Microfluidics Method and Design

In the same way that miniaturization has impacted the electronics industry, mi-crofluidic technologies promise to spark a revolution in the biological sciences by integrating ultra small-volume sample processing within a chip format. The use of nanoliter reaction volumes and highly scaleable parallel sample processing make microfluidic technologies ideally suited to protein crystallography, where the screening and processing of precious reagents is required. Beyond reduction in sample...

Selectivity of RAR Ligands and RAR Isotypes

A very elegant experiment has been reported where enantiomers of an unnatural RAR-y-selective ligand have been determined 28 . Of special significance is the observation that one of the enantiomers is effectively inactive in the biochemical assays. While acquiring co-crystal structures of many active ligands with the same protein is now commonplace, obtaining structures of inactive (i.e., weakly active) compounds is more challenging. This work provides a high-resolution structure (PDB entry...

Tertiary Structure

A great level of structural knowledge has accumulated for protein kinases over the past decade (for various reviews see 2-9 ). The minimal kinase domain consists of approximately 300 amino acids for example, Cyclin-Dependent Kinase 2 (CDK2) has 298 residues (Fig. 2.1) 10, 11 . The kinase domain can be further divided into two lobes or sub-domains, N-terminal and C-terminal, with the lobes connected via a five to six residue hinge or linker region. The N-terminal lobe is the smaller subunit and...

What is Ecotin

Ecotin (eco) is a potent inhibitor of serine proteases that is derived from Escherichia coli. It was originally named for its ability to inhibit trypsin (E. coli trypsin inhibitor), but it is known to interact with and inhibit virtually all characterized tryp-sin-fold serine proteases. It is insensitive to the active site P1 preference of the protease (the amino acid N-terminal to the cleaved or scissile bond1)) and inhibits proteases with specificity towards basic, large hydrophobic, small...

Removing Protein Heterogeneity by Point Mutation

It is well known that post-translational modifications are another source of heterogeneity for proteins. There are at least 150 different kinds of covalent modifications of proteins, such as glycosylation, phosphorylation, S-thiolation, etc. 15 . Although the modification itself may be very small, like the addition of a single phosphate group, it may serve a physiological role and cause substantial conformational changes in the protein. Post-translational modification of a protein usually...

Confirmation of the Binding Mode

In order to investigate the binding mode of6 more comprehensively, we performed a docking study between the Cdk4 model and 6. As a result, four patterns of binding modes were suggested 18 . Considering the structural requirements for Cdk2 4 inhibitors Fig. 6.2 and the SAR of the informer libraries, we selected the binding mode candidate shown in Fig. 6.9 B. In this binding mode, there is some degree of steric repulsion between the pyridine ring and the terminal benzene ring in the fluorenone....

Other Antibiotics that Bind to the 50S Subunit 4391 Sparsomycin

Sparsomycin inhibits protein synthesis in all organisms and consequently is not used as an antibiotic. However, it has been tested extensively for its antitumor activity 49 . Sparsomycin contains a pseudouracil base with a conjugated link to a sulfur containing tail Fig. 4.11 . This chemical structure has been compared with that of puromycin, and it has been suggested that sparsomycin binds to the A-site much like puromycin 50 . Sparsomycin does not bind to the ribosome unless a P-site...

Introduction

The use of structural information derived from protein X-ray crystallography for the design of therapeutically useful molecules has long been viewed as an ideal approach. We have had the opportunity to participate in a structure-based medicinal chemistry project directed at the development of inhibitors of the bone-specific cysteine protease cathepsin K, as a treatment for bone loss diseases such as osteoporosis 1 . The origins of this project were in a high-profile effort at Smith-Kline...

Crystallization and Analysis of Serine Proteases with Ecotin 171

Fletterick 7.2.1 Expression of Wild Type Ecotin 173 7.2.2 Purification of Wild Type Ecotin 174 7.2.3 Crystallization of Ecotin and Protease Complexes 175 7.3 Representative Examples of Ecotin and Protease Structures 175 7.3.1 Ecotin Defines the S7 Through S4' Subsites of Collagenase 176 Ecotin as a Tight Binding Substrate 177 Ecotin Defines Regions Distal to the Factor Xa Protease Domain 178 Crystallization and Structure Determination of E2P2 Complexes 180...

Crystallization and Analysis of Serine Proteases with Ecotin

Fletterick Abstract Ecotin is a macromolecular inhibitor of serine proteases. It is remarkable in binding and blocking activity of virtually all serine proteases with the canonical Asp-His-Ser catalytic triad, regardless of the amino acid sequence or substrate specificity. It forms a heterotetramer that inhibits the protease by presenting a substratelike, 11 amino acid loop to the active site. Since ecotin mimics a substrate, it defines the amino acids of the...

Summary

NHRs function as either activators or repressors of gene expression. Small molecule ligands hormones are the regulators of these proteins. Structural studies have characterized at least two distinct forms of ligated NHRs, agonist-bound NHR with a peptide derived from a co-activator and antagonist-bound NHR with a peptide derived from a co-repressor. These define two states for the NHR, the first one is the on state where genes are expressed and the second is the off state where gene expression...

Rational Drug Design of Macrolides

Understanding the mechanism of resistance conferred by a specific mutation is expected to assist in rational drug design experiments. As described above, crystal structures reveal the mechanisms by which the Ec A2058 to G2058 mutation confers resistance to macrolide antibiotics. However, knowledge of that specific resistance mechanism might not be useful in rational drug design experiments. Gua-nine at Ec 2058 is highly conserved in eukaryotes, including humans, and so a macrolide redesigned to...

Nuclear Hormone Receptors Ligand Binding Domains

Nuclear hormone receptors NHR are multidomain proteins that function as transcription factors. They contain a central DNA binding domain DBD responsible for targeting the receptor to highly specific DNA sequences comprising a response element. The DBD is surrounded by two activation domains the activation function 1 AF-1 domain that resides at the N-terminus and the activation function 2 AF-2 domain that resides at the C-terminal ligand-binding domain LBD 1, 2 . NHRs for which no natural ligand...

Chemical Inducers of Dimerization to Control Transcription

Rapamycin Analogs

Chemical inducers of dimerization CID are bivalent small molecules that bind two proteins simultaneously. The purpose of these molecules is to bring the proteins together to induce signal transduction 27 . For brevity, we will limit our discussion to CIDs that directly control transcription. The basic architecture of these systems consists of two chimeric proteins. The first contains a DNA-binding domain DBD fused to a ligand-binding domain LBD and the second chimera contains an LBD and an...