John T. Gooch

Structural Basis for the Assembly of the Smrt/Ncor Core Transcriptional Repression Machinery.

Eukaryotic transcriptional repressors function by recruiting large coregulatory complexes that target histone deacetylase enzymes to gene promoters and enhancers. Transcriptional repression complexes, assembled by the corepressor NCoR and its homolog SMRT, are crucial in many processes, including development and metabolic physiology. The core repression complex involves the recruitment of three proteins, HDAC3, GPS2 and TBL1, to a highly conserved repression domain within SMRT and NCoR. We have used structural and functional approaches to gain insight into the architecture and biological role of this complex. We report the crystal structure of the tetrameric oligomerization domain of TBL1, which interacts with both SMRT and GPS2, and the NMR structure of the interface complex between GPS2 and SMRT. These structures, together with computational docking, mutagenesis and functional assays, reveal the assembly mechanism and stoichiometry of the corepressor complex.

The Structural Basis for the Specificity of Retinoid-X Receptor-selective Agonists: New Insights Into the Role of Helix H12

Ligands that specifically target retinoid-X receptors (RXRs) are emerging as potentially powerful therapies for cancer, diabetes, and the lowering of circulatory cholesterol. To date, RXR has only been crystallized in the absence of ligand or with the promiscuous ligand 9-cis retinoic acid, which also activates retinoic acid receptors. Here we present the structure of hRXRβ in complex with the RXR-specific agonist LG100268 (LG268). The structure clearly reveals why LG268 is specific for the RXR ligand binding pocket and will not activate retinoic acid receptors. Intriguingly, in the crystals, the C-terminal “activation” helix (AF-2/helix H12) is trapped in a novel position not seen in other nuclear receptor structures such that it does not cap the ligand binding cavity. Mammalian two-hybrid assays indicate that LG268 is unable to release co-repressors from RXR unless co-activators are also present. Together these findings suggest that RXR ligands may be inefficient at repositioning helix H12.

An actin depolymerizing protein from pig plasma

Actin filaments are depolymerized by a protein component of plasma and serum [ l-31. This paper describes the isolation from pig plasma of a protein with actin depolymerizing activity (plasma ADF). It consists of a single polypeptide of -92 000 Mr. It interacts rapidly with actin in a stoichiometry close to 1: 1, without affecting the size or charge of the actin polypeptide. An abstract containing some of these data has been presented [4].

Identification of a novel co-regulator interaction surface on the ligand-binding domain of Nurr1 using NMR footprinting

The nuclear receptor Nurr1 is a transcription factor essential for the development of midbrain dopaminergic neurons in vertebrates. Recent crystal structures of the Nurr1 ligand binding domain (LBD) and the Drosophila orthologue dHR38 revealed that, although these receptors share the classical LBD architecture, they lack a ligand binding cavity. This volume is instead filled with bulky hydrophobic side chains. Furthermore the “canonical” non-polar co-regulator binding groove is filled with polar side chains; thus, the regulation of transcription by this sub-family of nuclear receptor LBDs may be mediated by some other interaction surface on the LBD. We report here the identification of a novel co-regulator interface on the LBD of Nurr1. We used an NMR footprinting strategy that facilitates the identification of an interaction surface without the need of a full assignment. We found that non-polar peptides derived from the co-repressors SMRT and NCoR bind to a hydrophobic patch on the LBD of Nurr1. This binding surface involves a groove between helices 11 and 12. Mutations in this site abolish activation by the Nurr1 LBD. These findings give insight into the unique mechanism of action of this class of nuclear receptors.

Variant plasma gelsolin responsible for familial amyloidosis (Finnish type) has defective actin severing activity

Familial amyloidosis, Finnish type is caused by a single base mutation in gelsolin, an actin filament severing and capping protein that is present in most tissues and in blood plasma. The mutation replaces aspartic acid with asparagine at residue 187 of the plasma sequence. This renders the gelsolin susceptible to proteolysis as a consequence of which amyloid protein is formed. Here it is shown that the mutant protein in plasma from a patient homozygous for this mutation lacks both actin severing and nucleating activities. Evidence is presented that the cleaved mutant gelsolin has dissociated under non‐denaturing conditions and that the resultant 65,000 and 55,000 M r C‐terminal fragments aggregate.

Gelsolin Domains 4–6 in Active, Actin-free Conformation Identifies Sites of Regulatory Calcium Ions

Structural analysis of gelsolin domains 4-6 demonstrates that the two highest-affinity calcium ions that activate the molecule are in domains 5 and 6, one in each. An additional calcium site in domain 4 depends on subsequent actin binding and is seen only in the complex. The uncomplexed structure is primed to bind actin. Since the disposition of the three domains is similar in different crystal environments, either free or in complex with actin, the conformation in calcium is intrinsic to active gelsolin itself. Thus the actin-free structure shows that the structure with an actin monomer is a good model for an actin filament cap. The last 13 residues of domain 6 have been proposed to be a calcium-activated latch that, in the inhibited form only, links two halves of gelsolin. Comparison with the active structure shows that loosening of the latch contributes but is not central to activation. Calcium binding in domain 6 invokes a cascade of swapped ion-pairs. A basic residue swaps acidic binding partners to stabilise a straightened form of a helix that is kinked in inhibited gelsolin. The other end of the helix is connected by a loop to an edge beta-strand. In active gelsolin, an acidic residue in this helix breaks with its loop partner to form a new intrahelical ion-pairing, resulting in the breakage of the continuous sheet between domains 4 and 6, which is central to the inhibited conformation. A structural alignment of domain sequences provides a rationale to understand why the two calcium sites found here have the highest affinity amongst the five different candidate sites found in other gelsolin structures.

Identification of the trapped calcium in the gelsolin segment 1—actincomplex: Implications for the role of calcium in the control of gelsolin activity

The X‐ray structure of the complex of actin with gelsolin segment 1 revealed the presence of two calcium ions, one bound at an intramolecular site within segment 1 and the other bridging the segment directly to actin. Although earlier calcium binding studies at pH 8.0 revealed only a single calcium trapped in the complex (and also in the binary gelsolin‐actin complex), it is here shown that two calcium ions are bound under the conditions of crystallization at physiological pH. Mutation of acidic residues in either actin or segment 1 involved in ligation of the intermolecular calcium ion resulted in loss of one of the bound calcium ions at pH <7, but not at pH 8. Thus the calcium ion trapped in the segment 1‐actin complex is that located at the intramolecular site. The implications of this for gelsolin function are discussed.

Crystallization of the complex of actin with gelsolin segment 1.

Crystals of a 1:1 complex between human gelsolin segment 1 and actin have been grown from solutions containing polyethylene glycol 6000. The crystals are orthorhombic, space group P2(1)2(1)2(1); the axes are a = 57.4 A, b = 70.4 A, c = 184.5 A. They are moderately stable to X-rays and diffract to beyond 2.5 A. There is one molecule of complex in the asymmetric unit.

Loss of calcium sensitivity of plasma gelsolin is associated with the presence of calcium ions during preparation

Gelsolin is a calcium‐dependent actin severing and capping protein. Calcium ‘opens’ the molecule to make actin binding sites accessible, but removal of calcium from the medium does not necessarily fully reverse this process. The calcium sensitivity of actin monomer binding and actin filament severing is here shown to vary considerably with the source of gelsolin and conditions of preparation. Plasma gelsolin undergoes irreversible loss of calcium sensitivity when prepared in the presence of calcium ions. This is not due solely to effects of bound calcium, because purified human plasma gelsolin expressed in E. coli and stored in calcium shows no comparable loss of calcium sensitivity when prepared or stored in calcium. These results suggest the presence of factors in plasma which, in the presence of calcium, promote an irreversible structural change in gelsolin resulting in permanent loss of calcium sensitivity.

Crystallization of human gelsolin

Human gelsolin has been crystallized by microdialysis techniques to give single crystals that diffract to 3.5 Å resolution. The crystals belong to space group P4212 and have cell dimensions a = 175.0 Å, c = 151.6 Å. They contain two gelsolin molecules in the asymmetric unit.