Biomedical subjects

K P Locher

Publications and source records attributed to K P Locher.

7 recordsLinked to original sources

PubMed2006

An inward-facing conformation of a putative metal-chelate-type ABC transporter.

The crystal structure of a putative metal-chelate-type adenosine triphosphate (ATP)-binding cassette (ABC) transporter encoded by genes HI1470 and HI1471 of Haemophilus influenzae has been solved at 2.4 angstrom resolution. The permeation pathway exhibits an inward-facing conformation, in contrast to the outward-facing state previously observed for the homologous vitamin B12 importer BtuCD. Although the structures of both HI1470/1 and BtuCD have been solved in nucleotide-free states, the pairs of ABC subunits in these two structures differ by a translational shift in the plane of the membrane that coincides with a repositioning of the membrane-spanning subunits. The differences observed between these ABC transporters involve relatively modest rearrangements and may serve as structural models for inward- and outward-facing conformations relevant to the alternating access mechanism of substrate translocation.

ATP-Binding Cassette Transporters↗

PubMed2001

Crystal structure of the Acidaminococcus fermentans 2-hydroxyglutaryl-CoA dehydratase component A.

Acidaminococcus fermentans degrades glutamate via the hydroxyglutarate pathway, which involves the syn-elimination of water from (R)-2-hydroxyglutaryl-CoA in a key reaction of the pathway. This anaerobic process is catalyzed by 2-hydroxyglutaryl-CoA dehydratase, an enzyme with two components (A and D) that reversibly associate during reaction cycles. Component A (CompA), a homodimeric protein of 2x27 kDa, contains a single, bridging [4Fe-4S] cluster and uses the hydrolysis of ATP to deliver an electron to the dehydratase component (CompD), where the electron is used catalytically. The structure of the extremely oxygen-sensitive CompA protein was solved by X-ray crystallography to 3 A resolution. The protein was found to be a member of the actin fold family, revealing a similar architecture and nucleotide-binding site. The key differences between CompA and other members of the actin fold family are: (i) the presence of a cluster binding segment, the "cluster helix"; (ii) the [4Fe-4S] cluster; and (iii) the location of the homodimer interface, which involves the bridging cluster. Possible reaction mechanisms are discussed in light of the close structural similarity to members of the actin-fold family and the functional similarity to the nitrogenase Fe- protein.

Actins↗

PubMed2000

Structure and function of bacterial outer membrane proteins: barrels in a nutshell.

The outer membrane protects Gram-negative bacteria against a harsh environment. At the same time, the embedded proteins fulfil a number of tasks that are crucial to the bacterial cell, such as solute and protein translocation, as well as signal transduction. Unlike membrane proteins from all other sources, integral outer membrane proteins do not consist of transmembrane alpha-helices, but instead fold into antiparallel beta-barrels. Over recent years, the atomic structures of several outer membrane proteins, belonging to six families, have been determined. They include the OmpA membrane domain, the OmpX protein, phospholipase A, general porins (OmpF, PhoE), substrate-specific porins (LamB, ScrY) and the TonB-dependent iron siderophore transporters FhuA and FepA. These crystallographic studies have yielded invaluable insight into and decisively advanced the understanding of the functions of these intriguing proteins. Our review is aimed at discussing their common principles and peculiarities as well as open questions associated with them.

Bacterial Outer Membrane Proteins↗

PubMed1998

Transmembrane signaling across the ligand-gated FhuA receptor: crystal structures of free and ferrichrome-bound states reveal allosteric changes.

FhuA protein facilitates ligand-gated transport of ferrichrome-bound iron across Escherichia coli outer membranes. X-ray analysis at 2.7 A resolution reveals two distinct conformations in the presence and absence of ferrichrome. The monomeric protein consists of a hollow, 22-stranded, antiparallel beta barrel (residues 160-714), which is obstructed by a plug (residues 19-159). The binding site of ferrichrome, an aromatic pocket near the cell surface, undergoes minor changes upon association with the ligand. These are propagated and amplified across the plug, eventually resulting in substantially different protein conformations at the periplasmic face. Our findings reveal the mechanism of signal transmission and suggest how the energy-transducing TonB complex senses ligand binding.

Allosteric Regulation↗

PubMed1998

Inactivation in vitro of the Escherichia coli outer membrane protein FhuA by a phage T5-encoded lipoprotein.

Bacteriophage T5-encoded lipoprotein, synthesized by infected Escherichia coli cells, prevents superinfection of the host cell by this virus. The molecular basis of its ability to inactivate the receptor of phage T5, the FhuA protein, was investigated in vitro. Fully competent T5 lipoprotein, with a His tag attached to the C-terminus, was purified in detergent solution. Coreconstitution with homogeneous FhuA protein into liposomes revealed that the lipoprotein inhibited the irreversible inactivation of phage T5 by FhuA protein. This phenomenon correlated with the inhibition of phage DNA ejection determined by fluorescence monitoring. Addition of detergent abolished the interaction between T5 lipoprotein and FhuA protein. When the signal sequence and N-terminal cysteinyl residue of the lipoprotein were removed by genetic truncation, the soluble polypeptide could be refolded and purified from inclusion bodies. The truncated lipoprotein interfered with infection of E. coli by phage T5, but only at very high concentrations. Circular dichroism spectra of both forms of T5 lipoprotein exhibited predominantly beta-structure. T5 lipoprotein is sufficient for inactivation of the FhuA protein, presumably by inserting the N-terminal acyl chains into the membrane, thus increasing its local concentration. An in vitro stoichiometry of 10:1 has been calculated for the phage-encoded T5 lipoprotein to FhuA protein complex.

Bacterial Outer Membrane Proteins↗

PubMed1997

Oligomeric states and siderophore binding of the ligand-gated FhuA protein that forms channels across Escherichia coli outer membranes.

The channel-forming FhuA protein, which translocates ferrichrome across Escherichia coli outer membranes, binds 1 mol ligand/mol monomer in detergent solution. The protein is homogenous and migrates as a single band with a mobility corresponding to 77 kDa in SDS/PAGE electrophoresis. Analytical ultracentrifugation revealed a monodisperse species (s(20,w) = 3.8 S) with a mass of 77,800 +/- 3200 Da. The properties of ligand binding, determined by two independent methods, revealed one binding site/monomer, but are complicated by a pronounced convexity of the Scatchard plot and a Hill coefficient calculated to be 2.5. This strongly suggests that oligomeric species are present. Cross-linking agents revealed the existence of possibly transient, mostly dimeric and trimeric species. The difference between the FhuA protein in detergent solution and in its native membrane environment may be related to the removal of lateral pressure that exists in situ.

Bacterial Outer Membrane Proteins↗

PubMed1997

Modeling ligand-gated receptor activity. FhuA-mediated ferrichrome efflux from lipid vesicles triggered by phage T5.

An in vitro assay of iron-ferrichrome translocation across the FhuA protein of outer membranes from Escherichia coli has been devised. Upon reconstitution into large lipid vesicles, bacteriophage T5 binds to this polyvalent receptor, triggering a conformational change that resulted in channel opening. This facilitates the translocation of an iron(III)-siderophore, without the complexities involved in the in vivo process. Efflux of 55Fe(III)-ferrichrome across FhuA channels was determined quantitatively by monitoring the release of trapped radioactivity. The assay is rapid, reliable, and specific, because other bacteriophages, such as Phi80, fail to trigger channel opening of the FhuA receptor.

Bacterial Outer Membrane Proteins↗