Lan-Fen Li

DNA binding mechanism revealed by high resolution crystal structure of Arabidopsis thaliana WRKY1 protein

WRKY proteins, defined by the conserved WRKYGQK sequence, are comprised of a large superfamily of transcription factors identified specifically from the plant kingdom. This superfamily plays important roles in plant disease resistance, abiotic stress, senescence as well as in some developmental processes. In this study, the Arabidopsis WRKY1 was shown to be involved in the salicylic acid signaling pathway and partially dependent on NPR1; a C-terminal domain of WRKY1, AtWRKY1-C, was constructed for structural studies. Previous investigations showed that DNA binding of the WRKY proteins was localized at the WRKY domains and these domains may define novel zinc-binding motifs. The crystal structure of the AtWRKY1-C determined at 1.6 Å resolution has revealed that this domain is composed of a globular structure with five β strands, forming an antiparallel β-sheet. A novel zinc-binding site is situated at one end of the β-sheet, between strands β4 and β5. Based on this high-resolution crystal structure and site-directed mutagenesis, we have defined and confirmed that the DNA-binding residues of AtWRKY1-C are located at β2 and β3 strands. These results provided us with structural information to understand the mechanism of transcriptional control and signal transduction events of the WRKY proteins.

Crystal structures of human caspase 6 reveal a new mechanism for intramolecular cleavage self‐activation

Dimeric effectors caspase 3 and caspase 7 are activated by initiator caspase processing. In this study, we report the crystal structures of effector caspase 6 (CASP6) zymogen and N‐Acetyl‐Val‐Glu‐Ile‐Asp‐al‐inhibited CASP6. Both of these forms of CASP6 have a dimeric structure, and in CASP6 zymogen the intersubunit cleavage site 190TEVD193 is well structured and inserts into the active site. This positions residue Asp 193 to be easily attacked by the catalytic residue Cys 163. We demonstrate biochemically that intramolecular cleavage at Asp 193 is a prerequisite for CASP6 self‐activation and that this activation mechanism is dependent on the length of the L2 loop. Our results indicate that CASP6 can be activated and regulated through intramolecular self‐cleavage.

Intermolecular recognition revealed by the complex structure of human CLOCK-BMAL1 basic helix-loop-helix domains with E-box DNA.

CLOCK (circadian locomotor output cycles kaput) and BMAL1 (brain and muscle ARNT-like 1) are both transcription factors of the circadian core loop in mammals. Recently published mouse CLOCK-BMAL1 bHLH (basic helix-loop-helix)-PAS (period-ARNT-single-minded) complex structure sheds light on the mechanism for heterodimer formation, but the structural details of the protein-DNA recognition mechanisms remain elusive. Here we have elucidated the crystal structure of human CLOCK-BMAL1 bHLH domains bound to a canonical E-box DNA. We demonstrate that CLOCK and BMAL1 bHLH domains can be mutually selected, and that hydrogen-bonding networks mediate their E-box recognition. We identified a hydrophobic contact between BMAL1 Ile80 and a flanking thymine nucleotide, suggesting that CLOCK-BMAL1 actually reads 7-bp DNA and not the previously believed 6-bp DNA. To find potential non-canonical E-boxes that could be recognized by CLOCK-BMAL1, we constructed systematic single-nucleotide mutations on the E-box and measured their relevant affinities. We defined two non-canonical E-box patterns with high affinities, AACGTGA and CATGTGA, in which the flanking A7-T7′ base pair is indispensable for recognition. These results will help us to identify functional CLOCK-BMAL1-binding sites in vivo and to search for clock-controlled genes. Furthermore, we assessed the inhibitory role of potential phosphorylation sites in bHLH regions. We found that the phospho-mimicking mutation on BMAL1 Ser78 could efficiently block DNA binding as well as abolish normal circadian oscillation in cells. We propose that BMAL1 Ser78 should be a key residue mediating input signal-regulated transcriptional inhibition for external cues to entrain the circadian clock by kinase cascade.

Parallel cloning, expression, purification and crystallization of human proteins for structural genomics

54 human genes were selected as test targets for parallel cloning, expression, purification and crystallization. Proteins from these genes were selected to have a molecular weight of between 14 and 50 kDa, not to have a high percentage of hydrophobic residues (i.e. more likely to be soluble) and to have no known crystal structures and were not known to be subunits of heterocomplexes. Four proteins containing transmembrane regions were selected for comparative tests. To date, 44 expression clones have been constructed with the Gateway cloning system (Invitrogen, The Netherlands). Of these, 35 clones were expressed as recombinant proteins in Escherichia coli strain BL21 (DE3)-pLysS, of which 12 were soluble and four have been purified to homogeneity. Crystallization conditions were screened for the purified proteins in 96-well plates under oil. After further refinement with the same device or by the hanging-drop method, crystals were grown, with needle, plate and prism shapes. A 2.12 A data set was collected for protein NCC27. The results provide insights into the high-throughput target selection, cloning, expression and crystallization of human genomic proteins.

High-resolution structure of a new crystal form of BamA POTRA4-5 from Escherichia coli.

In Escherichia coli, the BAM complex is employed to mediate correct folding of the outer membrane (OM) proteins into β-barrels and their insertion into the OM. BamA, which is an essential component of the complex, consists of a C-terminal transmembrane region and five N-terminal polypeptide transport-associated (POTRA) domains. Although deletion studies have shown that each of the POTRA domains plays an important role in the process of BAM complex formation, only POTRA5 is essential for cell viability. Here, the crystal structure of POTRA4-5 has been determined to 1.50 Å resolution with an R factor of 14.7% and an Rfree of 18.9%.

The crystal structure of human chloride intracellular channel protein 2: A disulfide bond with functional implications

Human chloride intracellular channel proteins (CLICs) are intracellular ion channels based on sequence homology. Human CLICs include six members (CLIC1-6), and all of them contain a conserved core about 240 amino acids. The most distinct feature of CLICs is that they can exist in two states: the soluble cytosolic state and the integral membrane ion channel state. Purified water-soluble CLICs adopt the glutathione S-transferase (GST) fold and can insert into a lipid membrane to form chloride channel.1,2 Among the human CLICs, CLIC1 and CLIC4 have been well studied with the crystal structures solved.1,3,4 The CLIC1 and CLIC4 have important functions in regulating cell cycle, apoptosis, and so forth, and have become potential drug targets for cancer therapy.5–8 Human CLIC2 has a molecular weight of 28.4 kDa and a calculated isoelectric point of 5.44. The CLIC2 gene locates in the telomeric region of Xq28 and is composed of six coding exons and five introns. Since this region of the X chromosome is closely associated with many hereditary diseases, CLIC2 has thus been proposed as a candidate gene for some genetic disorders linked to Xq28.9 Northern blot has demonstrated that the gene of CLIC2 could be expressed in lung, spleen, heart, and skeletal muscle.10 Compared with the CLIC1 or CLIC4, little functional knowledge is known about CLIC2 so far. The only documented function of CLIC2 is to regulate the cardiac calcium ion channel, ryanodine receptor 2 (RyR2).11 In this work, we present the crystal structure of the soluble form of human CLIC2 and tried to correlate some structural features to its potential function. MATERIALS AND METHODS

The crystal structure of the MPN domain from the COP9 signalosome subunit CSN6

CSN6 and CSN6 bind by x ray scattering ( View interaction )

Inhibitory Mechanism of Caspase-6 Phosphorylation Revealed by Crystal Structures, Molecular Dynamics Simulations, and Biochemical Assays

Background: Caspase-6 is a drug target against neurodegenerative diseases and is suppressed by phosphorylation at Ser257. Results: S257E mutation inhibits caspase-6 activation by locking the protein in the “inhibited state” and inhibits caspase-6 activity by steric hindrance. Conclusion: Phosphorylation inhibits caspase-6 through the same mechanism. Significance: The study revealed the inhibition mechanism of caspase-6 phosphorylation and provided new strategies for drug discovery. The apoptotic effector caspase-6 (CASP6) has been clearly identified as a drug target due to its strong association with neurodegeneration and axonal pruning events as well as its crucial roles in Huntington disease and Alzheimer disease. CASP6 activity is suppressed by ARK5-mediated phosphorylation at Ser257 with an unclear mechanism. In this work, we solved crystal structures of ΔproCASP6S257E and p20/p10S257E, which mimicked the phosphorylated CASP6 zymogen and activated CASP6, respectively. The structural investigation combined with extensive biochemical assay and molecular dynamics simulation studies revealed that phosphorylation on Ser257 inhibited self-activation of CASP6 zymogen by “locking” the enzyme in the TEVD193-bound “inhibited state.” The structural and biochemical results also showed that phosphorylation on Ser257 inhibited the CASP6 activity by steric hindrance. These results disclosed the inhibition mechanism of CASP6 phosphorylation and laid the foundation for a new strategy of rational CASP6 drug design.

Incidence and survival of symptomatic lacunar infarction in a Beijing population: a 6-year prospective study.

Background and purpose:  The incidence of ischaemic stroke has increased or remained high in China; however, little population‐based evidence is available on the incidence and survival of lacunar infarction (LAC). We examined the incidence of LAC in a northern Chinese (Beijing) population and monitored survival.

Get Phases from Arsenic Anomalous Scattering: de novo SAD Phasing of Two Protein Structures Crystallized in Cacodylate Buffer

The crystal structures of two proteins, a putative pyrazinamidase/nicotinamidase from the dental pathogen Streptococcus mutans (SmPncA) and the human caspase-6 (Casp6), were solved by de novo arsenic single-wavelength anomalous diffraction (As-SAD) phasing method. Arsenic (As), an uncommonly used element in SAD phasing, was covalently introduced into proteins by cacodylic acid, the buffering agent in the crystallization reservoirs. In SmPncA, the only cysteine was bound to dimethylarsinoyl, which is a pentavalent arsenic group (As (V)). This arsenic atom and a protein-bound zinc atom both generated anomalous signals. The predominant contribution, however, was from the As anomalous signals, which were sufficient to phase the SmPncA structure alone. In Casp6, four cysteines were found to bind cacodyl, a trivalent arsenic group (As (III)), in the presence of the reducing agent, dithiothreitol (DTT), and arsenic atoms were the only anomalous scatterers for SAD phasing. Analyses and discussion of these two As-SAD phasing examples and comparison of As with other traditional heavy atoms that generate anomalous signals, together with a few arsenic-based de novo phasing cases reported previously strongly suggest that As is an ideal anomalous scatterer for SAD phasing in protein crystallography.