the encoded protein. Additionally, analysis of mixed NDE1-NDEL1 complexes demonstrates that NDE1 and NDEL1 can interact directly.

Solution Structure of Factor I-like Modules from Complement C7 Reveals a Pair of Follistatin Domains in Compact Pseudosymmetric Arrangement

Factor I-like modules (FIMs) of complement proteins C6, C7, and factor I participate in protein-protein interactions critical to the progress of a complement-mediated immune response to infections and other trauma. For instance, the carboxyl-terminal FIM pair of C7 (C7-FIMs) binds to the C345C domain of C5 and its activated product, C5b, during self-assembly of the cytolytic membrane-attack complex. FIMs share sequence similarity with follistatin domains (FDs) of known three-dimensional structure, suggesting that FIM structures could be reliably modeled. However, conflicting disulfide maps, inconsistent orientations of subdomains within FDs, and the presence of binding partners in all FD structures led us to determine the three-dimensional structure of C7-FIMs by NMR spectroscopy. The solution structure reveals that each FIM within C7 contains a small amino-terminal FOLN subdomain connected to a larger carboxyl-terminal KAZAL domain. The open arrangement of the subdomains within FIMs resembles that of first FDs within structures of tandem FDs but differs from the more compact subdomain arrangement of second or third FDs. Unexpectedly, the two C7-FIMs pack closely together with an approximate 2-fold rotational symmetry that is rarely seen in module pairs and has not been observed in FD-containing proteins. Interfaces between subdomains and between modules include numerous hydrophobic and electrostatic contributions, suggesting that this is a physiologically relevant conformation that persists in the context of the parent protein. Similar interfaces were predicted in a homology-based model of the C6-FIM pair. The C7-FIM structures also facilitated construction of a model of the single FIM of factor I.