Changlin Tian

Discovery of a siderophore export system essential for virulence of Mycobacterium tuberculosis.

Iron is an essential nutrient for most bacterial pathogens, but is restricted by the host immune system. Mycobacterium tuberculosis (Mtb) utilizes two classes of small molecules, mycobactins and carboxymycobactins, to capture iron from the human host. Here, we show that an Mtb mutant lacking the mmpS4 and mmpS5 genes did not grow under low iron conditions. A cytoplasmic iron reporter indicated that the double mutant experienced iron starvation even under high-iron conditions. Loss of mmpS4 and mmpS5 did not change uptake of carboxymycobactin by Mtb. Thin layer chromatography showed that the ΔmmpS4/S5 mutant was strongly impaired in biosynthesis and secretion of siderophores. Pull-down experiments with purified proteins demonstrated that MmpS4 binds to a periplasmic loop of the associated transporter protein MmpL4. This interaction was corroborated by genetic experiments. While MmpS5 interacted only with MmpL5, MmpS4 interacted with both MmpL4 and MmpL5. These results identified MmpS4/MmpL4 and MmpS5/MmpL5 as siderophore export systems in Mtb and revealed that the MmpL proteins transport small molecules other than lipids. MmpS4 and MmpS5 resemble periplasmic adapter proteins of tripartite efflux pumps of Gram-negative bacteria, however, they are not only required for export but also for efficient siderophore synthesis. Membrane association of MbtG suggests a link between siderophore synthesis and transport. The structure of the soluble domain of MmpS4 (residues 52–140) was solved by NMR and indicates that mycobacterial MmpS proteins constitute a novel class of transport accessory proteins. The bacterial burden of the mmpS4/S5 deletion mutant in mouse lungs was lower by 10,000-fold and none of the infected mice died within 180 days compared to wild-type Mtb. This is the strongest attenuation observed so far for Mtb mutants lacking genes involved in iron utilization. In conclusion, this study identified the first components of novel siderophore export systems which are essential for virulence of Mtb.

Structural insight into the type-II mitochondrial NADH dehydrogenases

The single-component type-II NADH dehydrogenases (NDH-2s) serve as alternatives to the multisubunit respiratory complex I (type-I NADH dehydrogenase (NDH-1), also called NADH:ubiquinone oxidoreductase; EC 1.6.5.3) in catalysing electron transfer from NADH to ubiquinone in the mitochondrial respiratory chain. The yeast NDH-2 (Ndi1) oxidizes NADH on the matrix side and reduces ubiquinone to maintain mitochondrial NADH/NAD+ homeostasis. Ndi1 is a potential therapeutic agent for human diseases caused by complex I defects, particularly Parkinson’s disease, because its expression restores the mitochondrial activity in animals with complex I deficiency. NDH-2s in pathogenic microorganisms are viable targets for new antibiotics. Here we solve the crystal structures of Ndi1 in its substrate-free, NADH-, ubiquinone- and NADH–ubiquinone-bound states, to help understand the catalytic mechanism of NDH-2s. We find that Ndi1 homodimerization through its carboxy-terminal domain is critical for its catalytic activity and membrane targeting. The structures reveal two ubiquinone-binding sites (UQI and UQII) in Ndi1. NADH and UQI can bind to Ndi1 simultaneously to form a substrate–protein complex. We propose that UQI interacts with FAD to act as an intermediate for electron transfer, and that NADH transfers electrons through this FAD–UQI complex to UQII. Together our data reveal the regulatory and catalytic mechanisms of Ndi1 and may facilitate the development or targeting of NDH-2s for potential therapeutic applications.

Diaminodiacid-Based Solid-Phase Synthesis of Peptide Disulfide Bond Mimics**

These disulfide surrogates wereusually synthesized through thiol alkylation, azide–alkynecycloaddition, or olefin metathesis reactions occurring at thepeptide side chains after the peptide skeletons are fullyassembled (Figure 1). Limited types of disulfide surrogatescan be generated with the above post-chain-assembly cycli-zation strategy, because many bridge formation reactions arenot readily compatible with the peptide backbones. More-over, when more than one disulfide surrogate need to beinstalled, the post-chain-assembly cyclization method maysometimes encounter difficulties in controlling the regiose-lectivity of the cross-linking reactions.

Structural basis for activity regulation of MLL family methyltransferases.

The mixed lineage leukaemia (MLL) family of proteins (including MLL1–MLL4, SET1A and SET1B) specifically methylate histone 3 Lys4, and have pivotal roles in the transcriptional regulation of genes involved in haematopoiesis and development. The methyltransferase activity of MLL1, by itself severely compromised, is stimulated by the three conserved factors WDR5, RBBP5 and ASH2L, which are shared by all MLL family complexes. However, the molecular mechanism of how these factors regulate the activity of MLL proteins still remains poorly understood. Here we show that a minimized human RBBP5–ASH2L heterodimer is the structural unit that interacts with and activates all MLL family histone methyltransferases. Our structural, biochemical and computational analyses reveal a two-step activation mechanism of MLL family proteins. These findings provide unprecedented insights into the common theme and functional plasticity in complex assembly and activity regulation of MLL family methyltransferases, and also suggest a universal regulation mechanism for most histone methyltransferases.

PtdIns(4)P regulates retromer–motor interaction to facilitate dynein–cargo dissociation at the trans-Golgi network

The molecular mechanisms for the retrograde motor dynein–dynactin to unload its cargoes at their final destination remain to be elucidated. In this study, we have investigated the regulatory mechanism underlying release of retromer-associated cargoes at the trans-Golgi network (TGN). We report that phosphotidylinositol-4-phosphate (PtdIns(4)P), a Golgi-enriched phosphoinositide, negatively regulates the protein–protein interaction between the p150Glued subunit of dynein–dynactin and the retromer component SNX6. We show that PtdIns(4)P specifically facilitates dissociation of retromer-mediated membranous cargoes from the motor at the TGN and uncover an important function for PtdIns(4)P in the spatial control of retrograde vesicular trafficking to the TGN membrane. PtdIns(4)P also regulates SNX4-mediated retrograde vesicular trafficking to the endocytic recycling compartment by modulating its interaction with dynein. These results establish organelle-specific phosphoinositide regulation of motor–cargo interaction as a mechanism for cargo release by molecular motors at target membrane.

Diaminodiacid Bridges to Improve Folding and Tune the Bioactivity of Disulfide‐Rich Peptides

Disulfide-rich peptides containing three or more disulfide bonds are promising therapeutic and diagnostic agents, but their preparation is often limited by the tedious and low-yielding folding process. We found that a single cystine-to-diaminodiacid replacement could significantly increase the folding efficiency of disulfide-rich peptides and thus improve their production yields. The practicality of this strategy was demonstrated by the synthesis and folding of derivatives of the μ-conotoxin SIIIA, the preclinical hormone hepcidin, and the trypsin inhibitor EETI-II. NMR and X-ray crystallography studies confirmed that these derivatives of disulfide-rich peptide retained the correct three-dimensional conformations. Moreover, the cystine-to-diaminodiacid replacement enabled structural tuning, thereby leading to an EETI-II derivative with higher bioactivity than the native peptide.

Copper binding promotes the interaction of cisplatin with human copper chaperone Atox1

Cu(I) binding promotes the platination of Atox1, although cisplatin binds to the copper coordination sites. In addition, Cu(I) binding enhances the competition of Atox1 with DTT in the reaction of cisplatin. These results indicate that cuprous ions could regulate the cellular trafficking of cisplatin.

A genetically encoded 19F NMR probe for tyrosine phosphorylation.

Tyrosine phosphorylation is a pivotal post-translational modification (PTM) which regulates enzymatic activity, protein conformation, and protein–protein interactions. While the eukaryotic protein tyrosine kinases (PTKs) have been intensively studied in the past three decades because of their great importance in cellular signaling and diseases, the prokaryotic PTKs, which play ubiquitous roles in bacterial virulence, were found only recently, and their activation mechanism is controversial. F NMR spectroscopy has recently emerged as a powerful tool for characterizing enzyme mechanisms and protein motions over a range of time scales, because of the high intrinsic sensitivity of fluorine, 100% natural abundance of the NMR-active isotope, the absence of any natural background in proteins, and the exquisite sensitivity of the F chemical shift to environment. Numerous chemical and biosynthetic methods have been developed for incorporating fluorinated amino acids into proteins. Among these methods, the genetic code expansion technique has the unique advantages that fluorinated amino acids can be directly incorporated into specific sites of any protein of interest in living cells, and that the mutant protein can be easily obtained in milligram quantities. However, this powerful method has not been applied to investigate tyrosine phosphorylation. Here we report the highly efficient genetic incorporation of the unnatural amino acid (UAA) 3,5-difluorotyrosine 1 (hereafter termed F2Y, Scheme 1), which mimics the

Crystal structures of the Arabidopsis thaliana abscisic acid receptor PYL10 and its complex with abscisic acid

Abscisic acid (ABA) is one of the most essential phytohormones, and plays an important role in growth and development regulation, as well as in stress responses. The PYR/PYL/RCAR family (PYL for short)-comprised of 14 proteins in Arabidopsis-was recently identified as soluble ABA receptors that function in the perception and transduction of ABA signaling. In this work, the crystal structures of PYL10 were determined in the apo- and ABA-bound states, with respective resolutions of 3.0 and 2.7Å. Surprisingly, a closed CL2 conformation was observed in the apo-PYL10 structure, which was different from a previously reported open CL2 conformation. A putative two-conformation dynamical equilibrium model was proposed to explain PYL10s constitutive binding to PP2Cs in the apo-state and its increased PP2C binding ability in the ABA-bound state.

In situ 19F NMR studies of an E. coli membrane protein

In this report, 19F spin incorporation in a specific site of a specific membrane protein in E. coli was accomplished via trifluoromethyl‐phenylalanine (19F‐tfmF). Site‐specific 19F chemical shifts and longitudinal relaxation times of diacylglycerol kinase (DAGK), an E. coli membrane protein, were measured in its native membrane using in situ magic angle spinning (MAS) solid state nuclear magnetic resonance (NMR). Comparing with solution NMR data of the purified DAGK in detergent micelles, the in situ MAS‐NMR data illustrated that 19F chemical shift values of residues at different membrane protein locations were influenced by interactions between membrane proteins and their surrounding lipid or lipid mimic environments, while 19F side chain longitudinal relaxation values were probably affected by different interactions of DAGK with planar lipid bilayer versus globular detergent micelles.