Biomedical subjects

Christina Sizun

Publications and source records attributed to Christina Sizun.

6 recordsLinked to original sources

PubMed2006

Structure and dynamics of membrane-associated ICP47, a viral inhibitor of the MHC I antigen-processing machinery.

To evade the host's immune response, herpes simplex virus employs the immediate early gene product ICP47 (IE12) to suppress antigen presentation to cytotoxic T-lymphocytes by inhibition of the ATP-binding cassette transporter associated with antigen processing (TAP). ICP47 is a membrane-associated protein adopting an alpha-helical conformation. Its active domain was mapped to residues 3-34 and shown to encode all functional properties of the full-length protein. The active domain of ICP47 was reconstituted into oriented phospholipid bilayers and studied by proton-decoupled 15N and 2H solid-state NMR spectroscopy. In phospholipid bilayers, the protein adopts a helix-loop-helix structure, where the average tilt angle of the helices relative to the membrane surface is approximately 15 degrees (+/- 7 degrees ). The alignment of both structured domains exhibits a mosaic spread of approximately 10 degrees . A flexible dynamic loop encompassing residues 17 and 18 separates the two helices. Refinement of the experimental data indicates that helix 1 inserts more deeply into the membrane. These novel insights into the structure of ICP47 represent an important step toward a molecular understanding of the immune evasion mechanism of herpes simplex virus and are instrumental for the design of new therapeutics.

Cell Membrane↗

PubMed2006

Structure of the human multidrug resistance protein 1 nucleotide binding domain 1 bound to Mg2+/ATP reveals a non-productive catalytic site.

Human multidrug resistance protein 1 (MRP1) is a membrane protein that belongs to the ATP-binding cassette (ABC) superfamily of transport proteins. MRP1 contributes to chemotherapy failure by exporting a wide range of anti-cancer drugs when over expressed in the plasma membrane of cells. Here, we report the first high-resolution crystal structure of human MRP1-NBD1. Drug efflux requires energy resulting from hydrolysis of ATP by nucleotide binding domains (NBDs). Contrary to the prokaryotic NBDs, the extremely low intrinsic ATPase activity of isolated MRP1-NBDs allowed us to obtain the structure of wild-type NBD1 in complex with Mg2+/ATP. The structure shows that MRP1-NBD1 adopts a canonical fold, but reveals an unexpected non-productive conformation of the catalytic site, providing an explanation for the low intrinsic ATPase activity of NBD1 and new hypotheses on the cooperativity of ATPase activity between NBD1 and NBD2 upon heterodimer formation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2006

Proton-decoupled 15N and 31P solid-state NMR investigations of the Pf3 coat protein in oriented phospholipid bilayers.

The coat proteins of filamentous phage are first synthesized as transmembrane proteins and then assembled onto the extruding viral particles. We investigated the transmembrane conformation of the Pseudomonas aeruginosa Pf3 phage coat protein using proton-decoupled 15N and 31P solid-state NMR spectroscopy. The protein was either biochemically purified and uniformly labelled with 15N or synthesized chemically and labelled at specific sites. The proteins were then reconstituted into oriented phospholipid bilayers and the resulting samples analysed. The data suggest a model in which the protein adopts a tilted helix with an angle of approximately 30 degrees and an N-terminal 'swinging arm' at the membrane surface.

Capsid Proteins↗

PubMed2005

Attempts to characterize the NBD heterodimer of MRP1: transient complex formation involves Gly771 of the ABC signature sequence but does not enhance the intrinsic ATPase activity.

MRP1 (multidrug-resistance-associated protein 1; also known as ABCC1) is a member of the human ABC (ATP-binding cassette) transporter superfamily that confers cell resistance to chemotherapeutic agents. Considering the structural and functional similarities to the other ABC proteins, the interaction between its two NBDs (nucleotide-binding domains), NBD1 (N-terminal NBD) and NBD2 (C-terminal NBD), is proposed to be essential for the regulation of the ATP-binding/ATP-hydrolysis cycle of MRP1. We were interested in the ability of recombinant NBD1 and NBD2 to interact with each other and to influence ATPase activity. We purified NBD1 (Asn642-Ser871) and NBD2 (Ser1286-Val1531) as soluble monomers under native conditions. We measured extremely low intrinsic ATPase activity of NBD1 (10(-5) s(-1)) and NBD2 (6x10(-6) s(-1)) and no increase in the ATP-hydrolysis rate could be detected in an NBD1+NBD2 mixture, with concentrations up to 200 microM. Despite the fact that both monomers bind ATP, no stable NBD1.NBD2 heterodimer could be isolated by gel-filtration chromatography or native-PAGE, but we observed some significant modifications of the heteronuclear single-quantum correlation NMR spectrum of 15N-NBD1 in the presence of NBD2. This apparent NBD1.NBD2 interaction only occurred in the presence of Mg2+ and ATP. Partial sequential assignment of the NBD1 backbone resonances shows that residue Gly771 of the LSGGQ sequence is involved in NBD1.NBD2 complex formation. This is the first NMR observation of a direct interaction between the ABC signature and the opposite NBD. Our study also reveals that the NBD1.NBD2 heterodimer of MRP1 is a transient complex. This labile interaction is not sufficient to induce an ATPase co-operativity of the NBDs and suggests that other structures are required for the ATPase activation mechanism.

Adenosine Triphosphatases↗

PubMed2004

NMR methods for studying the structure and dynamics of oncogenic and antihistaminic peptides in biomembranes.

We present several applications of both wide-line and magic angle spinning (MAS) solid-state NMR of bicelles in which are embedded fragments of a tyrosine kinase receptor or enkephalins. The magnetically orientable bicelle membranes are shown to be of particular interest for studying the functional properties of lipids and proteins in a state that is very close to their natural environment. Quadrupolar, dipolar and chemical shielding interactions can be used to determine minute alterations of internal membrane dynamics and the orientation of peptides with respect to the membrane plane. MAS of bicelles can in turn lead to high-resolution proton spectra of hydrated membranes. Using deuterium-proton contrast methods one can then obtain pseudo-high-resolution proton spectra of peptides or proteins embedded in deuterated membranes and determine their atomic 3D structure using quasi-conventional liquid-state NMR methods.

Chlorobenzenes↗

PubMed2002

Bilayer sample for fast or slow magic angle oriented sample spinning solid-state NMR spectroscopy.

An alternative setup for Magic Angle Oriented Spinning Spectroscopy is proposed. Samples were prepared by orienting lipid bilayers onto polymer films, which were wrapped into a spiral so as to fit into 4 or 7 mm MAS rotors. This geometry resulted in narrow line widths and a higher upper spinning limit when compared to the conventional MAOSS setup with stacked glass plates. Whereas orientational information was extracted from low spinning spectra, fast spinning will be applicable to high-resolution multidimensional NMR pulse sequences.

Lipid Bilayers↗