The 3D structures of human therapeutic targets are enabling for drug discovery. However, their purification and crystallization remain rate determining. In individual cases, ligands have been used to increase the success rate of protein purification and crystallization, but the broad applicability of this approach is unknown. We implemented two screening platforms, based on either fluorimetry or static light scattering, to measure the increase in protein thermal stability upon binding of a ligand without the need to monitor enzyme activity. In total, 221 different proteins from humans and human parasites were screened against one or both of two sorts of small-molecule libraries. The first library comprised different salts, pH conditions, and commonly found small molecules and was applicable to all proteins. The second comprised compounds specific for protein families of particular interest (e.g., protein kinases). In 20 cases, including nine unique human protein kinases, a small molecule was identified that stabilized the proteins and promoted structure determination. The methods are cost-effective, can be implemented in any laboratory, promise to increase the success rates of purifying and crystallizing human proteins significantly, and identify new ligands for these proteins.chemical biology ͉ crystallography ͉ human S tructural, functional, and chemical genomics (proteomics) are disciplines that aim to determine the biochemical, cellular, and physiological functions of proteins on a genome scale. Many of the central, important experimental approaches that are involved, such as protein-based screens for small-molecule inhibitors, depend on the availability of purified and active proteins. To meet this demand, many large projects are devoted to developing methods to generate large numbers of purified proteins. However, the task is proving challenging: on average, for proteins from prokaryotes, only 50-70% of soluble proteins and 30% of membrane proteins can be readily expressed in recombinant form, and only 30-50% of these expressed proteins can be purified to homogeneity (1, 2). The success rates for human proteins are predicted to be significantly lower.To improve the general rates of protein purification, efforts have focused largely on alterations of the recombinant host, the expression conditions, changes of the construct encoding the protein, and the purification conditions. It is also known that the expression and purification of a protein can be improved significantly by the addition of a specific ligand, which serves to stabilize the protein, thereby reducing its propensity to unfold, aggregate, or succumb to proteolysis. This parameter has not been studied systematically, although in individual cases the addition of a specific ligand has had dramatic effects. For example, the recombinant expression of the guinea pig and human forms of the enzyme 11␤-hydroxysteroid dehydrogenase-1 in bacteria was increased dramatically by the addition of an inhibitor of the enzyme to the growing cells (3) Wu, K. L. Kav...

Peptidyl-prolyl isomerases catalyze the conversion between cis and trans isomers of proline. The cyclophilin family of peptidyl-prolyl isomerases is well known for being the target of the immunosuppressive drug cyclosporin, used to combat organ transplant rejection. There is great interest in both the substrate specificity of these enzymes and the design of isoform-selective ligands for them. However, the dearth of available data for individual family members inhibits attempts to design drug specificity; additionally, in order to define physiological functions for the cyclophilins, definitive isoform characterization is required. In the current study, enzymatic activity was assayed for 15 of the 17 human cyclophilin isomerase domains, and binding to the cyclosporin scaffold was tested. In order to rationalize the observed isoform diversity, the high-resolution crystallographic structures of seven cyclophilin domains were determined. These models, combined with seven previously solved cyclophilin isoforms, provide the basis for a family-wide structure∶function analysis. Detailed structural analysis of the human cyclophilin isomerase explains why cyclophilin activity against short peptides is correlated with an ability to ligate cyclosporin and why certain isoforms are not competent for either activity. In addition, we find that regions of the isomerase domain outside the proline-binding surface impart isoform specificity for both in vivo substrates and drug design. We hypothesize that there is a well-defined molecular surface corresponding to the substrate-binding S2 position that is a site of diversity in the cyclophilin family. Computational simulations of substrate binding in this region support our observations. Our data indicate that unique isoform determinants exist that may be exploited for development of selective ligands and suggest that the currently available small-molecule and peptide-based ligands for this class of enzyme are insufficient for isoform specificity.Enhanced version This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3-D representations and animated transitions. Please note that a Web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

Calcium-dependent protein kinases (CDPKs) play pivotal roles in the calcium-signaling pathway in plants, ciliates and apicomplexan parasites, and comprise a CaMK-like kinase domain regulated by a calcium-binding domain in the C-terminus. To understand this intramolecular mechanism of activation, we solved the structures of the autoinhibited (apo) and activated (calcium-bound) conformations of CDPKs from the apicomplexan parasites Toxoplasma gondii and Cryptosporidium parvum. In the apo form, the C-terminal CDPK activation domain (CAD) resembles a calmodulin protein with an unexpected long helix in the N-terminus that inhibits the kinase domain in the same manner as CaMKII. Calcium binding triggers the reorganization of the CAD into a highly intricate fold, leading to its relocation around the base of the kinase domain to a site remote from the substrate-binding site. This large conformational change constitutes a distinct mechanism in calcium signal transduction pathways.

We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.

Ubiquitin-specific protease 8 (USP8) hydrolyzes mono and polyubiquitylated targets such as epidermal growth factor receptors and is involved in clathrin-mediated internalization. In 1182 residues, USP8 contains multiple domains, including coiled-coil, rhodanese, and catalytic domains. We report the first high-resolution crystal structures of these domains and discuss their implications for USP8 function. The amino-terminal domain is a homodimer with a novel fold. It is composed of two five-helix bundles, where the first helices are swapped, and carboxyl-terminal helices are extended in an antiparallel fashion. The structure of the rhodanese domain, determined in complex with the E3 ligase NRDP1, reveals the canonical rhodanese fold but with a distorted primordial active site. The USP8 recognition domain of NRDP1 has a novel protein fold that interacts with a conserved peptide loop of the rhodanese domain. A consensus sequence of this loop is found in other NRDP1 targets, suggesting a common mode of interaction. The structure of the carboxyl-terminal catalytic domain of USP8 exhibits the conserved tripartite architecture but shows unique traits. Notably, the active site, including the ubiquitin binding pocket, is in a closed conformation, incompatible with substrate binding. The presence of a zinc ribbon subdomain near the ubiquitin binding site further suggests a polyubiquitin-specific binding site and a mechanism for substrate induced conformational changes.The post-translational ubiquitylation system is composed of a cascade of E1, 2 E2, and E3 enzymes that activate and transfer ubiquitin or ubiquitin homologs to target proteins, including those targets that are membrane-anchored (1, 2). Modification by ubiquitin or ubiquitin-like molecules typically affects localization of the protein within the cell. For example, Lys-48-polyubiquitylated targets are recognized by the proteasome, which catalyzes complete target proteolysis (3). By affecting the abundance and/or half-life of signaling molecules, the ubiquitylation system can have dramatic effects on signal transduction pathways and play major roles in cellular biology. Degradation of integral membrane proteins can also be initiated by the ubiquitylation system but subsequent steps differ (4). Ubiquitylated receptors for example are rounded up by multivalent adaptors (e.g. AP2, epsin), which in turn recruit clathrin molecules that induce internalization through membrane budding. Dissociation of clathrin coats releases endosomes, which are either recycled back to the plasma membrane or fused with lysosomes, where membrane proteins undergo proteolytic degradation. Budding, uncoating, trafficking, and fusion events are influenced by the ubiquitylation state of the various intermediates, which is regulated not just by E3 ligases but by deubiquitylases as well.The human genome contains over 90 deubiquitylases, the specific biological roles of which are mostly unknown. Some deubiquitylases have been implicated in ubiquitin recycling, which typically can occur at the ...

The significant increase in the demand for purified protein for crystallization and structural studies has made necessary the development of multi-sample methods for identifying solution conditions that affect protein stability and aggregation. Conditions that stabilize proteins can improve protein purification and crystallization. These methods can be used to identify small molecule compounds or inhibitors that interact with the purified proteins, and might serve as starting points for drug discovery. In this article three methods for measuring protein stability and aggregation are described and discussed: differential scanning fluorimetry (DSF), differential static light scattering (DSLS), and isothermal denaturation (ITD).

The Eph family of receptor tyrosine kinases has drawn growing attention due to their role in regulating diverse biological phenomena. However, pharmacological interrogation of Eph kinase function has been hampered by a lack of potent and selective Eph kinase inhibitors. Here we report the discovery of compounds 6 and 9 using a rationally designed kinase-directed library which potently inhibit Eph receptor tyrosine kinases, particularly EphB2 with cellular EC 50 s of 40 nM. Crystallographic data of EphA3 and EphA7 in complex with the inhibitors show that they bind to the "DFG-out" inactive kinase conformation and provide valuable information for structure-based design of second generation inhibitors.The Eph/ephrin family is the largest among tyrosine kinases and is unique in that the ligands and receptors are both membrane bound providing the possibility for bidirectional cell-cell signaling. Genome analysis reveals that there are 14 Eph receptors and eight ephrin ligands. Eph receptor signaling is responsible for arguably the most diverse set of biological phenomena of any tyrosine kinase family including organ development, tissue remodeling, neuronal signaling, insulin secretion, and bone metabolism. 1, 2 Not surprisingly, deregulation of ephrindependent signaling has been implicated in pathological conditions related to all of these systems. 3 The involvement of Eph/ephrin-signaling in tumorigenesis has been the most extensively investigated due to frequent upregulation of Eph receptor or ligand expression in numerous tumor types. [4][5][6] The emerging picture is complicated by the diversity of biological function that is associated with individual receptors and ligands, including oncogenic or tumor suppressor functions. The possibility of targeting Eph/ephrins therapeutically may be the most straightforward in the context of inhibiting Eph/ephrin-signaling in the vasculature as a means of preventing tumor angiogenesis. 2,6 Currently only a few small molecule Eph kinase © 2009 Elsevier Ltd. All rights reserved. * Corresponding Authors: Sirano Dhe-Paganon, Tel: 416-946-3876, E-mail: sirano.dhepaganon(@)utoronto.ca Nathanael Gray, Tel: 617-582-8590, E-mail: nathanael_gray(@)dfci.harvard.edu . † These authors contributed equally to this work Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ¶ Once inside PyMol, press the play button (at the bottom right) in order to start the 3D movie NIH Public Access To date the majority of investigation into functions of Eph receptor tyrosine kinases has been accomplished using genetic and biochemical methods. Pharmacological app...

Proteins containing C 2 H 2 type zinc ®nger motifs represent one of the largest classes of nucleic acid-binding proteins found in nature. We describe a novel zinc ®nger protein, dsRBP-ZFa, isolated by screening an expression library with dsRNA. The dsRBP-ZFa cDNA encodes a protein containing seven zinc ®nger motifs and an acidic C-terminal domain. Mobility shift experiments demonstrate that dsRBP-ZFa binds dsRNA and RNA-DNA hybrids with nanomolar dissociation constants and in a sequence independent manner. We also show that DNA and single stranded RNA fail to compete with dsRNA for binding suggesting dsRBP-ZFa prefers to bind an A-form helix. Using western analyses we have localized dsRBP-ZFa primarily to the nucleus of Xenopus laevis oocytes. The identi®cation of dsRBP-ZFa provides the ®rst example of a zinc ®nger protein that is speci®c for dsRNA. In addition, dsRBP-ZFa does not contain the previously described dsRNA binding motif, suggesting certain zinc ®ngers may provide an alternative way to recognize the A-form helix.# 1997 Academic Press Limited