Huihao Zhou

Ribosome stalling induced by mutation of a CNS-specific tRNA causes neurodegeneration

Problems making proteins kills nerve cells Neurodegeneration is associated with a variety of different diseases, but its cellular roots are often obscure. Ishimura et al. find that mutant mice whose brain cells start to die rapidly soon after birth have lost the function of two vital cellular components (see the Perspective by Darnell). The first is a protein that releases stalled ribosomes stuck on messenger RNA (mRNA); the second is a transfer RNA (tRNA), which reads the code for arginine in the mRNA. This tRNA is expressed predominantly in the central nervous system. The lack of the tRNA leads to increased ribosomal stalling at arginine codons, which, when left uncorrected, blocks protein synthesis and proves fatal. Science, this issue p. 455; see also p. 378 Mutations in a transfer RNA expressed in the nervous system stall ribosomes and can cause cell death if ribosome recycling fails. [Also see Perspective by Darnell] In higher eukaryotes, transfer RNAs (tRNAs) with the same anticodon are encoded by multiple nuclear genes, and little is known about how mutations in these genes affect translation and cellular homeostasis. Similarly, the surveillance systems that respond to such defects in higher eukaryotes are not clear. Here, we discover that loss of GTPBP2, a novel binding partner of the ribosome recycling protein Pelota, in mice with a mutation in a tRNA gene that is specifically expressed in the central nervous system causes ribosome stalling and widespread neurodegeneration. Our results not only define GTPBP2 as a ribosome rescue factor but also unmask the disease potential of mutations in nuclear-encoded tRNA genes.

ATP-directed capture of bioactive herbal-based medicine on human tRNA synthetase

Febrifugine is the active component of the Chinese herb Chang Shan (Dichroa febrifuga Lour.), which has been used for treating malaria-induced fever for about 2,000 years. Halofuginone (HF), the halogenated derivative of febrifugine, has been tested in clinical trials for potential therapeutic applications in cancer and fibrotic disease. Recently, HF was reported to inhibit TH17 cell differentiation by activating the amino acid response pathway, through inhibiting human prolyl-transfer RNA synthetase (ProRS) to cause intracellular accumulation of uncharged tRNA. Curiously, inhibition requires the presence of unhydrolysed ATP. Here we report an unusual 2.0 Å structure showing that ATP directly locks onto and orients two parts of HF onto human ProRS, so that one part of HF mimics bound proline and the other mimics the 3′ end of bound tRNA. Thus, HF is a new type of ATP-dependent inhibitor that simultaneously occupies two different substrate binding sites on ProRS. Moreover, our structure indicates a possible similar mechanism of action for febrifugine in malaria treatment. Finally, the elucidation here of a two-site modular targeting activity of HF raises the possibility that substrate-directed capture of similar inhibitors might be a general mechanism that could be applied to other synthetases.

CMT2D neuropathy is linked to the neomorphic binding activity of glycyl-tRNA synthetase

Selective neuronal loss is a hallmark of neurodegenerative diseases, which, counterintuitively, are often caused by mutations in widely expressed genes. Charcot–Marie–Tooth (CMT) diseases are the most common hereditary peripheral neuropathies, for which there are no effective therapies. A subtype of these diseases—CMT type 2D (CMT2D)—is caused by dominant mutations in GARS, encoding the ubiquitously expressed enzyme glycyl-transfer RNA (tRNA) synthetase (GlyRS). Despite the broad requirement of GlyRS for protein biosynthesis in all cells, mutations in this gene cause a selective degeneration of peripheral axons, leading to deficits in distal motor function. How mutations in GlyRS (GlyRSCMT2D) are linked to motor neuron vulnerability has remained elusive. Here we report that GlyRSCMT2D acquires a neomorphic binding activity that directly antagonizes an essential signalling pathway for motor neuron survival. We find that CMT2D mutations alter the conformation of GlyRS, enabling GlyRSCMT2D to bind the neuropilin 1 (Nrp1) receptor. This aberrant interaction competitively interferes with the binding of the cognate ligand vascular endothelial growth factor (VEGF) to Nrp1. Genetic reduction of Nrp1 in mice worsens CMT2D symptoms, whereas enhanced expression of VEGF improves motor function. These findings link the selective pathology of CMT2D to the neomorphic binding activity of GlyRSCMT2D that antagonizes the VEGF–Nrp1 interaction, and indicate that the VEGF–Nrp1 signalling axis is an actionable target for treating CMT2D.

Structural Insights into the Down-regulation of Overexpressed p185her2/neu Protein of Transformed Cells by the Antibody chA21

p185her2/neu belongs to the ErbB receptor tyrosine kinase family, which has been associated with human breast, ovarian, and lung cancers. Targeted therapies employing ectodomain-specific p185her2/neu monoclonal antibodies (mAbs) have demonstrated clinical efficacy for breast cancer. Our previous studies have shown that p185her2/neu mAbs are able to disable the kinase activity of homomeric and heteromeric kinase complexes and induce the conversion of the malignant to normal phenotype. We previously developed a chimeric antibody chA21 that specifically inhibits the growth of p185her2/neu-overexpressing cancer cells in vitro and in vivo. Herein, we report the crystal structure of the single-chain Fv of chA21 in complex with an N-terminal fragment of p185her2/neu, which reveals that chA21 binds a region opposite to the dimerization interface, indicating that chA21 does not directly disrupt the dimerization. In contrast, the bivalent chA21 leads to internalization and down-regulation of p185her2/neu. We propose a structure-based model in which chA21 cross-links two p185her2/neu molecules on separate homo- or heterodimers to form a large oligomer in the cell membrane. This model reveals a mechanism for mAbs to drive the receptors into the internalization/degradation path from the inactive hypophosphorylated tetramers formed dynamically by active dimers during a “physiologic process.”

Crystal structure of NusG N‐terminal (NGN) domain from Methanocaldococcus jannaschii and its interaction with rpoE″

Transcription in archaea employs a eukaryotic‐type transcription apparatus but uses bacterial‐type transcription factors. NusG is one of the few archaeal transcription factors whose orthologs are essential in both bacteria and eukaryotes. Archaeal NusG is composed of only an NusG N‐terminal (NGN) domain and a KOW domain, which is similar to bacterial NusG but not to the eukaryotic ortholog, Spt5. However, archaeal NusG was confirmed recently to form a complex with rpoE″ that was similar to the Spt5‐Spt4 complex. Thus, archaeal NusG presents hybrid features of Spt5 and bacterial NusG. Here we report the crystal structure of NGN from the archaea Methanocaldococcus jannaschii (MjNGN). MjNGN folds to an α‐β‐α sandwich without the appendant domain of bacterial NGNs, and forms a unique homodimer in crystal and solution. MjNGN alone was found to be sufficient for rpoE″ binding and an MjNGN‐rpoE″ model has been constructed by rigid docking. Proteins 2009.

Crystal structure of a membrane-bound l-amino acid deaminase from Proteus vulgaris

l-amino acid oxidases/deaminases (LAAOs/LAADs) are a class of oxidoreductases catalyzing the oxidative deamination of l-amino acids to α-keto acids. They are widely distributed in eukaryotic and prokaryotic organisms, and exhibit diverse substrate specificity, post-translational modifications and cellular localization. While LAAOs isolated from snake venom have been extensively characterized, the structures and functions of LAAOs from other species are largely unknown. Here, we reported crystal structure of a bacterial membrane-bound LAAD from Proteus vulgaris (pvLAAD) in complex with flavin adenine dinucleotide (FAD). We found that the overall fold of pvLAAD does not resemble typical LAAOs. Instead it, is similar to d-amino acid oxidases (DAAOs) with an additional hydrophobic insertion module on protein surface. Structural analysis and liposome-binding assays suggested that the hydrophobic module serves as an extra membrane-binding site for LAADs. Bacteria from genera Proteus and Providencia were found to encode two classes of membrane-bound LAADs. Based on our structure, the key roles of residues Q278 and L317 in substrate selectivity were proposed and biochemically analyzed. While LAADs on the membrane were proposed to transfer electrons to respiratory chain for FAD re-oxidization, we observed that the purified pvLAAD could generate a significant amount of hydrogen peroxide in vitro, suggesting it could use dioxygen to directly re-oxidize FADH2 as what typical LAAOs usually do. These findings provide a novel insights for a better understanding this class of enzymes and will help developing biocatalysts for industrial applications.

Crystal structure of human osteoclast stimulating factor.

Osteoclast is the main cell responsible for the degradation of bone matrix.1 Its differentiation and activity requires a variety of factors. For example, macrophage colony stimulating factor (M-CSF) prevents apoptosis of early osteoclast precursors,2 and receptor activator of nuclear factor jB ligand (RANKL) is very important for osteoclast formation and bone remodeling.3,4 Tyrosine protein kinase Src, involved in both M-CSF and RANKL mediated signal networks,4,5 was also found to be essential for osteoclast function.6,7 In addition, factors produced by osteoclasts themselves (such as interleukin-6, transforming growth factor beta, etc.) also play important roles.8–10 Osteoclast stimulating factor (OSF), composed of a proline-rich region, a SH3 domain and ankyrin repeats, is a intracellular protein produced by osteoclasts and shown to indirectly enhance osteoclast formation and activity.11 A mouse homolog of OSF called SH3P2 (95% sequence identity with OSF) can bind proline-rich fragment of Cbl and form an OSF-Cbl-Src triple protein complex,12 suggesting that OSF was involved in Srcand Cblmediated pathways. OSF-SH3 domain was also found to bind exon 6 region of survival motor neuron (a product of spinal muscular atrophy disease-determining gene).13 SH3 domains recognize proline-rich sequences with a PxxP motif, which was further classified into 1xuPxuP (class I) and uPxuPx1 (class II)14,15 (where x is any amino acid, u is usually a hydrophobic residue, and 1 is usually an arginine residue). This motif adopts a lefthanded polyproline type II helical conformation,14 known as a collagen chain conformation, and fits the ligand-binding groove on the SH3 surface. Ankyrin repeats are common protein scaffolds and have been found in many proteins spanning a wide range of functions.16 They mediate protein–protein interactions mostly via a concave inner surface formed by a loop region and inner short helices through hydrophilic contacts, such as hydrogen bonds and salt bridges.17 Here, we present crystal structures of human OSF in two space groups grown from the same condition. One is determined by multiwavelength anomalous diffraction (MAD) at 2.57 Å resolution in space group P212121, and the other is determined by molecular replacement at 1.95 Å resolution in space group P1. Models in both crystal forms contain one SH3 domain and four ankyrin repeats, but the relative position of the two domains is different in two forms. It is the first reported protein structure of such a domain sequence. A prolonged and more standard helix was found to be unique in OSF-SH3 domain, and residues that may be responsible for the binding specificity of OSF ankyrin repeats were also discussed.

Structural and biochemical insights into the DNA-binding mode of MjSpt4p:Spt5 complex at the exit tunnel of RNAPII

Spt5 (NusG in bacteria) is the only RNA polymerase-associated factor known to be conserved in all three domains of life. In archaea and eukaryotes, Spt5 associates with Spt4, an elongation factor that is absent in bacteria, to form a functional heterodimeric complex. Previous studies suggest that the Spt4:Spt5 complex interacts directly with DNA at the double-stranded DNA exit tunnel of RNA polymerase to regulate gene transcription. In this study, the DNA-binding ability of Spt4:Spt5 from the archaeon Methanocaldococcus jannaschii was confirmed via nuclear magnetic resonance chemical shift perturbation and fluorescence polarization assays. Crystallographic analysis of the full-length MjSpt4:Spt5 revealed two distinct conformations of the C-terminal KOW domain of Spt5. A similar alkaline region was found on the Spt4:Spt5 surface in both crystal forms, and identified as double-stranded DNA binding patch through mutagenesis-fluorescence polarization assays. Based on these structural and biochemical data, the Spt4:Spt5-DNA binding model was built for the first time.

Crystallization and preliminary crystallographic studies of the single-chain variable fragment of antibody chA21 in complex with an N-terminal fragment of ErbB2

ErbB2 is a transmembrane tyrosine kinase, the overexpression of which causes abnormality and disorder in cell signalling and leads to cell transformation. Previously, an anti-ErbB2 single-chain chimeric antibody chA21 that specifically inhibits the growth of ErbB2-overexpressing cancer cells in vitro and in vivo was developed. Here, an antibody-antigen complex consisting of the single-chain variable fragment (scFv) of chA21 and an N-terminal fragment (residues 1-192, named EP I) of the ErbB2 extracellular domain was crystallized using the sitting-drop vapour-diffusion method. An X-ray diffraction data set was collected to 2.45 A resolution from a single flash-cooled crystal; the crystal belonged to space group P2(1)2(1)2(1).

ASDB: a resource for probing protein functions with small molecules

UNLABELLED : Identifying chemical probes or seeking scaffolds for a specific biological target is important for protein function studies. Therefore, we create the Annotated Scaffold Database (ASDB), a computer-readable and systematic target-annotated scaffold database, to serve such needs. The scaffolds in ASDB were derived from public databases including ChEMBL, DrugBank and TCMSP, with a scaffold-based classification approach. Each scaffold was assigned with an InChIKey as its unique identifier, energy-minimized 3D conformations, and other calculated properties. A scaffold is also associated with drugs, natural products, drug targets and medical indications. The database can be retrieved through text or structure query tools. ASDB collects 333 601 scaffolds, which are associated with 4368 targets. The scaffolds consist of 3032 scaffolds derived from drugs and 5163 scaffolds derived from natural products. For given scaffolds, scaffold-target networks can be generated from the database to demonstrate the relations of scaffolds and targets. AVAILABILITY AND IMPLEMENTATION ASDB is freely available at http://www.rcdd.org.cn/asdb/with the major web browsers. CONTACT junxu@biochemomes.com or xujun9@mail.sysu.edu.cn SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.