Olivier Bornet

TRF2 promotes, remodels and protects telomeric Holliday junctions

The ability of the telomeric DNA‐binding protein, TRF2, to stimulate t‐loop formation while preventing t‐loop deletion is believed to be crucial to maintain telomere integrity in mammals. However, little is known on the molecular mechanisms behind these properties of TRF2. In this report, we show that TRF2 greatly increases the rate of Holliday junction (HJ) formation and blocks the cleavage by various types of HJ resolving activities, including the newly identified human GEN1 protein. By using potassium permanganate probing and differential scanning calorimetry, we reveal that the basic domain of TRF2 induces structural changes to the junction. We propose that TRF2 contributes to t‐loop stabilisation by stimulating HJ formation and by preventing resolvase cleavage. These findings provide novel insights into the interplay between telomere protection and homologous recombination and suggest a general model in which TRF2 maintains telomere integrity by controlling the turnover of HJ at t‐loops and at regressed replication forks.

Solution structure and backbone dynamics of an antigen‐free heavy chain variable domain (VHH) from Llama

Camelids, (dromedaries, camels, and llamas) produce heavy‐chains antibodies, with their antigen recognition sites composed of a single VH‐like domain, referred to as VHH. The solution structure of one of these VHHs domains (VHH‐H14), raised against the α subunit of the human chorionic gonadotropin hormone (hCG), has been determined by 15N heteronuclear three‐dimensional NMR spectroscopy. The framework is well resolved within the set of 20 best‐calculated NMR structures and is close to that of classical VH domains from vertebrate antibodies, consisting of two antiparallel β‐sheets organized in a β‐barrel. Loops display a lower precision, especially the Complementarity Determining Regions (CDRs), involved in antigen recognition. Comparison of the three‐dimensional VHH‐H14 solution structure with its previously solved crystal structure (Spinelli et al., Nature Struct. Biol. 1996;3:752–757) reveals a high similarity to the framework, whereas significant conformational differences occur on CDRs, leading to the assumption that the antigen recognition site is a more mobile part. In order to deepen our insights into the dynamics of VHH‐H14 in solution, 15N relaxation was measured with longitudinal R1 and transverse R2 self‐relaxation rates, and 15N steady‐state heteronuclear nuclear Overhauser enhancements (NOE), making it possible to probe picosecond‐to‐millisecond internal motions. Determination of dynamic parameters (S2, τe, and Rex) through the Lipari‐Szabo Model‐free approach enables the identification of several regions with enhanced dynamics. Especially, the mobility measurements from NMR confirm that the antigen recognition site is the most mobile part of the VHH‐H14 domain on picosecond‐to‐nanosecond fast time scales. Several residues belonging to the three CDRs are submitted to chemical exchange processes occurring on slow microsecond‐to‐millisecond time scales, suggesting that the formation of the VHH/antigen complex should be accompanied by structural changes. Proteins 2002;47:546–555.

The cytochrome c3–[Fe]-hydrogenase electron-transfer complex: structural model by NMR restrained docking

Cytochrome c3 (M r 13 000) is a low redox potential cytochrome specific of the anaerobic metabolism in sulfate‐reducing bacteria. This tetrahemic cytochrome is an intermediate between the [Fe]‐hydrogenase and the cytochrome Hmc in Desulfovibrio vulgaris Hildenborough strain. The present work describes the structural model of the cytochrome c3–[Fe]‐hydrogenase complex obtained by nuclear magnetic resonance restrained docking. This model connects the distal cluster of the [Fe]‐hydrogenase to heme 4 of the cytochrome, the same heme found in the interaction with cytochrome Hmc. This result gives evidence that cytochrome c3 is an electron shuttle between the periplasmic hydrogenase and the Hmc membrane‐bound complex.

Solution structure of BmKTX, a K+ blocker toxin from the Chinese scorpion Buthus Martensi

BmKTX is a toxin recently purified from the venom of Buthus Martensi, which belongs to the kaliotoxin family. We have determined its solution structure by use of conventional two‐dimensional NMR techniques followed by distance‐geometry and energy minimization. The calculated structure is composed of a short α‐helix (residues 14 to 20) connected by a tight turn to a two‐stranded antiparallel β‐sheet (sequences 25–27 and 32–34). The β‐turn connecting these strands belongs to type I. The N‐terminal segment (sequence 1 to 8) runs parallel to the β‐sheet although it cannot be considered as a third strand. Comparison of the conformation of BmKTX and toxins of the kaliotoxin familyclearly demonstrates that they are highly related. Therefore, analysis of the residues belonging to the interacting surface of those toxins allows us to propose a functional map of BmKTX slightly different from the one of KTX and AgTX2, which may explain the variations in affinities of these toxins towardsthe Kv1.3 channels. Proteins 2000;38:70–78.

Pre-B cell receptor binding to galectin-1 modifies galectin-1/carbohydrate affinity to modulate specific galectin-1/glycan lattice interactions

Galectins are glycan-binding proteins involved in various biological processes including cell/cell interactions. During B-cell development, bone marrow stromal cells secreting galectin-1 (GAL1) constitute a specific niche for pre-BII cells. Besides binding glycans, GAL1 is also a pre-B cell receptor (pre-BCR) ligand that induces receptor clustering, the first checkpoint of B-cell differentiation. The GAL1/pre-BCR interaction is the first example of a GAL1/unglycosylated protein interaction in the extracellular compartment. Here we show that GAL1/pre-BCR interaction modifies GAL1/glycan affinity and particularly inhibits binding to LacNAc containing epitopes. GAL1/pre-BCR interaction induces local conformational changes in the GAL1 carbohydrate-binding site generating a reduction in GAL1/glycan affinity. This fine tuning of GAL1/glycan interactions may be a strategic mechanism for allowing pre-BCR clustering and pre-BII cells departure from their niche. Altogether, our data suggest a novel mechanism for a cell to modify the equilibrium of the GAL1/glycan lattice involving GAL1/unglycosylated protein interactions.

Interactions of bile salt micelles and colipase studied through intermolecular nOes

Colipase is a small protein (10 kDa), which acts as a protein cofactor for the pancreatic lipase. Various models of the activated ternary complex (lipase–colipase–bile salt micelles) have been proposed using detergent micelles, but no structural information has been established with bile salt micelles. We have investigated the organization of sodium taurodeoxycholate (NaTDC) micelles and their interactions with pig and horse colipases by homonuclear nuclear magnetic resonance (NMR) spectroscopy. The NMR data supply evidence that the folding of horse colipase is similar to that already described for pig colipase. Intermolecular nuclear Overhauser effects have shown that two conserved aromatic residues interact with NaTDC micelles.

TM0486 from the hyperthermophilic anaerobe Thermotoga maritima is a thiamin-binding protein involved in response of the cell to oxidative conditions.

The COG database was used for a comparative genome analysis with genomes from anaerobic and aerobic microorganisms with the aim of identifying proteins specific to the anaerobic way of life. A total of 33 COGs were identified, five of which correspond to proteins of unknown function. We focused our study on TM0486 from Thermotoga maritima, which belongs to one of these COGs of unknown function, namely COG0011. The crystal structure of the protein was determined at 2 A resolution. The structure adopts a beta alpha beta beta alpha beta ferredoxin-like fold and assembles as a homotetramer. The structure also revealed the presence of a pocket in each monomer that bound an unidentified ligand. NMR and calorimetry revealed that TM0486 specifically bound thiamin with a K(d) of 1.58 microM, but not hydroxymethyl pyrimidine (HMP), which has been implicated as a potential ligand. We demonstrated that the TM0486 gene belongs to the same multicistronic unit as TM0483, TM0484 and TM0485. Although these three genes have been assigned to the transport of HMP, with TM0484 being the periplasmic thiamin/HMP-binding protein and TM0485 and TM0483 the transmembrane and the ATPase components, respectively, our results led us to conclude that this operon encodes an ABC transporter dedicated to thiamin, with TM0486 transporting charged thiamin in the cytoplasm. Given that this transcriptional unit was up-regulated when T. maritima was exposed to oxidative conditions, we propose that, by chelating cytoplasmic thiamin, TM0486 and, by extension, proteins belonging to COG0011 are involved in the response mechanism to stress that could arise during aerobic conditions.

Selectively 13C-enriched DNA: 13C and 1H assignments of a triple helix by two-dimensional relayed HMQC experiments.

SummaryWe present NMR studies of an intramolecular triple helix, the three strands of which have been linked by a hexaethylene glycol chain. To overcome the generally encountered difficulties of assignment in the homopyrimidine strands, the carbon C1′ of the pyrimidines were selectively 13C-enriched. Assignments of the aromatic and sugar protons were obtained from NOESY-HMQC and TOCSY-HMQC spectra. We show that the recognition of a DNA duplex by a third strand via triplex formation is easily carried out in solution by observing the changes of the 1H1′−13C1′ connectivities as a function of pH. Furthermore, the conformation of the sugars has been found to be C2′-endo, on the basis of the coupling constant values directly measured in an HSQC spectrum.

Solution structure of HndAc: A thioredoxin-like domain involved in the NADP-reducing hydrogenase complex

The NADP‐reducing hydrogenase complex from Desulfovibrio fructosovorans is a heterotetramer encoded by the hndABCD operon. Sequence analysis indicates that the HndC subunit (52 kDa) corresponds to the NADP‐reducing unit, and the HndD subunit (63.5 kDa) is homologous to Clostridium pasteurianum hydrogenase. The role of HndA and HndB subunits (18.8 kDa and 13.8 kDa, respectively) in the complex remains unknown. The HndA subunit belongs to the [2Fe‐2S] ferredoxin family typified by C. pasteurianum ferredoxin. HndA is organized into two independent structural domains, and we report in the present work the NMR structure of its C‐terminal domain, HndAc. HndAc has a thioredoxin‐like fold consisting in four β‐strands and two relatively long helices. The [2Fe‐2S] cluster is located near the surface of the protein and bound to four cysteine residues particularly well conserved in this class of proteins. Electron exchange between the HndD N‐terminal [2Fe‐2S] domain (HndDN) and HndAc has been previously evidenced, and in the present studies we have mapped the binding site of the HndDN domain on HndAc. A structural analysis of HndB indicates that it is a FeS subunit with 41% similarity with HndAc and it contains a possible thioredoxin‐like fold. Our data let us propose that HndAc and HndB can form a heterodimeric intermediate in the electron transfer between the hydrogenase (HndD) active site and the NADP reduction site in HndC.

Identification of a Src kinase SH3 binding site in the C-terminal domain of the human ErbB2 receptor tyrosine kinase

Overexpression of the ErbB2 receptor tyrosine kinase is associated with most aggressive tumors in breast cancer patients and is thus one of the main investigated therapeutic targets. Human ErbB2 C‐terminal domain is an unstructured anchor that recruits specific adaptors for signaling cascades resulting in cell growth, differentiation and migration. Herein, we report the presence of a SH3 binding motif in the proline rich unfolded ErbB2 C‐terminal region. NMR analysis of this motif supports a PPII helix conformation and the binding to Fyn‐SH3 domain. The interaction of a kinase of the Src family with ErbB2 C‐terminal domain could contribute to synergistic intracellular signaling and enhanced oncogenesis.