Jonathan D. Taylor

Human estrogen receptor beta binds DNA in a manner similar to and dimerizes with estrogen receptor alpha.

The cloning of a novel estrogen receptor β (denoted ERβ) has recently been described (Kuiper, G. G. J. M., Enmark, E., Pelto-Huikko, M., Nilsson, S., and Gustafsson, J-A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5925–5930 and Mosselman, S., Polman, J., and Dijkema, R. (1996)FEBS Lett. 392, 49–53). ERβ is highly homologous to the “classical” estrogen receptor α (here referred to as ERα), has been shown to bind estrogens with an affinity similar to that of ERα, and activates expression of reporter genes containing estrogen response elements in an estrogen-dependent manner. Here we describe functional studies comparing the DNA binding abilities of human ERα and β in gel shift assays. We show that DNA binding by ERα and β are similarly affected by elevated temperature in the absence of ligand or in the presence of 17β-estradiol and the partial estrogen agonist 4-hydroxy-tamoxifen. In the absence of ligand, DNA binding by ERα and β is rapidly lost at 37 °C, while in the presence of 17β-estradiol and 4-hydroxy-tamoxifen, the loss in DNA binding at elevated temperature is much more gradual. We show that the loss in DNA binding is not due to degradation of the receptor proteins. However, while the complete antagonist ICI 182,780 does not “protect” human ERα (hERα) from loss of DNA binding at elevated temperaturein vitro, it does appear to protect human ERβ (hERβ), suggestive of differences in the way ICI 182,780 acts on hERα and β. We further report that ERα and β can dimerize with each other, the DNA binding domain of hERα being sufficient for dimerization with hERβ. Cell and promoter-specific transcription activation by ERα has been shown to be dependent on the differential action of the N- and C-terminal transcription activation functions AF-1 and AF-2, respectively. The existence of a second estrogen receptor gene and the dimerization of ERα and β add greater levels of complexity to transcription activation in response to estrogens.

The bacterial curli system possesses a potent and selective inhibitor of amyloid formation.

Curli are extracellular functional amyloids that are assembled by enteric bacteria during biofilm formation and host colonization. An efficient secretion system and chaperone network ensures that the major curli fiber subunit, CsgA, does not form intracellular amyloid aggregates. We discovered that the periplasmic protein CsgC was a highly effective inhibitor of CsgA amyloid formation. In the absence of CsgC, CsgA formed toxic intracellular aggregates. In vitro, CsgC inhibited CsgA amyloid formation at substoichiometric concentrations and maintained CsgA in a non-β-sheet-rich conformation. Interestingly, CsgC inhibited amyloid assembly of human α-synuclein, but not Aβ42, in vitro. We identified a common D-Q-Φ-X0,1-G-K-N-ζ-E motif in CsgC client proteins that is not found in Aβ42. CsgC is therefore both an efficient and selective amyloid inhibitor. Dedicated functional amyloid inhibitors may be a key feature that distinguishes functional amyloids from disease-associated amyloids.

Atomic Resolution Insights into Curli Fiber Biogenesis

Summary Bacteria produce functional amyloid fibers called curli in a controlled, noncytotoxic manner. These extracellular fimbriae enable biofilm formation and promote pathogenicity. Understanding curli biogenesis is important for appreciating microbial lifestyles and will offer clues as to how disease-associated human amyloid formation might be ameliorated. Proteins encoded by the curli specific genes (csgA-G) are required for curli production. We have determined the structure of CsgC and derived the first structural model of the outer-membrane subunit translocator CsgG. Unexpectedly, CsgC is related to the N-terminal domain of DsbD, both in structure and oxido-reductase capability. Furthermore, we show that CsgG belongs to the nascent class of helical outer-membrane macromolecular exporters. A cysteine in a CsgG transmembrane helix is a potential target of CsgC, and mutation of this residue influences curli assembly. Our study provides the first high-resolution structural insights into curli biogenesis.

Structural Basis for the Broad Specificity to Host- Cell Ligands by the Pathogenic Fungus Candida Albicans.

Candida albicans is the most prevalent fungal pathogen in humans and a major source of life-threatening nosocomial infections. The Als (agglutinin-like sequence) glycoproteins are an important virulence factor for this fungus and have been associated with binding of host-cell surface proteins and small peptides of random sequence, the formation of biofilms and amyloid fibers. High-resolution structures of N-terminal Als adhesins (NT-Als; up to 314 amino acids) show that ligand recognition relies on a motif capable of binding flexible C termini of peptides in extended conformation. Central to this mechanism is an invariant lysine that recognizes the C-terminal carboxylate of ligands at the end of a deep-binding cavity. In addition to several protein–peptide interactions, a network of water molecules runs parallel to one side of the ligand and contributes to the recognition of diverse peptide sequences. These data establish NT-Als adhesins as a separate family of peptide-binding proteins and an unexpected adhesion system for primary, widespread protein–protein interactions at the Candida/host-cell interface.

Structural insights into serine-rich fimbriae from Gram-positive bacteria.

The serine-rich repeat family of fimbriae play important roles in the pathogenesis of streptococci and staphylococci. Despite recent attention, their finer structural details and precise adhesion mechanisms have yet to be determined. Fap1 (Fimbriae-associated protein 1) is the major structural subunit of serine-rich repeat fimbriae from Streptococcus parasanguinis and plays an essential role in fimbrial biogenesis, adhesion, and the early stages of dental plaque formation. Combining multidisciplinary, high resolution structural studies with biological assays, we provide new structural insight into adhesion by Fap1. We propose a model in which the serine-rich repeats of Fap1 subunits form an extended structure that projects the N-terminal globular domains away from the bacterial surface for adhesion to the salivary pellicle. We also uncover a novel pH-dependent conformational change that modulates adhesion and likely plays a role in survival in acidic environments.

Identification of novel fragment compounds targeted against the pY pocket of v‐Src SH2 by computational and NMR screening and thermodynamic evaluation

Discovery of small molecule inhibitors of protein–protein interactions is a major challenge to pharmaceutical development. Fragment‐based approaches have begun to be widely adopted as an effective way of exploring chemical space on a protein surface with reduced library size. On completion of a fragment screen, the subsequent selection of appropriate “hit” molecules for development is a key decision point. Thermodynamic parameters can be used in this decision process. In this work, a fragment identification protocol based on a virtual fragment analysis and selection followed by 19F NMR screening was directed at the phosphotyrosine binding site of the Src SH2 domain. Three new ligands were identified. Isothermal titration calorimetry was used to provide thermodynamic parameters for the physiologically relevant ligand and the selected fragments. One of these fragments possesses a highly favorable enthalpic contribution to complex formation compared to other fragments and to the physiologically relevant ligand suggesting that it would make a good candidate for compound development. Proteins 2007.

Structure, dynamics, and binding thermodynamics of the v‐Src SH2 domain: Implications for drug design

SH2 domains provide fundamental recognition sites in tyrosine kinase‐mediated signaling pathways which, when aberrant, give rise to disease states such as cancer, diabetes, and immune deficiency. Designing specific inhibitors that target the SH2 domain‐binding site, however, have presented a major challenge. Despite well over a decade of intensive research, clinically useful SH2 domain inhibitors have yet to become available. A better understanding of the structural, dynamic, and thermodynamic contributions to ligand binding of individual SH2 domains will provide some insight as to whether inhibitor development is possible. We report the first high resolution solution structure of the apo‐v‐Src SH2 domain. This is accompanied by the analysis of backbone dynamics and pKa values within the apo‐ and peptide‐bound states. Our results indicate that the phosphotyrosine (pY) pocket is tightly structured and hence not adaptable to exogenous ligands. On the other hand, the pocket which accommodates residues proximal and C‐terminal of the pY (pY + 3) or so‐called specificity determining region, is a large dynamic‐binding surface. This appears to allow a high level of promiscuity in binding. Binding of a series of synthetic, phosphotyrosyl, peptidomimetic compounds designed to explore interactions in the pY + 3 pocket further demonstrates the ability of the SH2 domain to accommodate diverse ligands. The thermodynamic parameters of these interactions show dramatic enthalpy/entropy compensation. These data suggest that the v‐Src SH2 domain does not have a highly specific secondary‐binding site, which clearly presents a major hurdle to design selective inhibitors. Proteins 2008.

Electrostatically-guided inhibition of Curli amyloid nucleation by the CsgC-like family of chaperones

Polypeptide aggregation into amyloid is linked with several debilitating human diseases. Despite the inherent risk of aggregation-induced cytotoxicity, bacteria control the export of amyloid-prone subunits and assemble adhesive amyloid fibres during biofilm formation. An Escherichia protein, CsgC potently inhibits amyloid formation of curli amyloid proteins. Here we unlock its mechanism of action, and show that CsgC strongly inhibits primary nucleation via electrostatically-guided molecular encounters, which expands the conformational distribution of disordered curli subunits. This delays the formation of higher order intermediates and maintains amyloidogenic subunits in a secretion-competent form. New structural insight also reveal that CsgC is part of diverse family of bacterial amyloid inhibitors. Curli assembly is therefore not only arrested in the periplasm, but the preservation of conformational flexibility also enables efficient secretion to the cell surface. Understanding how bacteria safely handle amyloidogenic polypeptides contribute towards efforts to control aggregation in disease-causing amyloids and amyloid-based biotechnological applications.

New insight into the molecular control of bacterial functional amyloids.

Amyloid protein structure has been discovered in a variety of functional or pathogenic contexts. What distinguishes the former from the latter is that functional amyloid systems possess dedicated molecular control systems that determine the timing, location, and structure of the fibers. Failure to guide this process can result in cytotoxicity, as observed in several pathologies like Alzheimers and Parkinsons Disease. Many gram-negative bacteria produce an extracellular amyloid fiber known as curli via a multi-component secretion system. During this process, aggregation-prone, semi-folded curli subunits have to cross the periplasm and outer-membrane and self-assemble into surface-attached fibers. Two recent breakthroughs have provided molecular details regarding periplasmic chaperoning and subunit secretion. This review offers a combined perspective on these first mechanistic insights into the curli system.

Extending the usability of the phasing power of diselenide bonds: SeCys SAD phasing of CsgC using a non-auxotrophic strain

The CsgC protein is a component of the curli system in Escherichia coli. Reported here is the successful incorporation of selenocysteine (SeCys) and selenomethionine (SeMet) into recombinant CsgC, yielding derivatized crystals suitable for structural determination. Unlike in previous reports, a standard autotrophic expression strain was used and only single-wavelength anomalous dispersion (SAD) data were required for successful phasing. The level of SeCys/SeMet incorporation was estimated by mass spectrometry to be about 80%. The native protein crystallized in two different crystal forms (form 1 belonging to space group C222(1) and form 2 belonging to space group C2), which diffracted to 2.4 and 2.0 Å resolution, respectively, whilst Se-derivatized protein crystallized in space group C2 and diffracted to 1.7 Å resolution. The Se-derivatized crystals are suitable for SAD structure determination using only the anomalous signal derived from the SeCys residues. These results extend the usability of SeCys labelling to more general and less favourable cases, rendering it a suitable alternative to traditional phasing approaches.