Combining structural and coevolution information to unveil allosteric sites.

Understanding allosteric regulation in biomolecules is of great interest to pharmaceutical research and computational methods emerged during the last decades to characterize allosteric coupling. However, the prediction of allosteric sites in a protein structure remains a challenging task. Here, we integrate local binding site information, coevolutionary information, and information on dynamic allostery into a structure-based three-parameter model to identify potentially hidden allosteric sites in ensembles of protein structures with orthosteric ligands. When tested on five allosteric proteins (LFA-1, p38-α, GR, MAT2A, and BCKDK), the model successfully ranked all known allosteric pockets in the top three positions. Finally, we identified a novel druggable site in MAT2A confirmed by X-ray crystallography and SPR and a hitherto unknown druggable allosteric site in BCKDK validated by biochemical and X-ray crystallography analyses. Our model can be applied in drug discovery to identify allosteric pockets.

Discovery of USP7 small-molecule allosteric inhibitors.

Ubiquitin specific protease-7 (USP7) is considered an attractive target for cancer therapy by promoting degradation of the tumor suppressor p53 and negatively affecting the immune response to tumors. However, the development of selective non-covalent USP7 inhibitors has proven challenging. In this work we report the NMR characterization of a weak binder from SPR screening of an in-house fragment library which reveals that it binds to the allosteric palm site of the catalytic domain. Molecular modeling combined with 1HNMR saturation transfer difference and NOESY experiments enabled structure-based design of additional compounds showing IC50 values in the low-micromolar range with good selectivity over the closest homolog USP47. The most potent analogue represents a promising starting point for the development of novel, selective USP7 inhibitors.

Selective inhibitors of the protein tyrosine phosphatase SHP2 block cellular motility and growth of cancer cells in†vitro and in†vivo.

Selective inhibitors of the protein tyrosine phosphatase SHP2 (src homology region 2 domain phosphatase; PTPN11), an enzyme that is deregulated in numerous human tumors, were generated through a combination of chemical synthesis and structure-based rational design. Seventy pyridazolon-4-ylidenehydrazinyl benzenesulfonates were prepared and evaluated in enzyme assays. The binding modes of active inhibitors were simulated in silico using a newly generated crystal structure of SHP2. The most powerful compound, GS-493 (4-{(2Z)-2-[1,3-bis(4-nitrophenyl)-5-oxo-1,5-dihydro-4H-pyrazol-4-yliden]hydrazino}benzenesulfonic acid; 25) inhibited SHP2 with an IC50 value of 71±15 nM in the enzyme assay and was 29- and 45-fold more active toward SHP2 than against related SHP1 and PTP1B. In cell culture experiments compound 25 was found to block hepatocyte growth factor (HGF)-stimulated epithelial-mesenchymal transition of human pancreatic adenocarcinoma (HPAF) cells, as indicated by a decrease in the minimum neighbor distances of cells. Moreover, 25 inhibited cell colony formation in the non-small-cell lung cancer cell line LXFA 526L in soft agar. Finally, 25 was observed to inhibit tumor growth in a murine xenograft model. Therefore, the novel specific compound 25 strengthens the hypothesis that SHP2 is a relevant protein target for the inhibition of mobility and invasiveness of cancer cells.

Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide.

In the 1950s, the drug thalidomide, administered as a sedative to pregnant women, led to the birth of thousands of children with multiple defects. Despite the teratogenicity of thalidomide and its derivatives lenalidomide and pomalidomide, these immunomodulatory drugs (IMiDs) recently emerged as effective treatments for multiple myeloma and 5q-deletion-associated dysplasia. IMiDs target the E3 ubiquitin ligase CUL4-RBX1-DDB1-CRBN (known as CRL4(CRBN)) and promote the ubiquitination of the IKAROS family transcription factors IKZF1 and IKZF3 by CRL4(CRBN). Here we present crystal structures of the DDB1-CRBN complex bound to thalidomide, lenalidomide and pomalidomide. The structure establishes that CRBN is a substrate receptor within CRL4(CRBN) and enantioselectively binds IMiDs. Using an unbiased screen, we identified the homeobox transcription factor MEIS2 as an endogenous substrate of CRL4(CRBN). Our studies suggest that IMiDs block endogenous substrates (MEIS2) from binding to CRL4(CRBN) while the ligase complex is recruiting IKZF1 or IKZF3 for degradation. This dual activity implies that small molecules can modulate an E3 ubiquitin ligase and thereby upregulate or downregulate the ubiquitination of proteins.

The molecular basis of CRL4DDB2/CSA ubiquitin ligase architecture, targeting, and activation.

The DDB1-CUL4-RBX1 (CRL4) ubiquitin ligase family regulates a diverse set of cellular pathways through dedicated substrate receptors (DCAFs). The DCAF DDB2 detects UV-induced pyrimidine dimers in the genome and facilitates nucleotide excision repair. We provide the molecular basis for DDB2 receptor-mediated cyclobutane pyrimidine dimer recognition in chromatin. The structures of the fully assembled DDB1-DDB2-CUL4A/B-RBX1 (CRL4(DDB2)) ligases reveal that the mobility of the ligase arm creates a defined ubiquitination zone around the damage, which precludes direct ligase activation by DNA lesions. Instead, the COP9 signalosome (CSN) mediates the CRL4(DDB2) inhibition in a CSN5 independent, nonenzymatic, fashion. In turn, CSN inhibition is relieved upon DNA damage binding to the DDB2 module within CSN-CRL4(DDB2). The Cockayne syndrome A DCAF complex crystal structure shows that CRL4(DCAF(WD40)) ligases share common architectural features. Our data support a general mechanism of ligase activation, which is induced by CSN displacement from CRL4(DCAF) on substrate binding to the DCAF.

Detecting UV-lesions in the genome: The modular CRL4 ubiquitin ligase does it best!

The DDB1-DDB2-CUL4-RBX1 complex serves as the primary detection device for UV-induced lesions in the genome. It simultaneously functions as a CUL4 type E3 ubiquitin ligase. We review the current understanding of this dual function ubiquitin ligase and damage detection complex. The DDB2 damage binding module is merely one of a large family of possible DDB1-CUL4 associated factors (DCAF), most of which are substrate receptors for other DDB1-CUL4 complexes. DDB2 and the Cockayne-syndrome A protein (CSA) function in nucleotide excision repair, whereas the remaining receptors operate in a wide range of other biological pathways. We will examine the modular architecture of DDB1-CUL4 in complex with DDB2, CSA and CDT2 focusing on shared architectural, targeting and regulatory principles.

Crystal structure of Klebsiella sp. ASR1 phytase suggests substrate binding to a preformed active site that meets the requirements of a plant rhizosphere enzyme.

The extracellular phytase of the plant-associated Klebsiella sp. ASR1 is a member of the histidine-acid-phosphatase family and acts primarily as a scavenger of phosphate groups locked in the phytic acid molecule. The Klebsiella enzyme is distinguished from the Escherichia coli phytase AppA by its sequence and phytate degradation pathway. The crystal structure of the phytase from Klebsiella sp. ASR1 has been determined to 1.7 A resolution using single-wavelength anomalous-diffraction phasing. Despite low sequence similarity, the overall structure of Klebsiella phytase bears similarity to other histidine-acid phosphatases, such as E. coli phytase, glucose-1-phosphatase and human prostatic-acid phosphatase. The polypeptide chain is organized into an alpha and an alpha/beta domain, and the active site is located in a positively charged cleft between the domains. Three sulfate ions bound to the catalytic pocket of an inactive mutant suggest a unique binding mode for its substrate phytate. Even in the absence of substrate, the Klebsiella phytase is closer in structure to the E. coli phytase AppA in its substrate-bound form than to phytate-free AppA. This is taken to suggest a preformed substrate-binding site in Klebsiella phytase. Differences in habitat and substrate availability thus gave rise to enzymes with different substrate-binding modes, specificities and kinetics.

Functional genomics of stress response in Pseudomonas putida KT2440.

The metabolically versatile soil bacterium Pseudomonas putida has to cope with numerous abiotic stresses in its habitats. The stress responses of P. putida KT2440 to 4 degrees C, pH 4.5, 0.8 M urea, and 45 mM sodium benzoate were analyzed by determining the global mRNA expression profiles and screening for stress-intolerant nonauxotrophic Tn5 transposon mutants. In 392 regulated genes or operons, 36 gene regions were differentially expressed by more than 2.5-fold, and 32 genes in 23 operons were found to be indispensable for growth during exposure to one of the abiotic stresses. The transcriptomes of the responses to urea, benzoate, and 4 degrees C correlated positively with each other but negatively with the transcriptome of the mineral acid response. The CbrAB sensor kinase, the cysteine synthase CysM, PcnB and VacB, which control mRNA stability, and BipA, which exerts transcript-specific translational control, were essential to cope with cold stress. The cyo operon was required to cope with acid stress. A functional PhoP, PtsP, RelA/SpoT modulon, and adhesion protein LapA were necessary for growth in the presence of urea, and the outer membrane proteins OmlA and FepA and the phosphate transporter PstBACS were indispensable for growth in the presence of benzoate. A lipid A acyltransferase (PP0063) was a mandatory component of the stress responses to cold, mineral acid, and benzoate. Adaptation of the membrane barrier, uptake of phosphate, maintenance of the intracellular pH and redox status, and translational control of metabolism are key mechanisms of the response of P. putida to abiotic stresses.

Transforming growth factor-beta-1 variants are not associated with chronic nonalcoholic pancreatitis.

BACKGROUND: Pancreatic fibrosis is a key pathological feature of chronic pancreatitis. In vivo and in vitro data have demonstrated that pancreatic stellate cells (PSC) play a central role in pancreatic fibrosis. PSC activation and collagen synthesis are highly controlled by transforming growth factor-beta-1 (TGF-beta1). We evaluated whether functionally relevant genetic variants of TGF-beta1 are associated with chronic nonalcoholic pancreatitis. METHODS: The promotor as well as exon 1 variants of the TGF-beta1 gene (G-800A, Leu10Pro and Arg25Pro) were investigated. Forty-two CP patients with a family history of nonalcoholic chronic pancreatitis (group A) and 88 patients without a family history of nonalcoholic chronic pancreatitis (group B) were studied. One hundred blood donors served as controls (group C). RESULTS: The allelic frequencies of G-800A, Leu10Pro and Arg25Pro were 12, 38 and 6% in group A; 7, 40 and 6% in group B and 12, 29 and 3% in group C, respectively. The differences were not significant. CONCLUSION: Functionally relevant genetic variants of the TGF-beta1 gene are not associated with nonalcoholic chronic pancreatitis.