Yajun Tang

Structural and Biochemical Characterization of Yeast Monothiol Glutaredoxin Grx6

Glutaredoxins (Grxs) are a ubiquitous family of proteins that reduce disulfide bonds in substrate proteins using electrons from reduced glutathione (GSH). The yeast Saccharomyces cerevisiae Grx6 is a monothiol Grx that is localized in the endoplasmic reticulum and Golgi compartments. Grx6 consists of three segments, a putative signal peptide (M1-I36), an N-terminal domain (K37-T110), and a C-terminal Grx domain (K111-N231, designated Grx6C). Compared to the classic dithiol glutaredoxin Grx1, Grx6 has a lower glutathione disulfide reductase activity but a higher glutathione S-transferase activity. In addition, similar to human Grx2, Grx6 binds GSH via an iron-sulfur cluster in vitro. The N-terminal domain is essential for noncovalent dimerization, but not required for either of the above activities. The crystal structure of Grx6C at 1.5 A resolution revealed a novel two-strand antiparallel beta-sheet opposite the GSH binding groove. This extra beta-sheet might also exist in yeast Grx7 and in a group of putative Grxs in lower organisms, suggesting that Grx6 might represent the first member of a novel Grx subfamily.

Solution structure of human peptidyl prolyl isomerase like protein 1 and insights into its interaction with SKIP

The human PPIL1 (peptidyl prolyl isomerase-like protein 1) is a specific component of human 35 S U5 small nuclear ribonucleoprotein particle and 45 S activated spliceosome. It is recruited by SKIP, another essential component of 45 S activated spliceosome, into spliceosome just before the catalytic step 1. It stably associates with SKIP, which also exists in 35 S and activated spliceosome as a nuclear matrix protein. We report here the solution structure of PPIL1 determined by NMR spectroscopy. The structure of PPIL1 resembles other members of the cyclophilin family and exhibits PPIase activity. To investigate its interaction with SKIP in vitro, we identified the SKIP contact region by GST pulldown experiments and surface plasmon resonance. We provide direct evidence of PPIL1 stably associated with SKIP. The dissociation constant is 1.25 × 10–7 M for the N-terminal peptide of SKIP-(59–129) with PPIL1. We also used chemical shift perturbation experiments to show the possible SKIP binding interface on PPIL1. These results illustrated that a novel cyclophilin-protein contact mode exists in the PPIL1-SKIP complex during activation of the spliceosome. The biological implication of this binding with spliceosome rearrangement during activation is discussed.

Crystal structure of the yeast cytoplasmic thioredoxin Trx2

Rui Bao, Yuxing Chen,* Ya-Jun Tang, Joel Janin, and Cong-Zhao Zhou* Protein Research Institute, Tongji University, Shanghai 200092, People’s Republic of China Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, Hefei, Anhui 230026, People’s Republic of China School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, People’s Republic of China Yeast Structural Genomics, IBBMC UMR 8619, Universite Paris-Sud, 91405-Orsay, France

NMR structure and regulated expression in APL cell of human SH3BGRL3

SH3 domain binding glutamic acid‐rich protein like 3 (SH3BGRL3) is the new member of thioredoxin (TRX) super family, whose posttranslational modified form was identified as tumor necrosis factor α (TNF‐α) inhibitory protein, TIP‐B1. In this paper, we determined its solution structure by multi‐dimensional nuclear magnetic resonance spectroscopy. The overall structure of human SH3BGRL3 conformed to a TRX‐like fold. To understand its function in vivo, the upregulated expression in acute promyelocytic leukemia cell line NB4 at both mRNA and protein level was elucidated. Immunofluorescence and immunohistochemistry staining with monoclonal antibody against SH3BGRL3 demonstrated that it was a cytoplasmic protein in both NB4 cell and human tissues. These results, as a whole, indicate that SH3BGRL3 may function as a regulator in all‐trans retinoic acid‐induced pathway.

Structural insights into the recognition of phosphorylated FUNDC1 by LC3B in mitophagy

Mitophagy is an essential intracellular process that eliminates dysfunctional mitochondria and maintains cellular homeostasis. Mitophagy is regulated by the post-translational modification of mitophagy receptors. Fun14 domain-containing protein 1 (FUNDC1) was reported to be a new receptor for hypoxia-induced mitophagy in mammalian cells and interact with microtubule-associated protein light chain 3 beta (LC3B) through its LC3 interaction region (LIR). Moreover, the phosphorylation modification of FUNDC1 affects its binding affinity for LC3B and regulates selective mitophagy. However, the structural basis of this regulation mechanism remains unclear. Here, we present the crystal structure of LC3B in complex with a FUNDC1 LIR peptide phosphorylated at Ser17 (pS17), demonstrating the key residues of LC3B for the specific recognition of the phosphorylated or dephosphorylated FUNDC1. Intriguingly, the side chain of LC3B Lys49 shifts remarkably and forms a hydrogen bond and electrostatic interaction with the phosphate group of FUNDC1 pS17. Alternatively, phosphorylated Tyr18 (pY18) and Ser13 (pS13) in FUNDC1 significantly obstruct their interaction with the hydrophobic pocket and Arg10 of LC3B, respectively. Structural observations are further validated by mutation and isothermal titration calorimetry (ITC) assays. Therefore, our structural and biochemical results reveal a working model for the specific recognition of FUNDC1 by LC3B and imply that the reversible phosphorylation modification of mitophagy receptors may be a switch for selective mitophagy.

Structural basis for the different activities of yeast Grx1 and Grx2

Yeast glutaredoxins Grx1 and Grx2 catalyze the reduction of both inter- and intra-molecular disulfide bonds using glutathione (GSH) as the electron donor. Although sharing the same dithiolic CPYC active site and a sequence identity of 64%, they have been proved to play different roles during oxidative stress and to possess different glutathione-disulfide reductase activities. To address the structural basis of these differences, we solved the crystal structures of Grx2 in oxidized and reduced forms, at 2.10 A and 1.50 A, respectively. With the Grx1 structures we previously reported, comparative structural analyses revealed that Grx1 and Grx2 share a similar GSH binding site, except for a single residue substitution from Asp89 in Grx1 to Ser123 in Grx2. Site-directed mutagenesis in combination with activity assays further proved this single residue variation is critical for the different activities of yeast Grx1 and Grx2.

Structure-guided activity enhancement and catalytic mechanism of yeast grx8.

Glutaredoxins (Grxs) are wide-spread oxidoreductases that are found in all kingdoms of life. The yeast Saccharomyces cerevisiae encodes eight Grxs, among which, Grx8 shares a sequence identity of 30 and 23% with typical dithiol Grx1 and Grx2, respectively, but it exhibits a much lower GSH-dependent oxidoreductase activity. To elucidate its catalytic mechanism, we solved the solution structure of Grx8, which displays a typical Grx fold. Structural analysis indicated that Grx8 possesses a negatively charged CXXC motif (Cys(33)-Pro(34)-Asp(35)-Cys(36)) and a GSH-recognition site, which are distinct from Grx1 and Grx2. Subsequent structure-guided site mutations revealed that the D35Y single mutant and N80T/L81V double mutant possess increased activity of 10- and 11-fold, respectively; moreover, the D35Y/N80T/L81V triple mutant has increased activity of up to 44-fold, which is comparable to that of canonical Grx. Biochemical analyses suggested that the increase in catalytic efficiency resulted from a decreased pKa value of catalytic cysteine Cys33 and/or enhancement of the putative GSH-recognition site. Moreover, NMR chemical shift perturbation analyses combined with GSH analogue inhibition assays enabled us to elucidate that wild-type Grx8 and all mutants adopt a ping-pong mechanism of catalysis. All together, these findings provide structural insights into the catalytic mechanism of dithiol Grxs.

Structural and Biochemical Insights into the Multiple Functions of Yeast Grx3.

The yeast Saccharomyces cerevisiae monothiol glutaredoxin Grx3 plays a key role in cellular defense against oxidative stress and more importantly, cooperates with BolA-like iron repressor of activation protein Fra2 to regulate the localization of the iron-sensing transcription factor Aft2. The interplay among Grx3, Fra2 and Aft2 responsible for the regulation of iron homeostasis has not been clearly described. Here we solved the crystal structures of the Trx domain (Grx3Trx) and Grx domain (Grx3Grx) of Grx3 in addition to the solution structure of Fra2. Structural analyses and activity assays indicated that the Trx domain also contributes to the glutathione S-transferase activity of Grx3, via an inter-domain disulfide bond between Cys37 and Cys176. NMR titration and pull-down assays combined with surface plasmon resonance experiments revealed that Fra2 could form a noncovalent heterodimer with Grx3 via an interface between the helix-turn-helix motif of Fra2 and the C-terminal segment of Grx3Grx, different from the previously identified covalent heterodimer mediated by Fe-S cluster. Comparative affinity assays indicated that the interaction between Fra2 and Aft2 is much stronger than that between Grx3 and Aft2, or Aft2 toward its target DNA. These structural and biochemical analyses enabled us to propose a model how Grx3 executes multiple functions to coordinate the regulation of Aft2-controlled iron metabolism.

Insights into the Structure of Dimeric RNA Helicase CsdA and Indispensable Role of Its C-Terminal Regions

CsdA has been proposed to be essential for the biogenesis of ribosome and gene regulation after cold shock. However, the structure of CsdA and the function of its long C-terminal regions are still unclear. Here, we solved all of the domain structures of CsdA and found two previously uncharacterized auxiliary domains: a dimerization domain (DD) and an RNA-binding domain (RBD). Small-angle X-ray scattering experiments helped to track the conformational flexibilities of the helicase core domains and C-terminal regions. Biochemical assays revealed that DD is indispensable for stabilizing the CsdA dimeric structure. We also demonstrate for the first time that CsdA functions as a stable dimer at low temperature. The C-terminal regions are critical for RNA binding and efficient enzymatic activities. CsdA_RBD could specifically bind to the regions with a preference for single-stranded G-rich RNA, which may help to bring the helicase core to unwind the adjacent duplex.

Structural and functional characterization of hMEX-3C Ring finger domain as an E3 ubiquitin ligase: hMEX-3C Ring Finger Domain as an E3 Ubiquitin Ligase

MEX‐3C, a novel RNA binding E3 ubiquitin ligases, contains two N‐terminal heterogeneous nuclear ribonucleoprotein K homology (KH) domains and C‐terminal Ring finger domain. Recent evidence has suggested that human MEX‐3C has a strong bondage with carcinogenesis and the MEX‐3C‐mediated ubiquitination of RIG‐I is essential for the antiviral innate immune response. Moreover, the Ring finger domain of MEX‐3C could regulate the degradation of HLA‐A2 (an MHC‐I allotype) mRNA with a novel mechanism. However, the structural basis for the ubiquitination catalyzed by hMEX‐3C Ring finger domain remains evasive. In this study, we solved the crystal structure of dimeric Ring finger domain of hMEX‐3C and compared it with the complex structure of MDM2/MDMX–UbcH5b–Ub. Our ubiquitination assay demonstrated that the Ring finger domain of hMEX‐3C acts as a ubiquitin E3 ligase in vitro, cooperating with specific E2 to mediate ubiquitination. Then, we identified several key residues in Ring finger domain of hMEX‐3C possibly involved in the interaction with E2–Ub conjugate and analyzed the E3 ligase activities of wild type and mutants at key sites. Additionally, zinc chelation experiments indicated that the intact structural stability is essential for the self‐ubiquitination activity of the Ring finger domain of hMEX‐3C. Taken together, our studies provided new insight into the mechanism of the Ring finger domain of hMEX‐3C that may play an important role in eliciting antiviral immune responses and therapeutic interventions.