Structure of a bacterial toxin-activating acyltransferase.

Secreted pore-forming toxins of pathogenic Gram-negative bacteria such as Escherichia coli hemolysin (HlyA) insert into host-cell membranes to subvert signal transduction and induce apoptosis and cell lysis. Unusually, these toxins are synthesized in an inactive form that requires posttranslational activation in the bacterial cytosol. We have previously shown that the activation mechanism is an acylation event directed by a specialized acyl-transferase that uses acyl carrier protein (ACP) to covalently link fatty acids, via an amide bond, to specific internal lysine residues of the protoxin. We now reveal the 2.15-Å resolution X-ray structure of the 172-aa ApxC, a toxin-activating acyl-transferase (TAAT) from pathogenic Actinobacillus pleuropneumoniae. This determination shows that bacterial TAATs are a structurally homologous family that, despite indiscernible sequence similarity, form a distinct branch of the Gcn5-like N-acetyl transferase (GNAT) superfamily of enzymes that typically use acyl-CoA to modify diverse bacterial, archaeal, and eukaryotic substrates. A combination of structural analysis, small angle X-ray scattering, mutagenesis, and cross-linking defined the solution state of TAATs, with intermonomer interactions mediated by an N-terminal α-helix. Superposition of ApxC with substrate-bound GNATs, and assay of toxin activation and binding of acyl-ACP and protoxin peptide substrates by mutated ApxC variants, indicates the enzyme active site to be a deep surface groove.This work was supported by UK Medical Research Council and the Wellcome Trust Grants (to C.H. and V.K.).This is the author accepted manuscript. The final version is available from PNAS via http://dx.doi.org/10.1073/pnas.150383211

The MASSIVE Survey - III. Molecular gas and a broken Tully-Fisher relation in the most massive early-type galaxies

In this work we present CO(1-0) and CO(2-1) observations of a pilot sample of 15 early-type galaxies (ETGs) drawn from the MASSIVE galaxy survey, a volume-limited integral-field spectroscopic study of the most massive ETGs ($M_* >10^{11.5}M_\odot$) within 108 Mpc. These objects were selected because they showed signs of an interstellar medium and/or star formation. A large amount of gas ($>$2$\times$10$^8$ M$_{\odot}$) is present in 10 out of 15 objects, and these galaxies have gas fractions higher than expected based on extrapolation from lower mass samples. We tentatively interpret this as evidence that stellar mass loss and hot halo cooling may be starting to play a role in fuelling the most massive galaxies. These MASSIVE ETGs seem to have lower star-formation efficiencies (SFE=SFR/M$_{\rm H2}$) than spiral galaxies, but the SFEs derived are consistent with being drawn from the same distribution found in other lower mass ETG samples. This suggests that the SFE is not simply a function of stellar mass, but that local, internal processes are more important for regulating star formation. Finally we used the CO line profiles to investigate the high-mass end of the Tully-Fisher relation (TFR). We find that there is a break in the slope of the TFR for ETGs at high masses (consistent with previous studies). The strength of this break correlates with the stellar velocity dispersion of the host galaxies, suggesting it is caused by additional baryonic mass being present in the centre of massive ETGs. We speculate on the root cause of this change and its implications for galaxy formation theories.Comment: 13 pages, 7 figures, accepted by MNRA

Antibiotic Resistance Mediated by the MacB ABC Transporter Family: A Structural and Functional Perspective.

The MacB ABC transporter forms a tripartite efflux pump with the MacA adaptor protein and TolC outer membrane exit duct to expel antibiotics and export virulence factors from Gram-negative bacteria. Here, we review recent structural and functional data on MacB and its homologs. MacB has a fold that is distinct from other structurally characterized ABC transporters and uses a unique molecular mechanism termed mechanotransmission. Unlike other bacterial ABC transporters, MacB does not transport substrates across the inner membrane in which it is based, but instead couples cytoplasmic ATP hydrolysis with transmembrane conformational changes that are used to perform work in the extra-cytoplasmic space. In the MacAB-TolC tripartite pump, mechanotransmission drives efflux of antibiotics and export of a protein toxin from the periplasmic space via the TolC exit duct. Homologous tripartite systems from pathogenic bacteria similarly export protein-like signaling molecules, virulence factors and siderophores. In addition, many MacB-like ABC transporters do not form tripartite pumps, but instead operate in diverse cellular processes including antibiotic sensing, cell division and lipoprotein trafficking

Insights into bacterial lipoprotein trafficking from a structure of LolA bound to the LolC periplasmic domain.

In Gram-negative bacteria, outer-membrane lipoproteins are essential for maintaining cellular integrity, transporting nutrients, establishing infections, and promoting the formation of biofilms. The LolCDE ABC transporter, LolA chaperone, and LolB outer-membrane receptor form an essential system for transporting newly matured lipoproteins from the outer leaflet of the cytoplasmic membrane to the innermost leaflet of the outer membrane. Here, we present a crystal structure of LolA in complex with the periplasmic domain of LolC. The structure reveals how a solvent-exposed β-hairpin loop (termed the "Hook") and trio of surface residues (the "Pad") of LolC are essential for recruiting LolA from the periplasm and priming it to receive lipoproteins. Experiments with purified LolCDE complex demonstrate that association with LolA is independent of nucleotide binding and hydrolysis, and homology models based on the MacB ABC transporter predict that LolA recruitment takes place at a periplasmic site located at least 50 Å from the inner membrane. Implications for the mechanism of lipoprotein extraction and transfer are discussed. The LolA-LolC structure provides atomic details on a key protein interaction within the Lol pathway and constitutes a vital step toward the complete molecular understanding of this important system.This work was supported by grants from the UK Medical Research Council (MR/N000994/1) and the Wellcome Trust (101828/Z/13/Z)

The MASSIVE Survey - I. A Volume-Limited Integral-Field Spectroscopic Study of the Most Massive Early-Type Galaxies within 108 Mpc

Massive early-type galaxies represent the modern-day remnants of the earliest major star formation episodes in the history of the universe. These galaxies are central to our understanding of the evolution of cosmic structure, stellar populations, and supermassive black holes, but the details of their complex formation histories remain uncertain. To address this situation, we have initiated the MASSIVE Survey, a volume-limited, multi-wavelength, integral-field spectroscopic (IFS) and photometric survey of the structure and dynamics of the ~100 most massive early-type galaxies within a distance of 108 Mpc. This survey probes a stellar mass range M* > 10^{11.5} Msun and diverse galaxy environments that have not been systematically studied to date. Our wide-field IFS data cover about two effective radii of individual galaxies, and for a subset of them, we are acquiring additional IFS observations on sub-arcsecond scales with adaptive optics. We are also acquiring deep K-band imaging to trace the extended halos of the galaxies and measure accurate total magnitudes. Dynamical orbit modeling of the combined data will allow us to simultaneously determine the stellar, black hole, and dark matter halo masses. The primary goals of the project are to constrain the black hole scaling relations at high masses, investigate systematically the stellar initial mass function and dark matter distribution in massive galaxies, and probe the late-time assembly of ellipticals through stellar population and kinematical gradients. In this paper, we describe the MASSIVE sample selection, discuss the distinct demographics and structural and environmental properties of the selected galaxies, and provide an overview of our basic observational program, science goals and early survey results.Comment: 19 pages, 14 figures. ApJ (2014) vol. 795, in pres

The MASSIVE Survey II: Stellar Population Trends Out to Large Radius in Massive Early Type Galaxies

We examine stellar population gradients in ~100 massive early type galaxies spanning 180 < sigma* < 370 km/s and M_K of -22.5 to -26.5 mag, observed as part of the MASSIVE survey (Ma et al. 2014). Using integral-field spectroscopy from the Mitchell Spectrograph on the 2.7m telescope at McDonald Observatory, we create stacked spectra as a function of radius for galaxies binned by their stellar velocity dispersion, stellar mass, and group richness. With excellent sampling at the highest stellar mass, we examine radial trends in stellar population properties extending to beyond twice the effective radius (~2.5 R_e). Specifically, we examine trends in age, metallicity, and abundance ratios of Mg, C, N, and Ca, and discuss the implications for star formation histories and elemental yields. At a fixed physical radius of 3-6 kpc (the likely size of the galaxy cores formed at high redshift) stellar age and [alpha/Fe] increase with increasing sigma* and depend only weakly on stellar mass, as we might expect if denser galaxies form their central cores earlier and faster. If we instead focus on 1-1.5 R_e, the trends in abundance and abundance ratio are washed out, as might be expected if the stars at large radius were accreted by smaller galaxies. Finally, we show that when controlling for \sigmastar, there are only very subtle differences in stellar population properties or gradients as a function of group richness; even at large radius internal properties matter more than environment in determining star formation history.Comment: 17 pages, 9 figures, accepted by ApJ; resubmitted with updated reference

The MASSIVE Survey - VII. The Relationship of Angular Momentum, Stellar Mass and Environment of Early-Type Galaxies

We analyse the environmental properties of 370 local early-type galaxies (ETGs) in the MASSIVE and ATLAS3D surveys, two complementary volume-limited integral-field spectroscopic (IFS) galaxy surveys spanning absolute $K$-band magnitude $-21.5 > M_K > -26.6$, or stellar mass $8 \times 10^{9} < M_* < 2 \times 10^{12} M_\odot$. We find these galaxies to reside in a diverse range of environments measured by four methods: group membership (whether a galaxy is a brightest group/cluster galaxy, satellite, or isolated), halo mass, large-scale mass density (measured over a few Mpc), and local mass density (measured within the $N$th neighbour). The spatially resolved IFS stellar kinematics provide robust measurements of the spin parameter $\lambda_e$ and enable us to examine the relationship among $\lambda_e$, $M_*$, and galaxy environment. We find a strong correlation between $\lambda_e$ and $M_*$, where the average $\lambda_e$ decreases from $\sim 0.4$ to below 0.1 with increasing mass, and the fraction of slow rotators $f_{\rm slow}$ increases from $\sim 10$% to 90%. We show for the first time that at fixed $M_*$, there are almost no trends between galaxy spin and environment; the apparent kinematic morphology-density relation for ETGs is therefore primarily driven by $M_*$ and is accounted for by the joint correlations between $M_*$ and spin, and between $M_*$ and environment. A possible exception is that the increased $f_{\rm slow}$ at high local density is slightly more than expected based only on these joint correlations. Our results suggest that the physical processes responsible for building up the present-day stellar masses of massive galaxies are also very efficient at reducing their spin, in any environment.Comment: Accepted to MNRA

The MASSIVE Survey XIII -- Spatially Resolved Stellar Kinematics in the Central 1 kpc of 20 Massive Elliptical Galaxies with the GMOS-North Integral-Field Spectrograph

We use observations from the GEMINI-N/GMOS integral-field spectrograph (IFS) to obtain spatially resolved stellar kinematics of the central $\sim 1$ kpc of 20 early-type galaxies (ETGs) with stellar masses greater than $10^{11.7} M_\odot$ in the MASSIVE survey. Together with observations from the wide-field Mitchell IFS at McDonald Observatory in our earlier work, we obtain unprecedentedly detailed kinematic maps of local massive ETGs, covering a scale of $\sim 0.1-30$ kpc. The high ($\sim 120$) signal-to-noise of the GMOS spectra enable us to obtain two-dimensional maps of the line-of-sight velocity, velocity dispersion $\sigma$, as well as the skewness $h_3$ and kurtosis $h_4$ of the stellar velocity distributions. All but one galaxy in the sample have $\sigma(R)$ profiles that increase towards the center, whereas the slope of $\sigma(R)$ at one effective radius ($R_e$) can be of either sign. The $h_4$ is generally positive, with 14 of the 20 galaxies having positive $h_4$ within the GMOS aperture and 18 having positive $h_4$ within $1 R_e$. The positive $h_4$ and rising $\sigma(R)$ towards small radii are indicative of a central black hole and velocity anisotropy. We demonstrate the constraining power of the data on the mass distributions in ETGs by applying Jeans anisotropic modeling (JAM) to NGC~1453, the most regular fast rotator in the sample. Despite the limitations of JAM, we obtain a clear $\chi^2$ minimum in black hole mass, stellar mass-to-light ratio, velocity anisotropy parameters, and the circular velocity of the dark matter halo.Comment: Accepted to Ap

Structure of CYRI-B (FAM49B), a key regulator of cellular actin assembly.

In eukaryotes, numerous fundamental processes are controlled by the WAVE regulatory complex (WRC) that regulates cellular actin polymerization, crucial for cell motility, cell-cell adhesion and epithelial differentiation. Actin assembly is triggered by interaction of the small GTPase Rac1 with CYFIP1, a key component of the WRC. Previously known as FAM49B, CYRI-B is a protein that is highly conserved across the Eukaryota and has recently been revealed to be a key regulator of Rac1 activity. Mutation of CYRI-B or alteration of its expression therefore leads to altered actin nucleation dynamics, with impacts on lamellipodia formation, cell migration and infection by intracellular pathogens. In addition, knockdown of CYRI-B expression in cancer cell lines results in accelerated cell proliferation and invasiveness. Here, the structure of Rhincodon typus (whale shark) CYRI-B is presented, which is the first to be reported of any CYRI family member. Solved by X-ray crystallography, the structure reveals that CYRI-B comprises three distinct α-helical subdomains and is highly structurally related to a conserved domain present in CYFIP proteins. The work presented here establishes a template towards a better understanding of CYRI-B biological function

Structure of an atypical periplasmic adaptor from a multidrug efflux pump of the spirochete Borrelia burgdorferi.

Periplasmic adaptor proteins are essential components of bacterial tripartite multidrug efflux pumps. Here we report the 2.35 Å resolution crystal structure of the BesA adaptor from the spirochete Borrelia burgdorferi solved using selenomethionine derivatized protein. BesA shows the archetypal linear, flexible, multi-domain architecture evident among proteobacteria and retains the lipoyl, β-barrel and membrane-proximal domains that interact with the periplasmic domains of the inner membrane transporter. However, it lacks the α-hairpin domain shown to establish extensive coiled-coil interactions with the periplasmic entrance helices of the outer membrane-anchored TolC exit duct. This has implications for the modelling of assembled tripartite efflux pumps.This is the final published version. It is available from the publisher here: http://www.sciencedirect.com/science/article/pii/S0014579313005206