The primary structural component of the bacterial cell wall is peptidoglycan, which is essential for viability and the synthesis of which is the target for crucial antibiotics
. Peptidoglycan is a ...single macromolecule made of glycan chains crosslinked by peptide side branches that surrounds the cell, acting as a constraint to internal turgor
. In Gram-positive bacteria, peptidoglycan is tens of nanometres thick, generally portrayed as a homogeneous structure that provides mechanical strength
. Here we applied atomic force microscopy
to interrogate the morphologically distinct Staphylococcus aureus and Bacillus subtilis species, using live cells and purified peptidoglycan. The mature surface of live cells is characterized by a landscape of large (up to 60 nm in diameter), deep (up to 23 nm) pores constituting a disordered gel of peptidoglycan. The inner peptidoglycan surface, consisting of more nascent material, is much denser, with glycan strand spacing typically less than 7 nm. The inner surface architecture is location dependent; the cylinder of B. subtilis has dense circumferential orientation, while in S. aureus and division septa for both species, peptidoglycan is dense but randomly oriented. Revealing the molecular architecture of the cell envelope frames our understanding of its mechanical properties and role as the environmental interface
, providing information complementary to traditional structural biology approaches.
In photosynthesis, the harvesting of solar energy and its subsequent conversion into a stable charge separation are dependent upon an interconnected macromolecular network of membrane-associated ...chlorophyll-protein complexes. Although the detailed structure of each complex has been determined, the size and organization of this network are unknown. Here we show the use of atomic force microscopy to directly reveal a native bacterial photosynthetic membrane. This first view of any multi-component membrane shows the relative positions and associations of the photosynthetic complexes and reveals crucial new features of the organization of the network: we found that the membrane is divided into specialized domains each with a different network organization and in which one type of complex predominates. Two types of organization were found for the peripheral light-harvesting LH2 complex. In the first, groups of 10-20 molecules of LH2 form light-capture domains that interconnect linear arrays of dimers of core reaction centre (RC)-light-harvesting 1 (RC-LH1-PufX) complexes; in the second they were found outside these arrays in larger clusters. The LH1 complex is ideally positioned to function as an energy collection hub, temporarily storing it before transfer to the RC where photochemistry occurs: the elegant economy of the photosynthetic membrane is demonstrated by the close packing of these linear arrays, which are often only separated by narrow 'energy conduits' of LH2 just two or three complexes wide.
The effects of the PufX polypeptide on membrane architecture were investigated by comparing the composition and structures of photosynthetic membranes from PufX+ and PufX− strains of Rhodobacter ...sphaeroides. We show that this single polypeptide profoundly affects membrane morphology, leading to highly elongated cells containing extended tubular membranes. Purified tubular membranes contain helical arrays composed solely of dimeric RC–LH1–PufX (RC, reaction centre; LH, light harvesting) complexes with apparently open LH1 rings. PufX− cells contain crystalline membranes with a pseudo‐hexagonal packing of monomeric core complexes. Analysis of purified complexes by electron microscopy and atomic force microscopy shows that LH1 and PufX form a continuous ring of protein around each RC. A model of the tubular membrane is presented with PufX located adjacent to the stained region created by a vacant LH1β. This arrangement, coupled with a flexible ring, would give the RC QB site transient access to the interstices in the lattice, which might be of functional importance. We discuss the implications of our data for the export of quinol from the RC, for eventual reduction of the cytochrome bc1 complex.
The bacterium Rhodospirillum rubrum contains a simple photosynthetic system, in which the reaction center (RC) receives energy from the light-harvesting (LH1) complex. We have used high-resolution ...atomic force microscopy (AFM) to image two-dimensional crystals of the RC-LH1 complex of R. rubrum. The AFM topographs show that the RC-LH1 complex is ∼94 Å in height, the RC-H subunit protrudes from the cytoplasmic face of the membrane by 40 Å, and it sits 21 Å above the highest point of the surrounding LH1 ring. In contrast, the RC on the periplasmic side is at a lower level than LH1, which protrudes from the membrane by 12 Å. The RC-LH1 complex can adopt an irregular shape in regions of uneven packing forces in the crystal; this reflects a likely flexibility in the natural membrane, which might be functionally important by allowing the export of quinol formed as a result of RC photochemistry. Nanodissection of the RC by the AFM tip removes the RC-H subunit and reveals the underlying RC-L and -M subunits. LH1 complexes completely lacking the RC were also found, providing ideal conditions for imaging both rings of LH1 polypeptides for the first time by AFM. In addition, we demonstrate the ellipticity of the LH1 ring at the cytoplasmic and periplasmic sides of the membrane, in both the presence and absence of the RC. These AFM measurements have been reconciled with previous electron microscopy and NMR data to produce a model of the RC-LH1 complex.
Low pH induces a conformational change in the influenza virus haemagglutinin, which then mediates fusion of the viral and host cell membranes. The three-dimensional structure of a fragment of the ...haemagglutinin in this conformation reveals a major refolding of the secondary and tertiary structure of the molecule. The apolar fusion peptide moves at least 100 A to one tip of the molecule. At the other end a helical segment unfolds, a subdomain relocates reversing the chain direction, and part of the structure becomes disordered.
Two-dimensional crystals from light-harvesting complex I (LHC I) of the purple non-sulfur bacterium Rhodospirillum rubrum have been reconstituted from detergent-solubilized protein complexes. ...Frozen-hydrated samples have been analysed by electron microscopy. The crystals diffract beyond 8 angstrom and a projection map was calculated to 8.5 angstrom. The projection map shows 16 subunits in a 116 angstrom diameter ring with a 68 angstrom hole in the centre. These dimensions are sufficient to incorporate a reaction centre in vivo. Within each subunit, density for the alpha - and the beta -polypeptide chains is clearly resolved, and the density for the bacteriochlorophylls can be assigned. The experimentally determined structure contradicts models of the LHC I presented so far.
Hemolysin E (HlyE) is a novel pore-forming toxin of
Escherichia coli,
Salmonella typhi, and
Shigella flexneri. Here we report the X-ray crystal structure of the water-soluble form of
E. coli HlyE at ...2.0 Å resolution and the visualization of the lipid-associated form of the toxin in projection at low resolution by electron microscopy. The crystal structure reveals HlyE to be the first member of a new family of toxin structures, consisting of an elaborated helical bundle some 100 Å long. The electron micrographs show how HlyE oligomerizes in the presence of lipid to form transmembrane pores. Taken together, the data from these two structural techniques allow us to propose a simple model for the structure of the pore and for membrane interaction.
Two‐dimensional crystals from light‐harvesting complex I (LHC I) of the purple non‐sulfur bacterium Rhodospirillum rubrum have been reconstituted from detergent‐solubilized protein complexes. ...Frozen‐hydrated samples have been analysed by electron microscopy. The crystals diffract beyond 8 A and a projection map was calculated to 8.5 A. The projection map shows 16 subunits in a 116 A diameter ring with a 68 A hole in the centre. These dimensions are sufficient to incorporate a reaction centre in vivo. Within each subunit, density for the alpha‐ and the beta‐polypeptide chains is clearly resolved, and the density for the bacteriochlorophylls can be assigned. The experimentally determined structure contradicts models of the LHC I presented so far.
Contamination with the multidrug transporter AcrB represents a potential pitfall in the structural analysis of recombinant membrane proteins expressed in Escherichia coli, especially when ...high-throughput approaches are adopted. This can be a particular problem in two-dimensional (2-D) crystallization for electron cryomicroscopy since individual crystals are too small for compositional analysis. Using a broad ‘sparse matrix’ of buffer conditions typically used in 2-D crystallization, we have identified at least eight unique crystal forms of AcrB. Reference to images and projection maps of these different forms can greatly facilitate the early identification of false leads in 2-D crystallization trials of other membrane proteins of interest. We illustrate the usefulness of such data by highlighting two studies of membrane proteins in our laboratories. We show in one case (a bacterial sodium channel, NaChBac) how early crystallization ‘hits’ could be attributed to contaminating AcrB by comparison against our AcrB crystal image database. In a second case, involving a member of the monovalent cation/proton antiporter-1 family (MPSIL0171), a comparison with the observed AcrB crystal forms allowed easy identification of reconstituted AcrB particles, greatly facilitating the eventual purification and crystallization of the correct protein in pure form as ordered helical arrays. Our database of AcrB crystal images will be of general use in assisting future 2-D crystallization studies of other membrane proteins.
Background:
Bacillus stearothermophilus glycerol dehydrogenase (GlyDH) (glycerol:NAD
+ 2-oxidoreductase, EC 1.1.1.6) catalyzes the oxidation of glycerol to dihydroxyacetone (1,3-dihydroxypropanone) ...with concomitant reduction of NAD
+ to NADH. Analysis of the sequence of this enzyme indicates that it is a member of the so-called iron-containing alcohol dehydrogenase family. Despite this sequence similarity, GlyDH shows a strict dependence on zinc for activity. On the basis of this, we propose to rename this group the family III metal-dependent polyol dehydrogenases. To date, no structural data have been reported for any enzyme in this group.
Results: The crystal structure of
B. stearothermophilus glycerol dehydrogenase has been determined at 1.7 Å resolution to provide structural insights into the mechanistic features of this family. The enzyme has 370 amino acid residues, has a molecular mass of 39.5 kDa, and is a homooctamer in solution.
Conclusions: Analysis of the crystal structures of the free enzyme and of the binary complexes with NAD
+ and glycerol show that the active site of GlyDH lies in the cleft between the enzyme's two domains, with the catalytic zinc ion playing a role in stabilizing an alkoxide intermediate. In addition, the specificity of this enzyme for a range of diols can be understood, as both hydroxyls of the glycerol form ligands to the enzyme-bound Zn
2+ ion at the active site. The structure further reveals a previously unsuspected similarity to dehydroquinate synthase, an enzyme whose more complex chemistry shares a common chemical step with that catalyzed by glycerol dehydrogenase, providing a striking example of divergent evolution. Finally, the structure suggests that the NAD
+ binding domain of GlyDH may be related to that of the classical Rossmann fold by switching the sequence order of the two mononucleotide binding folds that make up this domain.
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