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1.  Molecular Architecture of the Ankyrin SOCS Box Family of Cul5-Dependent E3 Ubiquitin Ligases 
Journal of Molecular Biology  2013;425(17):3166-3177.
Multi-subunit Cullin–RING E3 ligases often use repeat domain proteins as substrate-specific adaptors. Structures of these macromolecular assemblies are determined for the F-box-containing leucine-rich repeat and WD40 repeat families, but not for the suppressor of cytokine signaling (SOCS)-box-containing ankyrin repeat proteins (ASB1–18), which assemble with Elongins B and C and Cul5. We determined the crystal structures of the ternary complex of ASB9–Elongin B/C as well as the interacting N-terminal domain of Cul5 and used structural comparisons to establish a model for the complete Cul5-based E3 ligase. The structures reveal a distinct architecture of the ASB9 complex that positions the ankyrin domain coaxial to the SOCS box–Elongin B/C complex and perpendicular to other repeat protein complexes. This alternative architecture appears favorable to present the ankyrin domain substrate-binding site to the E2-ubiquitin, while also providing spacing suitable for bulky ASB9 substrates, such as the creatine kinases. The presented Cul5 structure also differs from previous models and deviates from other Cullins via a rigid-body rotation between Cullin repeats. This work highlights the adaptability of repeat domain proteins as scaffolds in substrate recognition and lays the foundation for future structure–function studies of this important E3 family.
Graphical Abstract
•Crystal structures were solved for the ASB9–Elongin B/C complex and the interacting Cul5 domain.•A model for their assembly into a Cullin–RING E3 ligase was constructed.•The molecular architecture of these ankyrin-containing SOCS box proteins deviates from the related F-box proteins to maintain the precise geometry for substrate ubiquitylation.
PMCID: PMC3779351  PMID: 23806657
NTD, N-terminal domain; CTD, C-terminal domain; PDB, Protein Data Bank; SOCS, suppressor of cytokine signaling; SCF, Skp1–Cul1–F-box; ASB, ankyrin SOCS box; proteasome; signaling; ubiquitination; degradation; protein–protein interaction
3.  Squeezing the most from every crystal: the fine details of data collection 
This article gives an overview of techniques and procedures for efficient data collection at synchrotron sources.
Modern synchrotron beamlines offer instrumentation of unprecedented quality, which in turn encourages increasingly marginal experiments, and for these, as much as ever, the ultimate success of data collection depends on the experience, but especially the care, of the experimenter. A representative set of difficult cases has been encountered at the Structural Genomics Consortium, a worldwide structural genomics initiative of which the Oxford site currently deposits three novel human structures per month. Achieving this target relies heavily on frequent visits to the Diamond Light Source, and the variety of crystal systems still demand customized data collection, diligent checks and careful planning of each experiment. Here, an overview is presented of the techniques and procedures that have been refined over the years and that are considered synchrotron best practice.
PMCID: PMC3689534  PMID: 23793157
data collection; data-collection strategy; structural genomics
4.  Functional and structural characterization of a thermostable acetyl esterase from Thermotoga maritima 
Proteins  2012;80(6):1545-1559.
TM0077 from Thermotoga maritima is a member of the carbohydrate esterase family 7 and is active on a variety of acetylated compounds, including cephalosporin C. TM0077 esterase activity is confined to short-chain acyl esters (C2-C3), and is optimal around 100°C and pH 7.5. The positional specificity of TM0077 was investigated using 4-nitrophenyl-β-D-xylopyranoside monoacetates as substrates in a β-xylosidase-coupled assay. TM0077 hydrolyzes acetate at positions 2, 3 and 4 with equal efficiency. No activity was detected on xylan or acetylated xylan, which implies that TM0077 is an acetyl esterase and not an acetyl xylan esterase as currently annotated. Selenomethionine-substituted and native structures of TM0077 were determined at 2.1 Å and 2.5 Å resolution, respectively, revealing a classic α/β-hydrolase fold. TM0077 assembles into a doughnut-shaped hexamer with small tunnels on either side leading to an inner cavity, which contains the six catalytic centers. Structures of TM0077 with covalently bound phenylmethylsulfonyl fluoride (PMSF) and paraoxon were determined to 2.4 Å and 2.1 Å, respectively, and confirmed that both inhibitors bind covalently to the catalytic serine (Ser188). Upon binding of inhibitor, the catalytic serine adopts an altered conformation, as observed in other esterase and lipases, and supports a previously proposed catalytic mechanism in which this Ser hydroxyl rotation prevents reversal of the reaction and allows access of a water molecule for completion of the reaction.
PMCID: PMC3348966  PMID: 22411095
Acetyl esterase; Thermotoga maritima; crystal structure; α/β hydrolase; inhibitor; serine rotation
5.  Crystal Structures of the Catalytic Domain of Human Soluble Guanylate Cyclase 
PLoS ONE  2013;8(3):e57644.
Soluble guanylate cyclase (sGC) catalyses the synthesis of cyclic GMP in response to nitric oxide. The enzyme is a heterodimer of homologous α and β subunits, each of which is composed of multiple domains. We present here crystal structures of a heterodimer of the catalytic domains of the α and β subunits, as well as an inactive homodimer of β subunits. This first structure of a metazoan, heteromeric cyclase provides several observations. First, the structures resemble known structures of adenylate cyclases and other guanylate cyclases in overall fold and in the arrangement of conserved active-site residues, which are contributed by both subunits at the interface. Second, the subunit interaction surface is promiscuous, allowing both homodimeric and heteromeric association; the preference of the full-length enzyme for heterodimer formation must derive from the combined contribution of other interaction interfaces. Third, the heterodimeric structure is in an inactive conformation, but can be superposed onto an active conformation of adenylate cyclase by a structural transition involving a 26° rigid-body rotation of the α subunit. In the modelled active conformation, most active site residues in the subunit interface are precisely aligned with those of adenylate cyclase. Finally, the modelled active conformation also reveals a cavity related to the active site by pseudo-symmetry. The pseudosymmetric site lacks key active site residues, but may bind allosteric regulators in a manner analogous to the binding of forskolin to adenylate cyclase. This indicates the possibility of developing a new class of small-molecule modulators of guanylate cyclase activity targeting the catalytic domain.
PMCID: PMC3591389  PMID: 23505436
6.  Structural Basis for ASPP2 Recognition by the Tumor Suppressor p73 
Journal of Molecular Biology  2012;423(4):515-527.
Tumor suppressors p53, p63 and p73 comprise a family of stress-responsive transcription factors with distinct functions in development and tumor suppression. Most human cancers lose p53 function, yet all three proteins are capable of inducing apoptosis or cellular senescence. Mechanisms are therefore under investigation to activate p73-dependent apoptosis in p53-deficient cancer cells. Significantly, the DNA-binding domain (DBD) of p73 escapes viral oncoproteins and displays an enhanced thermal stability. To further understand the variant features of p73, we solved the high‐resolution crystal structure of the p73 DBD as well as its complex with the ankyrin repeat and SH3 domains of the pro-apoptotic factor ASPP2. The p73 structure exhibits the same conserved architecture as p53 but displays a divergent L2 loop, a known site of protein–protein interaction. The loop in p73 is changed by a two-residue insertion that also induces repacking around the site of the p53 mutational hotspot R175. Importantly, the binding of ASPP2 is preserved by conformational changes in both the ankyrin repeat and SH3 domains. These results further highlight the structural variation that impacts p53 family interactions within the p53 interactome.
Graphical Abstract
► p53 and p73 have distinct functions in development and tumor suppression. ► The 1.8‐Å structure of the p73 DBD reveals a variant L2 loop. ► The p73–ASPP2 structure shows a compensatory shift in the ASPP2 binding interface. ► Drugs have the potential to activate p73-dependent apoptosis in p53-deficient cancers.
PMCID: PMC3472557  PMID: 22917970
DBD, DNA-binding domain; OD, oligomerization domain; TEV, tobacco etch virus; TCEP, tris(2-carboxyethyl)phosphine; PDB, Protein Data Bank; TLS, translation/liberation/screw; TP73; 53BP2; transactivation; DNA damage; mutant p53
7.  Structure of the Bone Morphogenetic Protein Receptor ALK2 and Implications for Fibrodysplasia Ossificans Progressiva* 
The Journal of Biological Chemistry  2012;287(44):36990-36998.
Background: Mutations in the ALK2 kinase cause extraskeletal bone formation.
Results: We solved the structure of ALK2 in complex with the inhibitor FKBP12.
Conclusion: Disease mutations break critical interactions that stabilize the inactive ALK2-FKBP12 complex leading to kinase activation.
Significance: We offer an explanation for the effects of mutation and a structural template for the design of small molecule inhibitors.
Bone morphogenetic protein (BMP) receptor kinases are tightly regulated to control development and tissue homeostasis. Mutant receptor kinase domains escape regulation leading to severely degenerative diseases and represent an important therapeutic target. Fibrodysplasia ossificans progressiva (FOP) is a rare but devastating disorder of extraskeletal bone formation. FOP-associated mutations in the BMP receptor ALK2 reduce binding of the inhibitor FKBP12 and promote leaky signaling in the absence of ligand. To establish structural mechanisms of receptor regulation and to address the effects of FOP mutation, we determined the crystal structure of the cytoplasmic domain of ALK2 in complex with the inhibitors FKBP12 and dorsomorphin. FOP mutations break critical interactions that stabilize the inactive state of the kinase, thereby facilitating structural rearrangements that diminish FKBP12 binding and promote the correct positioning of the glycine-serine-rich loop and αC helix for kinase activation. The balance of these effects accounts for the comparable activity of R206H and L196P. Kinase activation in the clinically benign mutant L196P is far weaker than R206H but yields equivalent signals due to the stronger interaction of FKBP12 with R206H. The presented ALK2 structure offers a valuable template for the further design of specific inhibitors of BMP signaling.
PMCID: PMC3481300  PMID: 22977237
Activin; Bone Morphogenetic Protein (BMP); Phosphorylation; Signaling; SMAD Transcription Factor; Transforming Growth Factor β (TGFbeta); ACVR1; FKBP1A; Kinase; Smad
8.  Structure of human aspartyl aminopeptidase complexed with substrate analogue: insight into catalytic mechanism, substrate specificity and M18 peptidase family 
Aspartyl aminopeptidase (DNPEP), with specificity towards an acidic amino acid at the N-terminus, is the only mammalian member among the poorly understood M18 peptidases. DNPEP has implicated roles in protein and peptide metabolism, as well as the renin-angiotensin system in blood pressure regulation. Despite previous enzyme and substrate characterization, structural details of DNPEP regarding ligand recognition and catalytic mechanism remain to be delineated.
The crystal structure of human DNPEP complexed with zinc and a substrate analogue aspartate-β-hydroxamate reveals a dodecameric machinery built by domain-swapped dimers, in agreement with electron microscopy data. A structural comparison with bacterial homologues identifies unifying catalytic features among the poorly understood M18 enzymes. The bound ligands in the active site also reveal the coordination mode of the binuclear zinc centre and a substrate specificity pocket for acidic amino acids.
The DNPEP structure provides a molecular framework to understand its catalysis that is mediated by active site loop swapping, a mechanism likely adopted in other M18 and M42 metallopeptidases that form dodecameric complexes as a self-compartmentalization strategy. Small differences in the substrate binding pocket such as shape and positive charges, the latter conferred by a basic lysine residue, further provide the key to distinguishing substrate preference. Together, the structural knowledge will aid in the development of enzyme-/family-specific aminopeptidase inhibitors.
PMCID: PMC3472314  PMID: 22720794
Aspartyl aminopeptidase; Dodecameric tetrahedron; M18 peptidase; Metalloprotease; Domain swapping
9.  Assessment of radiation damage behaviour in a large collection of empirically optimized datasets highlights the importance of unmeasured complicating effects 
Journal of Synchrotron Radiation  2011;18(Pt 3):387-397.
A retrospective analysis of radiation damage behaviour in a statistically significant number of real-life datasets is presented, in order to gauge the importance of the complications not yet measured or rigorously evaluated in current experiments, and the challenges that remain before radiation damage can be considered a problem solved in practice.
The radiation damage behaviour in 43 datasets of 34 different proteins collected over a year was examined, in order to gauge the reliability of decay metrics in practical situations, and to assess how these datasets, optimized only empirically for decay, would have benefited from the precise and automatic prediction of decay now possible with the programs RADDOSE [Murray, Garman & Ravelli (2004 ▶). J. Appl. Cryst. 37, 513–522] and BEST [Bourenkov & Popov (2010 ▶). Acta Cryst. D66, 409–419]. The results indicate that in routine practice the diffraction experiment is not yet characterized well enough to support such precise predictions, as these depend fundamentally on three interrelated variables which cannot yet be determined robustly and practically: the flux density distribution of the beam; the exact crystal volume; the sensitivity of the crystal to dose. The former two are not satisfactorily approximated from typical beamline information such as nominal beam size and transmission, or two-dimensional images of the beam and crystal; the discrepancies are particularly marked when using microfocus beams (<20 µm). Empirically monitoring decay with the dataset scaling B factor (Bourenkov & Popov, 2010 ▶) appears more robust but is complicated by anisotropic and/or low-resolution diffraction. These observations serve to delineate the challenges, scientific and logistic, that remain to be addressed if tools for managing radiation damage in practical data collection are to be conveniently robust enough to be useful in real time.
PMCID: PMC3083914  PMID: 21525647
radiation damage; data collection; strategy; beamline software; datasets
10.  Structure of the CaMKIIδ/Calmodulin Complex Reveals the Molecular Mechanism of CaMKII Kinase Activation 
PLoS Biology  2010;8(7):e1000426.
Structural and biophysical studies reveal how CaMKII kinases, which are important for cellular learning and memory, are switched on by binding of Ca2+/calmodulin.
Long-term potentiation (LTP), a long-lasting enhancement in communication between neurons, is considered to be the major cellular mechanism underlying learning and memory. LTP triggers high-frequency calcium pulses that result in the activation of Calcium/Calmodulin (CaM)-dependent kinase II (CaMKII). CaMKII acts as a molecular switch because it remains active for a long time after the return to basal calcium levels, which is a unique property required for CaMKII function. Here we describe the crystal structure of the human CaMKIIδ/Ca2+/CaM complex, structures of all four human CaMKII catalytic domains in their autoinhibited states, as well as structures of human CaMKII oligomerization domains in their tetradecameric and physiological dodecameric states. All four autoinhibited human CaMKIIs were monomeric in the determined crystal structures but associated weakly in solution. In the CaMKIIδ/Ca2+/CaM complex, the inhibitory region adopted an extended conformation and interacted with an adjacent catalytic domain positioning T287 into the active site of the interacting protomer. Comparisons with autoinhibited CaMKII structures showed that binding of calmodulin leads to the rearrangement of residues in the active site to a conformation suitable for ATP binding and to the closure of the binding groove for the autoinhibitory helix by helix αD. The structural data, together with biophysical interaction studies, reveals the mechanism of CaMKII activation by calmodulin and explains many of the unique regulatory properties of these two essential signaling molecules.
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Author Summary
CaMKII enzymes transmit calcium ion (Ca2+) signals released inside the cell by regulating signal transduction pathways through phosphorylation: Ca2+ first binds to the small regulatory protein CaM; this Ca2+/CaM complex then binds to and activates the kinase, which phosphorylates other proteins in the cell. Since CaMKs remain active long after rapid Ca2+ pulses have dropped they function as molecular switches that turn on or off crucial cell functions in response to Ca2+ levels. The multifunctional CaMKII forms of this enzyme – of which there are four in human – are important in many processes including signaling in neurons and controlling of the heart rate. They are particularly abundant in the brain where they probably play a role in memory. CaMKII forms an exceptionally large, dodecameric complex. Here, we describe the crystal structure of this complex for each of the four human CaMKII catalytic domains in their autoinhibited states, a complex of CaMKII with Ca2+/CaM, as well as the structure of the oligomerization domain (the part of the protein that mediates complex formation) in its physiological dodecameric state and in a tetradecameric state. Detailed comparison of this large body of structural data together with biophysical studies has allowed us to better understand the structural mechanisms of CaMKII activation by CaM and to explain many of the complex regulatory features of these essential enzymes.
PMCID: PMC2910593  PMID: 20668654
11.  Crystal Structure of the PIM2 Kinase in Complex with an Organoruthenium Inhibitor 
PLoS ONE  2009;4(10):e7112.
The serine/threonine kinase PIM2 is highly expressed in human leukemia and lymphomas and has been shown to positively regulate survival and proliferation of tumor cells. Its diverse ATP site makes PIM2 a promising target for the development of anticancer agents. To date our knowledge of catalytic domain structures of the PIM kinase family is limited to PIM1 which has been extensively studied and which shares about 50% sequence identity with PIM2.
Principal Findings
Here we determined the crystal structure of PIM2 in complex with an organoruthenium complex (inhibition in sub-nanomolar level). Due to its extraordinary shape complementarity this stable organometallic compound is a highly potent inhibitor of PIM kinases.
The structure of PIM2 revealed several differences to PIM1 which may be explored further to generate isoform selective inhibitors. It has also demonstrated how an organometallic inhibitor can be adapted to the binding site of protein kinases to generate highly potent inhibitors.
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PMCID: PMC2743286  PMID: 19841674
12.  The scientific impact of the Structural Genomics Consortium: a protein family and ligand-centered approach to medically-relevant human proteins 
As many of the structural genomics centers have ended their first phase of operation, it is a good point to evaluate the scientific impact of this endeavour. The Structural Genomics Consortium (SGC), operating from three centers across the Atlantic, investigates human proteins involved in disease processes and proteins from Plasmodium falciparum and related organisms. We present here some of the scientific output of the Oxford node of the SGC, where the target areas include protein kinases, phosphatases, oxidoreductases and other metabolic enzymes, as well as signal transduction proteins. The SGC has aimed to achieve extensive coverage of human gene families with a focus on protein–ligand interactions. The methods employed for effective protein expression, crystallization and structure determination by X-ray crystallography are summarized. In addition to the cumulative impact of accelerated delivery of protein structures, we demonstrate how family coverage, generic screening methodology, and the availability of abundant purified protein samples, allow a level of discovery that is difficult to achieve otherwise. The contribution of NMR to structure determination and protein characterization is discussed. To make this information available to a wide scientific audience, a new tool for disseminating annotated structural information was created that also represents an interactive platform allowing for a continuous update of the annotation by the scientific community.
PMCID: PMC2140095  PMID: 17932789
High-throughput; Protein kinase; Dehydrogenase; Reductase; PDZ; 14-3-3; Binding specificity; Protein crystallography
13.  Molecular replacement 
PMCID: PMC2394778
preface; molecular replacement; CCP4 study weekend 2007
14.  The Crystal Structure of Shikimate Dehydrogenase (AroE) Reveals a Unique NADPH Binding Mode 
Journal of Bacteriology  2003;185(14):4144-4151.
Shikimate dehydrogenase catalyzes the NADPH-dependent reversible reduction of 3-dehydroshikimate to shikimate. We report the first X-ray structure of shikimate dehydrogenase from Haemophilus influenzae to 2.4-Å resolution and its complex with NADPH to 1.95-Å resolution. The molecule contains two domains, a catalytic domain with a novel open twisted α/β motif and an NADPH binding domain with a typical Rossmann fold. The enzyme contains a unique glycine-rich P-loop with a conserved sequence motif, GAGGXX, that results in NADPH adopting a nonstandard binding mode with the nicotinamide and ribose moieties disordered in the binary complex. A deep pocket with a narrow entrance between the two domains, containing strictly conserved residues primarily contributed by the catalytic domain, is identified as a potential 3-dehydroshikimate binding pocket. The flexibility of the nicotinamide mononucleotide portion of NADPH may be necessary for the substrate 3-dehydroshikimate to enter the pocket and for the release of the product shikimate.
PMCID: PMC164887  PMID: 12837789
15.  Crystal Structures of ABL-Related Gene (ABL2) in Complex with Imatinib, Tozasertib (VX-680), and a Type I Inhibitor of the Triazole Carbothioamide Class† 
Journal of Medicinal Chemistry  2011;54(7):2359-2367.
ABL2 (also known as ARG (ABL related gene)) is closely related to the well-studied Abelson kinase cABL. ABL2 is involved in human neoplastic diseases and is deregulated in solid tumors. Oncogenic gene translocations occur in acute leukemia. So far no structural information for ABL2 has been reported. To elucidate structural determinants for inhibitor interaction, we determined the cocrystal structure of ABL2 with the oncology drug imatinib. Interestingly, imatinib not only interacted with the ATP binding site of the inactive kinase but was also bound to the regulatory myristate binding site. This structure may therefore serve as a tool for the development of allosteric ABL inhibitors. In addition, we determined the structures of ABL2 in complex with VX-680 and with an ATP-mimetic type I inhibitor, which revealed an interesting position of the DFG motif intermediate between active and inactive conformations, that may also serve as a template for future inhibitor design.
PMCID: PMC3075623  PMID: 21417343
16.  Targeting Low-Druggability Bromodomains: Fragment Based Screening and Inhibitor Design against the BAZ2B Bromodomain 
Journal of Medicinal Chemistry  2013;56(24):10183-10187.
Bromodomains are epigenetic reader domains that have recently become popular targets. In contrast to BET bromodomains, which have proven druggable, bromodomains from other regions of the phylogenetic tree have shallower pockets. We describe successful targeting of the challenging BAZ2B bromodomain using biophysical fragment screening and structure-based optimization of high ligand-efficiency fragments into a novel series of low-micromolar inhibitors. Our results provide attractive leads for development of BAZ2B chemical probes and indicate the whole family may be tractable.
PMCID: PMC3905694  PMID: 24304323
17.  Crystal Structures of Malonyl-Coenzyme A Decarboxylase Provide Insights into Its Catalytic Mechanism and Disease-Causing Mutations 
Structure(London, England:1993)  2013;21(7):1182-1192.
Malonyl-coenzyme A decarboxylase (MCD) is found from bacteria to humans, has important roles in regulating fatty acid metabolism and food intake, and is an attractive target for drug discovery. We report here four crystal structures of MCD from human, Rhodopseudomonas palustris, Agrobacterium vitis, and Cupriavidus metallidurans at up to 2.3 Å resolution. The MCD monomer contains an N-terminal helical domain involved in oligomerization and a C-terminal catalytic domain. The four structures exhibit substantial differences in the organization of the helical domains and, consequently, the oligomeric states and intersubunit interfaces. Unexpectedly, the MCD catalytic domain is structurally homologous to those of the GCN5-related N-acetyltransferase superfamily, especially the curacin A polyketide synthase catalytic module, with a conserved His-Ser/Thr dyad important for catalysis. Our structures, along with mutagenesis and kinetic studies, provide a molecular basis for understanding pathogenic mutations and catalysis, as well as a template for structure-based drug design.
•Structures of human and bacterial MCDs were determined at up to 2.3 Å resolution•Distinct tetrameric and dimeric MCD oligomerizations were observed•Unexpected homology to the GNAT superfamily gives insights into catalytic mechanism•The structures provide the molecular basis for the disease-causing mutations in MCD
Malonyl-CoA decarboxylase (MCD) is important in fatty acid metabolism. Froese et al. report structures of several MCDs and show that the MCD catalytic domain shares structural homology with GNAT superfamily. The structures further our understanding of catalysis, pathogenic mutations, and drug design.
PMCID: PMC3701320  PMID: 23791943
18.  Structural Basis for Cul3 Protein Assembly with the BTB-Kelch Family of E3 Ubiquitin Ligases* 
The Journal of Biological Chemistry  2013;288(11):7803-7814.
Background: BTB-Kelch proteins, including KLHL11, are proposed to bind Cul3 through a “3-box” motif to form E3 ubiquitin ligases.
Results: We solved crystal structures of the KLHL11-Cul3 complex and four Kelch domains.
Conclusion: The 3-box forms a hydrophobic groove that binds a specific N-terminal extension of Cul3.
Significance: Dimeric BTB-Kelch proteins bind two Cul3 molecules and support a two-site model for substrate recognition.
Cullin-RING ligases are multisubunit E3 ubiquitin ligases that recruit substrate-specific adaptors to catalyze protein ubiquitylation. Cul3-based Cullin-RING ligases are uniquely associated with BTB adaptors that incorporate homodimerization, Cul3 assembly, and substrate recognition into a single multidomain protein, of which the best known are BTB-BACK-Kelch domain proteins, including KEAP1. Cul3 assembly requires a BTB protein “3-box” motif, analogous to the F-box and SOCS box motifs of other Cullin-based E3s. To define the molecular basis for this assembly and the overall architecture of the E3, we determined the crystal structures of the BTB-BACK domains of KLHL11 both alone and in complex with Cul3, along with the Kelch domain structures of KLHL2 (Mayven), KLHL7, KLHL12, and KBTBD5. We show that Cul3 interaction is dependent on a unique N-terminal extension sequence that packs against the 3-box in a hydrophobic groove centrally located between the BTB and BACK domains. Deletion of this N-terminal region results in a 30-fold loss in affinity. The presented data offer a model for the quaternary assembly of this E3 class that supports the bivalent capture of Nrf2 and reveals potential new sites for E3 inhibitor design.
PMCID: PMC3597819  PMID: 23349464
Proteasome; Protein-Protein Interactions; Signaling; Ubiquitination; X-ray Crystallography; β-Propeller; Degradation
19.  On the need for an international effort to capture, share and use crystallization screening data 
Development of an ontology for the description of crystallization experiments and results is proposed.
When crystallization screening is conducted many outcomes are observed but typically the only trial recorded in the literature is the condition that yielded the crystal(s) used for subsequent diffraction studies. The initial hit that was optimized and the results of all the other trials are lost. These missing results contain information that would be useful for an improved general understanding of crystallization. This paper provides a report of a crystallization data exchange (XDX) workshop organized by several international large-scale crystallization screening laboratories to discuss how this information may be captured and utilized. A group that administers a significant fraction of the world’s crystallization screening results was convened, together with chemical and structural data informaticians and computational scientists who specialize in creating and analysing large disparate data sets. The development of a crystallization ontology for the crystallization community was proposed. This paper (by the attendees of the workshop) provides the thoughts and rationale leading to this conclusion. This is brought to the attention of the wider audience of crystallographers so that they are aware of these early efforts and can contribute to the process going forward.
PMCID: PMC3310524  PMID: 22442216
crystallization screening data; crystallization ontology
20.  The Cryptosporidium parvum Kinome 
BMC Genomics  2011;12:478.
Hundreds of millions of people are infected with cryptosporidiosis annually, with immunocompromised individuals suffering debilitating symptoms and children in socioeconomically challenged regions at risk of repeated infections. There is currently no effective drug available. In order to facilitate the pursuit of anti-cryptosporidiosis targets and compounds, our study spans the classification of the Cryptosporidium parvum kinome and the structural and biochemical characterization of representatives from the CDPK family and a MAP kinase.
The C. parvum kinome comprises over 70 members, some of which may be promising drug targets. These C. parvum protein kinases include members in the AGC, Atypical, CaMK, CK1, CMGC, and TKL groups; however, almost 35% could only be classified as OPK (other protein kinases). In addition, about 25% of the kinases identified did not have any known orthologues outside of Cryptosporidium spp. Comparison of specific kinases with their Plasmodium falciparum and Toxoplasma gondii orthologues revealed some distinct characteristics within the C. parvum kinome, including potential targets and opportunities for drug design. Structural and biochemical analysis of 4 representatives of the CaMK group and a MAP kinase confirms features that may be exploited in inhibitor design. Indeed, screening CpCDPK1 against a library of kinase inhibitors yielded a set of the pyrazolopyrimidine derivatives (PP1-derivatives) with IC50 values of < 10 nM. The binding of a PP1-derivative is further described by an inhibitor-bound crystal structure of CpCDPK1. In addition, structural analysis of CpCDPK4 identified an unprecedented Zn-finger within the CDPK kinase domain that may have implications for its regulation.
Identification and comparison of the C. parvum protein kinases against other parasitic kinases shows how orthologue- and family-based research can be used to facilitate characterization of promising drug targets and the search for new drugs.
PMCID: PMC3227725  PMID: 21962082

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