Probing the spatial organization of bacteriochlorophyll C by solid-state nuclear magnetic resonance.

Green sulfur bacteria, which live in extremely low-light environments, use chlorosomes to harvest light. A chlorosome is the most efficient, and arguably the simplest, light-harvesting antenna complex, which contains hundreds of thousands of densely packed bacteriochlorophylls (BChls). To harvest light efficiently, BChls in a chlorosome form supramolecular aggregates; thus, it is of great interest to determine the organization of the BChls in a chlorosome. In this study, we conducted a (13)C solid-state nuclear magnetic resonance and Mg K-edge X-ray absorption analysis of chlorosomes from wild-type Chlorobaculum tepidum. The X-ray absorption results indicated that the coordination number of the Mg in the chlorosome must be >4, providing evidence that electrostatic interactions formed between the Mg of a BChl and the carbonyl group or the hydroxyl group of the neighboring BChl molecule. According to the intermolecular distance constraints obtained on the basis of (13)C homonuclear dipolar correlation spectroscopy, we determined that the molecular assembly of BChls is dimer-based and that the hydrogen bonds among the BChls are less extensive than commonly presumed because of the twist in the orientation of the BChl dimers. This paper also reports the first (13)C homonuclear correlation spectrum acquired for carotenoids and lipids-which are minor, but crucial, components of chlorosomes-extracted from wild-type Cba. tepidum.

Structural basis of a physical blockage mechanism for the interaction of response regulator PmrA with connector protein PmrD from Klebsiella pneumoniae.

In bacteria, the two-component system is the most prevalent for sensing and transducing environmental signals into the cell. The PmrA-PmrB two-component system, responsible for sensing external stimuli of high Fe(3+) and mild acidic conditions, can control the genes involved in lipopolysaccharide modification and polymyxin resistance in pathogens. In Klebsiella pneumoniae, the small basic connector protein PmrD protects phospho-PmrA and prolongs the expression of PmrA-activated genes. We previously determined the phospho-PmrA recognition mode of PmrD. However, how PmrA interacts with PmrD and prevents its dephosphorylation remains unknown. To address this question, we solved the x-ray crystal structure of the N-terminal receiver domain of BeF3(-)-activated PmrA (PmrA(N)) at 1.70 Å. With this structure, we applied the data-driven docking method based on NMR chemical shift perturbation to generate the complex model of PmrD-PmrA(N), which was further validated by site-directed spin labeling experiments. In the complex model, PmrD may act as a blockade to prevent phosphatase from contacting with the phosphorylation site on PmrA.

Purification of recombinant nacre-associated mineralization protein AP7 fused with maltose-binding protein.

Formation of biominerals often involves specific proteins that modulate the process of matrix assembly, nucleation, and crystal growth. AP7 is an aragonite-associated protein of 7 kDa and is intrinsically disordered. The structural disorder of AP7 makes it very difficult to express in Escherchiacoli. In this work, we report the first successful expression and purification of recombinant AP7 using the maltose-binding protein (MBP) fusion approach. We obtain a high-yield production of recombinant MBP-AP7 protein inE. coli (∼60 mg/L). We also establish an efficient protocol to remove the MBP fusion protein by Factor Xa, followed by purification using size-exclusion chromatography. Characterization of the recombinant AP7 protein has been carried out using MALDI-TOF, peptide mass fingerprinting, and circular dichroism (CD). The mass data confirm that the purified recombinant protein is AP7. The CD data suggest that the recombinant AP7 protein exists as partially disordered structure at neutral pH. The calcium carbonate precipitation assay shows that both MBP-AP7 and AP7 exhibit morphological modification on calcite crystallites. The co-precipitation of MBP-tagged AP7 derivatives and calcium carbonate generate different types of AP7 composite calcite and vaterite crystals. This system should be helpful to establish a model for understanding the structure/function relationship between the protein and inorganic mineral interaction.

A RAD51-ADP double filament structure unveils the mechanism of filament dynamics in homologous recombination.

ATP-dependent RAD51 recombinases play an essential role in eukaryotic homologous recombination by catalyzing a four-step process: 1) formation of a RAD51 single-filament assembly on ssDNA in the presence of ATP, 2) complementary DNA strand-exchange, 3) ATP hydrolysis transforming the RAD51 filament into an ADP-bound disassembly-competent state, and 4) RAD51 disassembly to provide access for DNA repairing enzymes. Of these steps, filament dynamics between the ATP- and ADP-bound states, and the RAD51 disassembly mechanism, are poorly understood due to the lack of near-atomic-resolution information of the ADP-bound RAD51-DNA filament structure. We report the cryo-EM structure of ADP-bound RAD51-DNA filaments at 3.1 Å resolution, revealing a unique RAD51 double-filament that wraps around ssDNA. Structural analysis, supported by ATP-chase and time-resolved cryo-EM experiments, reveals a collapsing mechanism involving two four-protomer movements along ssDNA for mechanical transition between RAD51 single- and double-filament without RAD51 dissociation. This mechanism enables elastic change of RAD51 filament length during structural transitions between ATP- and ADP-states.

Identification of fidelity-governing factors in human recombinases DMC1 and RAD51 from cryo-EM structures.

Both high-fidelity and mismatch-tolerant recombination, catalyzed by RAD51 and DMC1 recombinases, respectively, are indispensable for genomic integrity. Here, we use cryo-EM, MD simulation and functional analysis to elucidate the structural basis for the mismatch tolerance of DMC1. Structural analysis of DMC1 presynaptic and postsynaptic complexes suggested that the lineage-specific Loop 1 Gln244 (Met243 in RAD51) may help stabilize DNA backbone, whereas Loop 2 Pro274 and Gly275 (Val273/Asp274 in RAD51) may provide an open "triplet gate" for mismatch tolerance. In support, DMC1-Q244M displayed marked increase in DNA dynamics, leading to unobservable DNA map. MD simulation showed highly dispersive mismatched DNA ensemble in RAD51 but well-converged DNA in DMC1 and RAD51-V273P/D274G. Replacing Loop 1 or Loop 2 residues in DMC1 with RAD51 counterparts enhanced DMC1 fidelity, while reciprocal mutations in RAD51 attenuated its fidelity. Our results show that three Loop 1/Loop 2 residues jointly enact contrasting fidelities of DNA recombinases.

Solution structure and phospho-PmrA recognition mode of PmrD from Klebsiella pneumoniae.

In bacteria, the two-component system (TCS) is the most prevalent for sensing and transducing the environmental signals into the cell. In Salmonella, the small basic protein PmrD is found to protect phospho-PmrA and prolong the expression of PmrA-activated genes. In contrast, Escherichia coli PmrD fails to protect phospho-PmrA. Here, we show that Klebsiella pneumoniae PmrD (KP-PmrD) can inhibit the dephosphrylation of phospho-PmrA, and the interaction between KP-PmrD and the N-terminal receiver domain of PmrA (PmrA(N)) is much stronger in the presence than in the absence of the phosphoryl analog beryllofluoride (BeF(3)(-)) (K(D)=1.74 ± 0.81 µM vs. K(D)=236 ± 48 µM). To better understand the molecular interactions involved, the solution structure of KP-PmrD was found to comprise six ß-strands and a flexible C-terminal α-helix. Amide chemical shift perturbations of KP-PmrD in complex with BeF(3)(-)-activated PmrA(N) suggested that KP-PmrD may undergo a certain conformational rearrangement on binding to activated PmrA(N). Saturation transfer experiments revealed the binding surface to be located on one face of the ß-barrel. This finding was further verified by in vivo polymyxin B susceptibility assay of the mutants of KP-PmrD. The phospho-PmrA recognition surface of KP-PmrD, which involves two KP-PmrD proteins in complex with an activated-PmrA(N) dimer, is suggested to be a contiguous patch consisting of Trp3, Trp4, Ser23, Leu26, Glu27, Met28, Thr46, Leu48, Ala49, Asp50, Ala51, Arg52, Ile65, Asn67, Ala68, Thr69, His70, Tyr71, Ser73 and Glu74. Our study furthers the understanding of how PmrD protects phopho-PmrA in the PmrAB TCS.

Multi-enzyme one-pot strategy for the synthesis of sialyl Lewis X-containing PSGL-1 glycopeptide.

An enzymatic one-pot three-step glycosylation strategy was developed for the synthesis of sLex moiety of truncated PSGL-1 glycopeptide with and without sulfation. The method provided an efficient way to afford complex glycopeptides in a semi-preparative scale without further complicated and time-consuming purification process in each glycosylation step.

Human RegIV protein adopts a typical C-type lectin fold but binds mannan with two calcium-independent sites.

Human RegIV protein, which contains a sequence motif homologous to calcium-dependent (C-type) lectin-like domain, is highly expressed in mucosa cells of the gastrointestinal tract during pathogen infection and carcinogenesis and may be applied in both diagnosis and treatment of gastric and colon cancers. Here, we provide evidence that, unlike other C-type lectins, human RegIV binds to polysaccharides, mannan, and heparin in the absence of calcium. To elucidate the structural basis for carbohydrate recognition by NMR, we generated the mutant with Pro91 replaced by Ser (hRegIV-P91S) and showed that the structural property and carbohydrate binding ability of hRegIV-P91S are almost identical with those of wild-type protein. The solution structure of hRegIV-P91S was determined, showing that it adopts a typical fold of C-type lectin. Based on the chemical shift perturbations of amide resonances, two calcium-independent mannan-binding sites were proposed. One site is similar to the calcium-independent sugar-binding site on human RegIII and Langerin. Interestingly, the other site is adjacent to the conserved calcium-dependent site at position Ca-2 of typical C-type lectins. Moreover, model-free analysis of (15)N relaxation parameters and simplified Carr-Purcell-Meiboom-Gill relaxation dispersion experiments showed that a slow microsecond-to-millisecond time-scale backbone motion is involved in mannan binding by this site, suggesting a potential role for specific carbohydrate recognition. Our findings shed light on the sugar-binding mode of Reg family proteins, and we postulate that Reg family proteins evolved to bind sugar without calcium to keep the carbohydrate recognition activity under low-pH environments in the gastrointestinal tract.

Identification of Tbr-1/CASK complex target genes in neurons.

Tbr-1, a neuron-specific T-box transcription factor, plays a critical role in brain development. Here, we performed a computational search using the non-palindromic T-box binding sequence, namely the non-palindromic T-element, to determine the putative downstream target genes of Tbr-1. More than 20 identified genes containing the non-palindromic T-element in the 5' regulatory region were found expressed in brain. Luciferase reporter assays using cultured hippocampal neurons showed that overexpression of Tbr-1 and CASK-enhanced promoter activities of some of these putative target genes, including NMDAR subunit 2b (NR2b), glycine transporter, interleukin 7 receptor (IL-7R) and OX-2. Among these genes, NR2b promoter responded strongest to overexpression of Tbr-1 and CASK. Deletion of the non-palindromic T-elements from NR2b promoter impaired the induction by Tbr-1 and CASK. We also examined expression of these target genes in Tbr-1 knockout mice, it was found that NR2b expression was consistently downregulated. Similarly, both RNA and protein expression levels of NMDAR subunit 1 (NR1), which also contains the non-palindromic T-elements in its 5' regulatory region, were reduced in Tbr-1 knockout mice. We suggest that Tbr-1/CASK protein complex regulates expression of these downstream target genes and thus modulates neuronal activity and function.

Maturation processing and characterization of streptopain.

Streptopain is a cysteine protease expressed by Streptococcus pyogenes. To study the maturation mechanism of streptopain, wild-type and Q186N, C192S, H340R, N356D and W357A mutant proteins were expressed in Escherichia coli and purified to homogeneity. Proteolytic analyses showed that the maturation of prostreptococcal pyrogenic exotoxin B zymogen (pro-SPE B) involves eight intermediates with a combination of cis- and trans-processing. Based on the sequences of these intermediates, the substrate specificity of streptopain favors a hydrophobic residue at the P2 site. The relative autocatalytic rates of these mutants exhibited the order Q186N > W357A > N356D, C192S, H340R. Interestingly, the N356D mutant containing protease activity could not be converted into the 28-kDa form by autoprocessing. This observation suggested that Asn(356) might involve the cis-processing of the propeptide. In addition, the maturation rates of pro-SPE B with trypsin and plasmin were 10- and 60-fold slower than that with active mature streptopain. These findings indicate that active mature streptopain likely plays the most important role in the maturation of pro-SPE B under physiological conditions.