Structural and functional insights into a dodecameric molecular machine - the RuvBL1/RuvBL2 complex.

RuvBL1 (RuvB-like 1) and its homolog RuvBL2 are evolutionarily highly conserved AAA(+) ATPases essential for many cellular activities. They play an important role in chromatin remodeling, transcriptional regulation and DNA damage repair. RuvBL1 and RuvBL2 are overexpressed in different types of cancer and interact with major oncogenic factors, such as ß-catenin and c-Myc regulating their function. We solved the first three-dimensional crystal structure of the human RuvBL complex with a truncated domain II and show that this complex is competent for helicase activity. The structure reveals a dodecamer consisting of two heterohexameric rings with alternating RuvBL1 and RuvBL2 monomers bound to ADP/ATP, that interact with each other via the retained part of domain II. The dodecameric quaternary structure of the R1ΔDII/R2ΔDII complex observed in the crystal structure was confirmed by small-angle X-ray scattering analysis. Interestingly, truncation of domain II led to a substantial increase in ATP consumption of RuvBL1, RuvBL2 and their complex. In addition, we present evidence that DNA unwinding of the human RuvBL proteins can be auto-inhibited by domain II, which is not present in the homologous bacterial helicase RuvB. Our data give new insights into the molecular arrangement of RuvBL1 and RuvBL2 and strongly suggest that in vivo activities of these highly interesting therapeutic drug targets are regulated by cofactors inducing conformational changes via domain II in order to modulate the enzyme complex into its active state.

Off-target decoding of a multitarget kinase inhibitor by chemical proteomics.

Unbiased: Chemical proteomics was used to profile compound interactions in an unbiased fashion. We present here the application of different compound-immobilization routes for decoding nonprotein kinase off-targets of the multitarget kinase inhibitor C1, which interacts with distinct compound moieties. Since the approval of the first selective tyrosine kinase inhibitor, imatinib, various drugs have been developed to target protein kinases. However, due to a high degree of structural conservation of the ATP binding site, off-target effects have been reported for several drugs. Here, we report on off-target decoding for a multitarget protein kinase inhibitor by chemical proteomics, by focusing on interactions with nonprotein kinases. We tested two different routes for the immobilization of the inhibitor on a carrier matrix, and thus identified off-targets that interact with distinct compound moieties. Besides several of the kinases known to bind to the compound, the pyridoxal kinase (PDXK), which has been described to interact with the CDK inhibitor (R)-roscovitine, was captured. The PDXK-inhibitor interaction was shown to occur at the substrate binding site rather than at the ATP binding site. In addition, carbonic anhydrase 2 (CA2) binding was demonstrated, and the determination of the IC(50) revealed an enzyme inhibition in the submicromolar range. The data demonstrate that different compound immobilization routes for chemical proteomics approaches are a valuable method to improve the knowledge about the off-target profile of a compound.

Cloning, expression, purification, crystallization and preliminary X-ray analysis of the human RuvBL1-RuvBL2 complex.

The complex of RuvBL1 and its homologue RuvBL2, two evolutionarily highly conserved eukaryotic proteins belonging to the AAA(+) (ATPase associated with diverse cellular activities) family of ATPases, was co-expressed in Escherichia coli. For crystallization purposes, the flexible domains II of RuvBL1 and RuvBL2 were truncated. The truncated RuvBL1-RuvBL2 complex was crystallized using the hanging-drop vapour-diffusion method at 293 K. The crystals were hexagonal-shaped plates and belonged to either the orthorhombic space group C222(1), with unit-cell parameters a = 111.4, b = 188.0, c = 243.4 A and six monomers in the asymmetric unit, or the monoclinic space group P2(1), with unit-cell parameters a = 109.2, b = 243.4, c = 109.3 A, beta = 118.7 degrees and 12 monomers in the asymmetric unit. The crystal structure could be solved by molecular replacement in both possible space groups and the solutions obtained showed that the complex forms a dodecamer.

Structure of wild-type Plk-1 kinase domain in complex with a selective DARPin.

As a key regulator of mitosis, the Ser/Thr protein polo-like kinase-1 (Plk-1) is a well validated drug target in cancer therapy. In order to enable structure-guided drug design, determination of the crystal structure of the kinase domain of Plk-1 was attempted. Using a multi-parallel cloning and expression approach, a set of length variants were identified which could be expressed in large amounts from insect cells and which could be purified to high purity. However, all attempts to crystallize these constructs failed. Crystals were ultimately obtained by generating designed ankyrin-repeat proteins (DARPins) selective for Plk-1 and using them for cocrystallization. Here, the first crystal structure of the kinase domain of wild-type apo Plk-1, in complex with DARPin 3H10, is presented, underlining the power of selective DARPins as crystallization tools. The structure was refined to 2.3 A resolution and shows the active conformation of Plk-1. It broadens the basis for modelling and cocrystallization studies for drug design. The binding epitope of 3H10 is rich in arginine, glutamine and lysine residues, suggesting that the DARPin enabled crystallization by masking a surface patch which is unfavourable for crystal contact formation. Based on the packing observed in the crystal, a truncated DARPin variant was designed which showed improved binding characteristics.

Identifying protein construct variants with increased crystallization propensity--a case study.

This study describes an efficient multiparallel automated workflow of cloning, expression, purification, and crystallization of a large set of construct variants for isolated protein domains aimed at structure determination by X-ray crystallography. This methodology is applied to MAPKAP kinase 2, a key enzyme in the inflammation pathway and thus an attractive drug target. The study reveals a distinct subset of truncation variants with improved crystallization properties. These constructs distinguish themselves by increased solubility and stability during a parallel automated multistep purification process including removal of the recombinant tag. High-throughput protein melting point analysis characterizes this subset of constructs as particularly thermostable. Both parallel purification screening and melting point determination clearly identify residue 364 as the optimal C terminus for the kinase domain. Moreover, all three constructs that ultimately crystallized feature this C terminus. At the N terminus, only three amino acids differentiate a noncrystallizing from a crystallizing construct. This study addresses the very common issues associated with difficult to crystallize proteins, those of solubility and stability, and the crucial importance of particular residues in the formation of crystal contacts. A methodology is suggested that includes biophysical measurements to efficiently identify and produce construct variants of isolated protein domains which exhibit higher crystallization propensity.

Crystal structure of the human AAA+ protein RuvBL1.

RuvBL1 is an evolutionarily highly conserved eukaryotic protein belonging to the AAA(+)-family of ATPases (ATPase associated with diverse cellular activities). It plays important roles in essential signaling pathways such as the c-Myc and Wnt pathways in chromatin remodeling, transcriptional and developmental regulation, and DNA repair and apoptosis. Herein we present the three-dimensional structure of the selenomethionine variant of human RuvBL1 refined using diffraction data to 2.2A of resolution. The crystal structure of the hexamer is formed of ADP-bound RuvBL1 monomers. The monomers contain three domains, of which the first and the third are involved in ATP binding and hydrolysis. Although it has been shown that ATPase activity of RuvBL1 is needed for several in vivo functions, we could only detect a marginal activity with the purified protein. Structural homology and DNA binding studies demonstrate that the second domain, which is unique among AAA(+) proteins and not present in the bacterial homolog RuvB, is a novel DNA/RNA-binding domain. We were able to demonstrate that RuvBL1 interacted with single-stranded DNA/RNA and double-stranded DNA. The structure of the RuvBL1.ADP complex, combined with our biochemical results, suggest that although RuvBL1 has all the structural characteristics of a molecular motor, even of an ATP-driven helicase, one or more as yet undetermined cofactors are needed for its enzymatic activity.

Expression, purification, crystallization and preliminary X-ray analysis of the human RuvB-like protein RuvBL1.

RuvBL1, an evolutionary highly conserved protein related to the AAA+ family of ATPases, has been crystallized using the hanging-drop vapour-diffusion method at 293 K. The crystals are hexagonal and belong to space group P6, with unit-cell parameters a = b = 207.1, c = 60.7 A and three molecules in the asymmetric unit.

LRRK1 protein kinase activity is stimulated upon binding of GTP to its Roc domain.

Human leucine-rich repeat kinase 1 (LRRK1) is a multi-domain protein of unknown function belonging to the ROCO family of complex proteins. Here, we report the molecular characterization of human LRRK1 and show, for the first time, that LRRK1 is both a functional protein kinase and a GDP/GTP-binding protein. Binding of GTP to LRRK1 is specific, requires the GTPase-like Roc domain, and leads to a stimulation of LRRK1 kinase activity. LRRK1 is the first example of a GTP-regulated protein kinase harboring both the kinase effector domain and the GTP-binding regulatory domain. Hence, we propose a model in which LRRK1 cycles between a GTP-bound active and a GDP-bound inactive state. Moreover, we mutated LRRK1 to mimic mutations previously identified in LRRK2/dardarin, the only human paralogue of LRRK1, that have been linked to autosomal-dominant parkinsonism. We demonstrate that three of four mutations analyzed significantly downregulate LRRK1 kinase activity. Ultimately, the results presented for LRRK1 may contribute to the elucidation of LRRK2's role in the pathogenesis of Parkinson's disease.

Domain-specific characteristics of the bifunctional key enzyme of sialic acid biosynthesis, UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase.

UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase is a bifunctional enzyme, which initiates and regulates sialic acid biosynthesis. Sialic acids are important compounds of mammalian glycoconjugates, mediating several biological processes, such as cell-cell or cell-matrix interactions. In order to characterize the function of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, a number of deletion mutants were generated, lacking either parts of the N-terminal epimerase or the C-terminal kinase domain. N-terminal deletion of only 39 amino acids results in a complete loss of epimerase activity. Deletions in the C-terminal part result in a reduction or complete loss of kinase activity, depending on the size of the deletion. Deletions at either the N- or the C-terminus also result in a reduction of the other enzyme activity. These results indicate that a separate expression of both domains is possible, but that a strong intramolecular dependency of the two domains has arisen during evolution of the enzyme. N-terminal, as well as C-terminal, mutants tend to form trimers, in addition to the hexameric structure of the native enzyme. These results and yeast two-hybrid experiments show that structures required for dimerization are localized within the kinase domain, and a potential trimerization site is possibly located in a region between the two domains. In conclusion, our results reveal that the activities, as well as the oligomeric structure, of this bifunctional enzyme seem to be organized and regulated in a complex manner.

UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, functionally expressed in and purified from Escherichia coli, yeast, and insect cells.

UDP-GlcNAc 2-epimerase/ManNAc kinase is the key enzyme of sialic acid biosynthesis in mammals. Its functional expression is a prerequisite for early embryogenesis and for the synthesis of several cell recognition motifs in adult organism. This bifunctional enzyme is involved in the development of different diseases like sialuria or hereditary inclusion body myopathy. For a detailed understanding of the enzyme, large amounts of the pure active protein are needed. Different heterologous cell systems were therefore analyzed for the enzyme, which was found to be functionally expressed in Escherichia coli, the yeast strains Saccharomyces cerevisiae and Pichia pastoris, and insect cells. In all these cell types, the expressed enzyme displayed both epimerase and kinase activities. In E. coli, up to 2mg protein/l cell culture was expressed, in yeast cells only 0.4mg/L, while up to 100mg/L, were detected in insect cells. In all three cell systems, insoluble protein aggregates were also observed. Purification from E. coli resulted in 100microg/L pure and structurally intact protein. For insect cells, purification methods were established which resulted in up to 50mg/L pure, soluble, and active protein. In summary, expression and purification of the UDP-GlcNAc 2-epimerase/ManNAc kinase in Sf-900 cells can yield the milligram amounts of protein required for structural characterization of the enzyme. However, the easier expression in E. coli and yeast provides sufficient quantities for enzymatic and kinetic characterization.

Comparison of proteomic and genomic analyses of the human breast cancer cell line T47D and the antiestrogen-resistant derivative T47D-r.

In search of novel mechanisms leading to the development of antiestrogen-resistance in human breast tumors, we analyzed differences in the gene and protein expression pattern of the human breast carcinoma cell line T47D and its derivative T47D-r, which is resistant toward the pure antiestrogen ZM 182780 (Faslodex trade mark, fulvestrant). Affymetrix DNA chip hybridizations on the commercially available HuGeneFL and Hu95A arrays were carried out in parallel to the proteomics analysis where the total cellular protein content of T47D or T47D-r was separated on two-dimensional gels. Thirty-eight proteins were found to be reproducibly up- or down-regulated more than 2-fold in T47D-r versus T47D in the proteomics analysis. Comparison with differential mRNA analysis revealed that 19 of these were up- or down-regulated in parallel with the corresponding mRNA molecules, among which are the protease cathepsin D, the GTPases Rab11a and MxA, and the secreted protein hAG-2. For 11 proteins, the corresponding mRNA was not found to be differentially expressed, and for eight proteins an inverse regulation was found at the mRNA level. In summary, mRNA expression data, when combined with proteomic information, provide a more detailed picture of how breast cancer cells are altered in their antiestrogen-resistant compared with the antiestrogen-sensitive state.

Keratinocyte unresponsiveness towards interleukin-10: lack of specific binding due to deficient IL-10 receptor 1 expression.

Whereas the effects of interleukin (IL)-10 on several epithelial cell types are well established the capability of IL-10 to target keratinocytes (KC) is still a matter of debate. This, however, is of considerable importance, as IL-10 is a major anti-inflammatory, immunosuppressive cytokine with impact on the cutaneous homeostasis. Recently, IL-10 therapy has been proven to be clinically effective in psoriasis. Response to therapy is associated with normalization of typical parameters of keratinocyte pathology, but it is unclear whether this results from direct or indirect (secondary) effects. The purpose of the present study was to further investigate direct effects of IL-10 on keratinocytes and to address the reason for potential IL-10 unresponsiveness using keratinocytes such as the cell line HaCaT as well as primary foreskin keratinocytes. Using real time RT-PCR we demonstrated that IL-10 is neither able to induce its typical early gene product suppressor of cytokine signalling (SOCS) 3 nor to modulate the interferone (IFN)-gamma-induced expression of SOCS 1 and 3. Although flow cytometric analyses showed binding of biotin labelled IL-10 to HaCaT cells, blocking experiments indicated that this resulted from unspecific binding. Moreover, scatchard plot analyses excluded specific binding to primary KC and HaCaT cells. Finally, real time mRNA analyses and Western blot experiments demonstrated that the absence of any specific binding results from the absence of clear IL-10R1 (alpha chain) expression, whereas the IL-10R2 (beta chain) is strongly expressed. Our data indicates that IL-10 unresponsiveness of keratinocytes could be explained by the lacking of functional IL-10 receptor expression and suggest that any IL-10 effects on these cells observed are indirectly mediated.

A tumor-specific kinase activity regulates the viral death protein Apoptin.

Apoptin, a chicken anemia virus-encoded protein, is thought to be activated by a general tumor-specific pathway, because it induces apoptosis in a large number of human tumor or transformed cells but not in their normal, healthy counterparts. Here, we show that Apoptin is phosphorylated robustly both in vitro and in vivo in tumor cells but negligibly in normal cells, and we map the site to threonine 108. A gain-of-function point mutation (T108E) conferred upon Apoptin the ability to accumulate in the nucleus and kill normal cells, implying that phosphorylation is a key regulator of the tumor-specific properties of Apoptin. An activity that could phosphorylate Apoptin on threonine 108 was found specifically in tumor and transformed cells from a variety of tissue origins, suggesting that activation of this kinase is generally associated with the cancerous or pre-cancerous state. Moreover, analyses of human tissue samples confirm that Apoptin kinase activity is detectable in primary malignancies but not in tissue derived from healthy individuals. Taken together, our results support a model whereby the dysregulation of the cellular pathway leading to the phosphorylation of Apoptin contributes to human tumorigenesis.

Structural basis for the glucocorticoid response in a mutant human androgen receptor (AR(ccr)) derived from an androgen-independent prostate cancer.

The crystal structure of a mutant androgen receptor (AR) ligand-binding domain (LBD) in complex with the agonist 9alpha-fluorocortisol has been determined at 1.95 A resolution. This mutant AR contains two mutations (L701H and T877A) and was previously reported as a high-affinity cortisol/cortisone responsive AR (AR(ccr)) isolated from the androgen-independent human prostate cancer cell lines MDA PCa 2a and 2b (Zhao et al. Nature Med. 2000, 6, 703-6). The three-dimensional structure of the AR(ccr) LBD complexed with 9alpha-fluorocortisol shows the typical conformation of an agonist-bound nuclear receptor in which helix 12 is precisely positioned as a "lid" for the ligand-binding pocket. Binding of 9alpha-fluorocortisol to the AR(ccr) involves favorable hydrogen bond patterns on the C17 and C21 substituents of the ligand due to the mutations at 701 and 877 in the AR(ccr). Our studies provide the first structural explanation for the glucocorticoid activation of AR(ccr), which is important for the development of new therapeutic treatments for androgen-independent prostate cancer.