Human gut Bacteroides capture vitamin B12 via cell surface-exposed lipoproteins.

Human gut Bacteroides use surface-exposed lipoproteins to bind and metabolize complex polysaccharides. Although vitamins and other nutrients are also essential for commensal fitness, much less is known about how commensal bacteria compete with each other or the host for these critical resources. Unlike in Escherichia coli, transport loci for vitamin B12 (cobalamin) and other corrinoids in human gut Bacteroides are replete with conserved genes encoding proteins whose functions are unknown. Here we report that one of these proteins, BtuG, is a surface-exposed lipoprotein that is essential for efficient B12 transport in B. thetaiotaomicron. BtuG binds B12 with femtomolar affinity and can remove B12 from intrinsic factor, a critical B12 transport protein in humans. Our studies suggest that Bacteroides use surface-exposed lipoproteins not only for capturing polysaccharides, but also to acquire key vitamins in the gut

Oligomeric States and Hydrodynamic Properties of Lysyl Oxidase-Like 2

Lysyl oxidase-like 2 (LOXL2) has emerged as a promising therapeutic target against metastatic/invasive tumors and organ and tissue fibrosis. LOXL2 catalyzes the oxidative deamination of lysine and hydroxylysine residues in extracellular matrix (ECM) proteins to promote crosslinking of these proteins, and thereby plays a major role in ECM remodeling. LOXL2 secretes as 100-kDa full-length protein (fl-LOXL2) and then undergoes proteolytic cleavage of the first two scavenger receptor cysteine-rich (SRCR) domains to yield 60-kDa protein (Δ1-2SRCR-LOXL2). This processing does not affect the amine oxidase activity of LOXL2 in vitro. However, the physiological importance of this cleavage still remains elusive. In this study, we focused on characterization of biophysical properties of fl- and Δ1-2SRCR-LOXL2s (e.g., oligomeric states, molecular weights, and hydrodynamic radii in solution) to gain insight into the structural role of the first two SRCR domains. Our study reveals that fl-LOXL2 exists predominantly as monomer but also dimer to the lesser extent when its concentration is <~1 mM. The hydrodynamic radius (Rh) determined by multi-angle light scattering coupled with size exclusion chromatography (SEC-MALS) indicates that fl-LOXL2 is a moderately asymmetric protein. In contrast, Δ1-2SRCR-LOXL2 exists solely as monomer and its Rh is in good agreement with the predicted value. The Rh values calculated from a 3D modeled structure of fl-LOXL2 and the crystal structure of the precursor Δ1-2SRCR-LOXL2 are within a reasonable margin of error of the values determined by SEC-MALS for fl- and Δ1-2SRCR-LOXL2s in mature forms in this study. Based on superimposition of the 3D model and the crystal structure of Δ1-2SRCR-LOXL2 (PDB:5ZE3), we propose a configuration of fl-LOXL2 that explains the difference observed in Rh between fl- and Δ1-2SRCR-LOXL2s in solution

The RCK1 domain of the human BK_(Ca) channel transduces Ca^(2+) binding into structural rearrangements

Large-conductance voltage- and Ca^(2+)-activated K^+ (BK_(Ca)) channels play a fundamental role in cellular function by integrating information from their voltage and Ca2+ sensors to control membrane potential and Ca^(2+) homeostasis. The molecular mechanism of Ca^(2+)-dependent regulation of BKCa channels is unknown, but likely relies on the operation of two cytosolic domains, regulator of K^+ conductance (RCK)1 and RCK2. Using solution-based investigations, we demonstrate that the purified BK_(Ca) RCK1 domain adopts an α/β fold, binds Ca^(2+), and assembles into an octameric superstructure similar to prokaryotic RCK domains. Results from steady-state and time-resolved spectroscopy reveal Ca^(2+)-induced conformational changes in physiologically relevant [Ca^(2+)]. The neutralization of residues known to be involved in high-affinity Ca^(2+) sensing (D362 and D367) prevented Ca^(2+)-induced structural transitions in RCK1 but did not abolish Ca^(2+) binding. We provide evidence that the RCK1 domain is a high-affinity Ca^(2+) sensor that transduces Ca^(2+) binding into structural rearrangements, likely representing elementary steps in the Ca^(2+)-dependent activation of human BK_(Ca) channels

Caught in the act: the lifetime of synaptic intermediates during the search for homology on DNA

Homologous recombination plays pivotal roles in DNA repair and in the generation of genetic diversity. To locate homologous target sequences at which strand exchange can occur within a timescale that a cell’s biology demands, a single-stranded DNA-recombinase complex must search among a large number of sequences on a genome by forming synapses with chromosomal segments of DNA. A key element in the search is the time it takes for the two sequences of DNA to be compared, i.e. the synapse lifetime. Here, we visualize for the first time fluorescently tagged individual synapses formed by RecA, a prokaryotic recombinase, and measure their lifetime as a function of synapse length and differences in sequence between the participating DNAs. Surprisingly, lifetimes can be ∼10 s long when the DNAs are fully heterologous, and much longer for partial homology, consistently with ensemble FRET measurements. Synapse lifetime increases rapidly as the length of a region of full homology at either the 3′- or 5′-ends of the invading single-stranded DNA increases above 30 bases. A few mismatches can reduce dramatically the lifetime of synapses formed with nearly homologous DNAs. These results suggest the need for facilitated homology search mechanisms to locate homology successfully within the timescales observed in vivo

Thermodynamics and kinetics for base-pair opening in the P1 duplex of the Tetrahymena group I ribozyme

The thermodynamics and kinetics for base-pair opening of the P1 duplex of the Tetrahymena group I ribozyme were studied by NMR hydrogen exchange experiments. The apparent equilibrium constants for base pair opening were measured for most of the imino protons in the P1 duplex using the base catalysts NH3, HPO42− or TRIS. These equilibrium constants were also measured for several modified P1 duplexes, and the C-2·G23 base pair was the most stable base pair in all the duplexes. The conserved U-1·G22 base pair is required for activity of the ribozyme and the data here show that this wobble base pair destabilizes neighboring base pairs on only one side of the wobble. A 2′-OMe modification on the U-3 residue stabilized its own base pair but had little effect on the neighboring base pairs. Three base pairs, U-1·G22, C-2·G23 and A2·U21 showed unusual equilibrium constants for opening and possible implications of the opening thermodynamics of these base pairs on the undocking rates of the P1 helix with catalytic core are discussed

Mechanism of bacterial interference with TLR4 signaling by Brucella TIR-domain-containing protein TcpB

Upon activation of Toll-like receptors (TLRs), cytoplasmic Toll/interleukin-1 receptor (TIR) domains of the receptors undergo homo- or hetero-dimerization. This in turn leads to the recruitment of adaptor proteins, activation of transcription factors, and the secretion of pro-inflammatory cytokines. Recent studies have described the TIR-domain-containing protein from Brucella melitensis, TcpB (BtpA/Btp1), to be involved in virulence and suppression of host innate immune responses. TcpB interferes with TLR4 and TLR2 signaling pathways by a mechanism that remains controversial. In this study, we show using co-immunoprecipitation analyses that TcpB interacts with MAL, MyD88, and TLR4, but interferes only with the MAL:TLR4 interaction. We present the crystal structure of the TcpB TIR domain, which reveals significant structural differences in the loop regions compared to other TIR domain structures. We demonstrate that TcpB forms a dimer in solution, and the crystal structure reveals the dimerization interface, which we validate by mutagenesis and biophysical studies. Our study advances the understanding of the molecular mechanisms of host immunosuppression by bacterial pathogens

RecA homology search is promoted by mechanical stress along the scanned duplex DNA

A RecA–single-stranded DNA (RecA–ssDNA) filament searches a genome for sequence homology by rapidly binding and unbinding double-stranded DNA (dsDNA) until homology is found. We demonstrate that pulling on the opposite termini (3′ and 5′) of one of the two DNA strands in a dsDNA molecule stabilizes the normally unstable binding of that dsDNA to non-homologous RecA–ssDNA filaments, whereas pulling on the two 3′, the two 5′, or all four termini does not. We propose that the ‘outgoing’ strand in the dsDNA is extended by strong DNA–protein contacts, whereas the ‘complementary’ strand is extended by the tension on the base pairs that connect the ‘complementary’ strand to the ‘outgoing’ strand. The stress resulting from different levels of tension on its constitutive strands causes rapid dsDNA unbinding unless sufficient homology is present

Direct observation of the temperature-induced melting process of the Salmonella fourU RNA thermometer at base-pair resolution

In prokaryotes, RNA thermometers regulate a number of heat shock and virulence genes. These temperature sensitive RNA elements are usually located in the 5′-untranslated regions of the regulated genes. They repress translation initiation by base pairing to the Shine–Dalgarno sequence at low temperatures. We investigated the thermodynamic stability of the temperature labile hairpin 2 of the Salmonella fourU RNA thermometer over a broad temperature range and determined free energy, enthalpy and entropy values for the base-pair opening of individual nucleobases by measuring the temperature dependence of the imino proton exchange rates via NMR spectroscopy. Exchange rates were analyzed for the wild-type (wt) RNA and the A8C mutant. The wt RNA was found to be stabilized by the extraordinarily stable G14–C25 base pair. The mismatch base pair in the wt RNA thermometer (A8–G31) is responsible for the smaller cooperativity of the unfolding transition in the wt RNA. Enthalpy and entropy values for the base-pair opening events exhibit linear correlation for both RNAs. The slopes of these correlations coincide with the melting points of the RNAs determined by CD spectroscopy. RNA unfolding occurs at a temperature where all nucleobases have equal thermodynamic stabilities. Our results are in agreement with a consecutive zipper-type unfolding mechanism in which the stacking interaction is responsible for the observed cooperativity. Furthermore, remote effects of the A8C mutation affecting the stability of nucleobase G14 could be identified. According to our analysis we deduce that this effect is most probably transduced via the hydration shell of the RNA

RecO-mediated DNA homology search and annealing is facilitated by SsbA

Bacillus subtilis RecO plays a central role in recombinational repair and genetic recombination by (i) stimulating RecA filamentation onto SsbA-coated single-stranded (ss) DNA, (ii) modulating the extent of RecA-mediated DNA strand exchange and (iii) promoting annealing of complementary DNA strands. Here, we report that RecO-mediated strand annealing is facilitated by cognate SsbA, but not by a heterologous one. Analysis of non-productive intermediates reveals that RecO interacts with SsbA-coated ssDNA, resulting in transient ternary complexes. The self-interaction of ternary complexes via RecO led to the formation of large nucleoprotein complexes. In the presence of homology, SsbA, at the nucleoprotein, removes DNA secondary structures, inhibits spontaneous strand annealing and facilitates RecO loading onto SsbA–ssDNA complex. RecO relieves SsbA inhibition of strand annealing and facilitates transient and random interactions between homologous naked ssDNA molecules. Finally, both proteins lose affinity for duplex DNA. Our results provide a mechanistic framework for rationalizing protein release and dsDNA zippering as coordinated events that are crucial for RecA-independent plasmid transformation