Tianlong Zhang

Crystal structure of the Ego1-Ego2-Ego3 complex and its role in promoting Rag GTPase-dependent TORC1 signaling.

The target of rapamycin complex 1 (TORC1) integrates various hormonal and nutrient signals to regulate cell growth, proliferation, and differentiation. Amino acid-dependent activation of TORC1 is mediated via the yeast EGO complex (EGOC) consisting of Gtr1, Gtr2, Ego1, and Ego3. Here, we identify the previously uncharacterized Ycr075w-a/Ego2 protein as an additional EGOC component that is required for the integrity and localization of the heterodimeric Gtr1-Gtr2 GTPases, equivalent to mammalian Rag GTPases. We also report the crystal structure of the Ego1-Ego2-Ego3 ternary complex (EGO-TC) at 2.4 Å resolution, in which Ego2 and Ego3 form a heterodimer flanked along one side by Ego1. Structural data also reveal the structural conservation of protein components between the yeast EGO-TC and the human Ragulator, which acts as a GEF for Rag GTPases. Interestingly, however, artificial tethering of Gtr1-Gtr2 to the vacuolar membrane is sufficient to activate TORC1 in response to amino acids even in the absence of the EGO-TC. Our structural and functional data therefore support a model in which the EGO-TC acts as a scaffold for Rag GTPases in TORC1 signaling.

Crystal Structure of DNA Cytidine Deaminase ABOBEC3G Catalytic Deamination Domain Suggests a Binding Mode of Full-length Enzyme to Single-stranded DNA

Background: The mechanism for DNA cytidine deaminase APOBEC3G (A3G) interacting with single-stranded DNA (ssDNA) is not well characterized. Results: The crystal structure of a head-to-tail dimer of the A3G catalytic deamination domain (A3G-CD2) was obtained. Conclusion: The dimer structure of A3G-CD2 suggests a binding mode of full-length A3G to ssDNA. Significance: The dimer structure of A3G-CD2 may represent a structural model of full-length A3G. APOBEC3G (A3G) is a DNA cytidine deaminase (CD) that demonstrates antiviral activity against human immunodeficiency virus 1 (HIV-1) and other pathogenic virus. It has an inactive N-terminal CD1 virus infectivity factor (Vif) protein binding domain (A3G-CD1) and an actively catalytic C-terminal CD2 deamination domain (A3G-CD2). Although many studies on the structure of A3G-CD2 and enzymatic properties of full-length A3G have been reported, the mechanism of how A3G interacts with HIV-1 single-stranded DNA (ssDNA) is still not well characterized. Here, we reported a crystal structure of a novel A3G-CD2 head-to-tail dimer (in which the N terminus of the monomer H (head) interacts with the C terminus of monomer T (tail)), where a continuous DNA binding groove was observed. By constructing the A3G-CD1 structural model, we found that its overall fold was almost identical to that of A3G-CD2. We mutated the residues located in or along the groove in monomer H and the residues in A3G-CD1 that correspond to those seated in or along the groove in monomer T. Then, by performing enzymatic assays, we confirmed the reported key elements and the residues in A3G necessary to the catalytic deamination. Moreover, we identified more than 10 residues in A3G essential to DNA binding and deamination reaction. Therefore, this dimer structure may represent a structural model of full-length A3G, which indicates a possible binding mode of A3G to HIV-1 ssDNA.

Crystal structure of the ARL2-GTP-BART complex reveals a novel recognition and binding mode of small GTPase with effector

ARL2 is a member of the ADP-ribosylation factor family but has unique biochemical features. BART is an effector of ARL2 that is essential for nuclear retention of STAT3 and may also be involved in mitochondria transport and apoptosis. Here we report the crystal structure and biochemical characterization of human ARL2-GTP-BART complex. ARL2-GTP assumes a typical small GTPase fold with a unique N-terminal alpha helix conformation. BART consists of a six alpha helix bundle. The interactions between ARL2 and BART involve two interfaces: a conserved N-terminal LLXIL motif of ARL2 is embedded in a hydrophobic cleft of BART and the switch regions of ARL2 interact with helix alpha3 of BART. Both interfaces are essential for the binding as verified by mutagenesis study. This novel recognition and binding mode is different from that of other small GTPase-effector interactions and provides molecular basis for the high specificity of ARL2 for BART.

Instrumented indentation study of plastic deformation in bulk metallic glasses

Mechanical properties and micro-plastic deformation behavior of five bulk metallic Glasses (BMGs) were studied by instrumented indentation. These materials included La60Al10Ni10Cu20, Mg65Cu25Gd10, Zr52.5Al10Ni10Cu15Be12.5, Cu60Zr20Hf10Ti10, and Ni60Nb37Sn3 alloys. Remarkable difference in deformation behavior was found in the load-displacement curves of nanoindentation and pileup morphologies around the indents. Serrated plastic deformation depended on the loading rate was found in Mg-, Zr-, and Cu-based BMGs. The subsurface plastic deformation zone of typical alloys was investigated through bonded interface technique using depth-sensing microindentation. Large and widely spaced shear bands were observed in Mg-based BMG. The effect of loading rate on the indentation deformation behaviors in different BMGs was elucidated by the change of shear band pattern.

Structural insights into DndE from Escherichia coli B7A involved in DNA phosphorothioation modification

Structural insights into DndE from Escherichia coli B7A involved in DNA phosphorothioation modification

Molecular mechanism for Rabex-5 GEF activation by Rabaptin-5

Rabex-5 and Rabaptin-5 function together to activate Rab5 and further promote early endosomal fusion in endocytosis. The Rabex-5 GEF activity is autoinhibited by the Rabex-5 CC domain (Rabex-5CC) and activated by the Rabaptin-5 C2-1 domain (Rabaptin-5C21) with yet unknown mechanism. We report here the crystal structures of Rabex-5 in complex with the dimeric Rabaptin-5C21 (Rabaptin-5C212) and in complex with Rabaptin-5C212 and Rab5, along with biophysical and biochemical analyses. We show that Rabex-5CC assumes an amphipathic α-helix which binds weakly to the substrate-binding site of the GEF domain, leading to weak autoinhibition of the GEF activity. Binding of Rabaptin-5C21 to Rabex-5 displaces Rabex-5CC to yield a largely exposed substrate-binding site, leading to release of the GEF activity. In the ternary complex the substrate-binding site of Rabex-5 is completely exposed to bind and activate Rab5. Our results reveal the molecular mechanism for the regulation of the Rabex-5 GEF activity. DOI: http://dx.doi.org/10.7554/eLife.02687.001

Structure of human lysine methyltransferase Smyd2 reveals insights into the substrate divergence in Smyd proteins

The SET- and myeloid-Nervy-DEAF-1 (MYND)-domain containing (Smyd) lysine methyltransferases 1-3 share relatively high sequence similarity but exhibit divergence in the substrate specificity. Here we report the crystal structure of the full-length human Smyd2 in complex with S-adenosyl-L-homocysteine (AdoHcy). Although the Smyd1-3 enzymes are similar in the overall structure, detailed comparisons demonstrate that they differ substantially in the potential substrate-binding site. The binding site of Smyd3 consists mainly of a deep and narrow pocket, while those of Smyd1 and Smyd2 consist of a comparable pocket and a long groove. In addition, Smyd2, which has lysine methyltransferase activity on histone H3-lysine 36, exhibits substantial differences in the wall of the substrate-binding pocket compared with those of Smyd1 and Smyd3 which have activity specifically on histone H3-lysine 4. The differences in the substrate-binding site might account for the observed divergence in the specificity and methylation state of the substrates. Further modeling study of Smyd2 in complex with a p53 peptide indicates that mono-methylation of p53-Lys(372) might result in steric conflict of the methyl group with the surrounding residues of Smyd2, providing a structural explanation for the inhibitory effect of the SET7/9-mediated mono-methylation of p53-Lys(372) on the Smyd2-mediated methylation of p53-Lys(370).

Saccharomyces Cerevisiae MHF Complex Structurally Resembles the Histones (H3-H4)(2) Heterotetramer and Functions as a Heterotetramer

Fanconi anemia (FA) is a chromosomal instability disorder associated with deficiencies in the Fanconi anemia complementation group (FANC) network. A complex consisting of FANCM-associated histone-fold proteins 1 and 2 (MHF1 and MHF2) has been shown to act cooperatively with FANCM in DNA damage repair in the FA pathway. Here we report the structure of Saccharomyces cerevisiae MHF complex in which MHF1 and MHF2 assume a typical histone fold, and the complex has a heterotetrameric architecture similar to that of the histones (H3-H4)₂ heterotetramer. Loop L2 of MHF1 is probably involved in DNA binding, and loop L3 and helices α2 and α3 of one MHF1 subunit interact with those of the other to form two heterotetramer interfaces. Further genetic data demonstrate that the heterotetramer assembly is essential for the function of the complex in DNA repair. These results provide, to the best of our knowledge, new mechanistic insights into the function of the MHF complex.

Conotoxin αd-GeXXA utilizes a novel strategy to antagonize nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) play essential roles in transmitting acetylcholine-mediated neural signals across synapses and neuromuscular junctions, and are also closely linked to various diseases and clinical conditions. Therefore, novel nAChR-specific compounds have great potential for both neuroscience research and clinical applications. Conotoxins, the peptide neurotoxins produced by cone snails, are a rich reservoir of novel ligands that target receptors, ion channels and transporters in the nervous system. From the venom of Conus generalis, we identified a novel dimeric nAChR-inhibiting αD-conotoxin GeXXA. By solving the crystal structure and performing structure-guided dissection of this toxin, we demonstrated that the monomeric C-terminal domain of αD-GeXXA, GeXXA-CTD, retains inhibitory activity against the α9α10 nAChR subtype. Furthermore, we identified that His7 of the rat α10 nAChR subunit determines the species preference of αD-GeXXA, and is probably part of the binding site of this toxin. These results together suggest that αD-GeXXA cooperatively binds to two inter-subunit interfaces on the top surface of nAChR, thus allosterically disturbing the opening of the receptor. The novel antagonistic mechanism of αD-GeXXA via a new binding site on nAChRs provides a valuable basis for the rational design of new nAChR-targeting compounds.

Structural insight into the arginine-binding specificity of CASTOR1 in amino acid-dependent mTORC1 signaling.

The mechanistic Target Of Rapamycin Complex 1 (mTORC1) is central to the cellular response to changes in nutrient signals such as amino acids. CASTOR1 is shown to be an arginine sensor, which plays an important role in the activation of the mTORC1 pathway. In the deficiency of arginine, CASTOR1 interacts with GATOR2, which together with GATOR1 and Rag GTPases controls the relocalization of mTORC1 to lysosomes. The binding of arginine to CASTOR1 disrupts its association with GATOR2 and hence activates the mTORC1 signaling. Here, we report the crystal structure of CASTOR1 in complex with arginine at 2.5 Å resolution. CASTOR1 comprises of four tandem ACT domains with an architecture resembling the C-terminal allosteric domains of aspartate kinases. ACT1 and ACT3 adopt the typical βαββαβ topology and function in dimerization via the conserved residues from helices α1 of ACT1 and α5 of ACT3; whereas ACT 2 and ACT4, both comprising of two non-sequential regions, assume the unusual ββαββα topology and contribute an arginine-binding pocket at the interface. The bound arginine makes a number of hydrogen-bonding interactions and extensive hydrophobic contacts with the surrounding residues of the binding pocket. The functional roles of the key residues are validated by mutagenesis and biochemical assays. Our structural and functional data together reveal the molecular basis for the arginine-binding specificity of CASTOR1 in the arginine-dependent activation of the mTORC1 signaling.