The assembly of different types of virulence-associated surface fibers called pili in Gram-negative bacteria requires periplasmic chaperones. PapD is the prototype member of the periplasmic chaperone family, and the structural basis of its interactions with pilus subunits was investigated. Peptides corresponding to the carboxyl terminus of pilus subunits bound PapD and blocked the ability of PapD to bind to the pilus adhesin PapG in vitro. The crystal structure of PapD complexed to the PapG carboxyl-terminal peptide was determined to 3.0 A resolution. The peptide bound in an extended conformation with its carboxyl terminus anchored in the interdomain cleft of the chaperone via hydrogen bonds to invariant chaperone residues Arg8 and Lys112. Main chain hydrogen bonds and contacts between hydrophobic residues in the peptide and the chaperone stabilized the complex and may play a role in determining binding specificity. Site-directed mutations in Arg8 and Lys112 abolished the ability of PapD to bind pilus subunits and mediate pilus assembly in vivo, an indication that the PapD-peptide crystal structure is a reflection of at least part of the PapD-subunit interaction.
The nuclear receptor heterodimers of liver X receptor (LXR) and retinoid X receptor (RXR) are key transcriptional regulators of genes involved in lipid homeostasis and in¯ammation. We report the crystal structure of the ligand-binding domains (LBDs) of LXRa and RXRb complexed to the synthetic LXR agonist T-0901317 and the RXR agonist methoprene acid (Protein Data Base entry 1UHL). Both LBDs are in agonist conformation with GRIP-1 peptides bound at the coactivator binding sites. T-0901317 occupies the center of the LXR ligand-binding pocket and its hydroxyl head group interacts with H421 and W443, residues identi®ed by mutational analysis as critical for ligand-induced transcriptional activation by T-0901317 and various endogenous oxysterols. The topography of the pocket suggests a common anchoring of these oxysterols via their 22-, 24-or 27-hydroxyl group to H421 and W443. Polyunsaturated fatty acids act as LXR antagonists and an E267A mutation was found to enhance their transcriptional inhibition. The present structure provides a powerful tool for the design of novel modulators that can be used to characterize further the physiological functions of the LXR± RXR heterodimer.
Proteins of the bromodomain and extraterminal (BET) family, in particular bromodomain-containing protein 4 (BRD4), are of great interest as biological targets. BET proteins contain two separate bromodomains, and existing inhibitors bind to them monovalently. Here we describe the discovery and characterization of probe compound biBET, capable of engaging both bromodomains simultaneously in a bivalent, in cis binding mode. The evidence provided here was obtained in a variety of biophysical and cellular experiments. The bivalent binding results in very high cellular potency for BRD4 binding and pharmacological responses such as disruption of BRD4-mediator complex subunit 1 foci with an EC of 100 pM. These compounds will be of considerable utility as BET/BRD4 chemical probes. This work illustrates a novel concept in ligand design-simultaneous targeting of two separate domains with a drug-like small molecule-providing precedent for a potentially more effective paradigm for developing ligands for other multi-domain proteins.
Axonal growth cone guidance is a central process in nervous system development and repair. Collapsin response mediator protein 2 (CRMP-2) is a neurite extension-promoting neuronal cytosolic molecule involved in the signalling of growth inhibitory cues from external stimuli, such as semaphorin 3A and the myelin-associated glycoprotein. We have determined the crystal structure of human tetrameric CRMP-2, which is structurally related to the dihydropyriminidases; however, the active site is not conserved. The wealth of earlier functional mapping data for CRMP-2 are discussed in light of the threedimensional structure of the protein. The differences in oligomerisation interfaces between CRMP-1 and CRMP-2 are used to model CRMP-1/2 heterotetramers.
The class of proteins collectively known as periplasmic immunoglobulin-like chaperones play an essential role in the assembly of a diverse set of adhesive organelles used by pathogenic strains of Gram-negative bacteria. Herein, we present a combination of genetic and structural data that sheds new light on chaperone-subunit and subunit-subunit interactions in the prototypical P pilus system, and provides new insights into how PapD controls pilus biogenesis. New crystallographic data of PapD with the C-terminal fragment of a subunit suggest a mechanism for how periplasmic chaperones mediate the extraction of pilus subunits from the inner membrane, a prerequisite step for subunit folding. In addition, the conserved N- and C-terminal regions of pilus subunits are shown to participate in the quaternary interactions of the mature pilus following their uncapping by the chaperone. By coupling the folding of subunit proteins to the capping of their nascent assembly surfaces, periplasmic chaperones are thereby able to protect pilus subunits from premature oligomerization until their delivery to the outer membrane assembly site.
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