Xavier Robert

ESPript/ENDscript: extracting and rendering sequence and 3D information from atomic structures of proteins

The fortran program ESPript was created in 1993, to display on a PostScript figure multiple sequence alignments adorned with secondary structure elements. A web server was made available in 1999 and ESPript has been linked to three major web tools: ProDom which identifies protein domains, PredictProtein which predicts secondary structure elements and NPS@ which runs sequence alignment programs. A web server named ENDscript was created in 2002 to facilitate the generation of ESPript figures containing a large amount of information. ENDscript uses programs such as BLAST, Clustal and PHYLODENDRON to work on protein sequences and such as DSSP, CNS and MOLSCRIPT to work on protein coordinates. It enables the creation, from a single Protein Data Bank identifier, of a multiple sequence alignment figure adorned with secondary structure elements of each sequence of known 3D structure. Similar 3D structures are superimposed in turn with the program PROFIT and a final figure is drawn with BOBSCRIPT, which shows sequence and structure conservation along the Calpha trace of the query. ESPript and ENDscript are available at http://genopole.toulouse.inra.fr/ESPript.

The Structure of Barley α-Amylase Isozyme 1 Reveals a Novel Role of Domain C in Substrate Recognition and Binding: A Pair of Sugar Tongs

Though the three-dimensional structures of barley alpha-amylase isozymes AMY1 and AMY2 are very similar, they differ remarkably from each other in their affinity for Ca(2+) and when interacting with substrate analogs. A surface site recognizing maltooligosaccharides, not earlier reported for other alpha-amylases and probably associated with the different activity of AMY1 and AMY2 toward starch granules, has been identified. It is located in the C-terminal part of the enzyme and, thus, highlights a potential role of domain C. In order to scrutinize the possible biological significance of this domain in alpha-amylases, a thorough comparison of their three-dimensional structures was conducted. An additional role for an earlier-identified starch granule binding surface site is proposed, and a new calcium ion is reported.

Oligosaccharide binding to barley α-amylase 1

Enzymatic subsite mapping earlier predicted 10 binding subsites in the active site substrate binding cleft of barley α-amylase isozymes. The three-dimensional structures of the oligosaccharide complexes with barley α-amylase isozyme 1 (AMY1) described here give for the first time a thorough insight into the substrate binding by describing residues defining 9 subsites, namely -7 through +2. These structures support that the pseudotetrasaccharide inhibitor acarbose is hydrolyzed by the active enzymes. Moreover, sugar binding was observed to the starch granule-binding site previously determined in barley α-amylase isozyme 2 (AMY2), and the sugar binding modes are compared between the two isozymes. The “sugar tongs” surface binding site discovered in the AMY1-thio-DP4 complex is confirmed in the present work. A site that putatively serves as an entrance for the substrate to the active site was proposed at the glycone part of the binding cleft, and the crystal structures of the catalytic nucleophile mutant (AMY1D180A) complexed with acarbose and maltoheptaose, respectively, suggest an additional role for the nucleophile in the stabilization of the Michaelis complex. Furthermore, probable roles are outlined for the surface binding sites. Our data support a model in which the two surface sites in AMY1 can interact with amylose chains in their naturally folded form. Because of the specificities of these two sites, they may locate/orient the enzyme in order to facilitate access to the active site for polysaccharide chains. Moreover, the sugar tongs surface site could also perform the unraveling of amylose chains, with the aid of Tyr-380 acting as “molecular tweezers.”

Trehalulose synthase native and carbohydrate complexed structures provide insights into sucrose isomerization

Various diseases related to the overconsumption of sugar make a growing need for sugar substitutes. Because sucrose is an inexpensive and readily available d-glucose donor, the industrial potential for enzymatic synthesis of the sucrose isomers trehalulose and/or isomaltulose from sucrose is large. The product specificity of sucrose isomerases that catalyze this reaction depends essentially on the possibility for tautomerization of sucrose, which is required for trehalulose formation. For optimal use of the enzyme, targeting controlled synthesis of these functional isomers, it is necessary to minimize the side reactions. This requires an extensive analysis of substrate binding modes and of the specificity-determining sites in the structure. The 1.6-2.2-Å resolution three-dimensional structures of native and mutant complexes of a trehalulose synthase from Pseudomonas mesoacidophila MX-45 mimic successive states of the enzyme reaction. Combined with mutagenesis studies they give for the first time thorough insights into substrate recognition and processing and reaction specificities of these enzymes. Among the important outcomes of this study is the revelation of an aromatic clamp defined by Phe256 and Phe280 playing an essential role in substrate recognition and in controlling the reaction specificity, which is further supported by mutagenesis studies. Furthermore, this study highlights essential residues for binding the glucosyl and fructosyl moieties. The introduction of subtle changes informed by comparative three-dimensional structural data observed within our study can lead to fundamental modifications in the mode of action of sucrose isomerases and hence provide a template for industrial catalysts.

Assessing Quaternary reactivation of the Main Central thrust zone (central Nepal Himalaya): New thermochronologic data and numerical modeling

We study the recent dynamics of the central Nepal Himalaya, focusing on possible reactivation of the footwall of the Main Central thrust, which is marked by an abrupt topographic transition. Different tectonic mechanisms, such as overthrusting of a major crustal ramp, underplating, or out-of-sequence thrusting, have been suggested to explain the morphology and exhumation patterns in this area. We present 25 new apatite fission-track ages collected along a north-south transect in central Nepal, as well as two age-elevation profiles. Ages are consistently younger than 3 Ma old in the Main Central thrust zone and increase continuously to 4–6 Ma old in the south. No jump in apatite fission-track ages is observed across the topographic transition. Apparent exhumation rates from age-elevation relationships vary from 0.46 +0.13/−0.09 km/Ma in the Palung granite south of Kathmandu to 4.4 +4.8/−1.5 km/Ma in the Main Central thrust zone; the latter rate is probably overestimated by a factor of two due to topographic effects. As shown by a new numerical model, these strongly varying exhumation rates can be explained by overthrusting of a crustal ramp, which exerts a primary control on age patterns, and do not require out-of-sequence reactivation of thrusts in the Main Central thrust zone.

Substrate recognition by the bacterial type II secretion system: more than a simple interaction.

Type II secretion system (T2SS) is a multiprotein trans‐envelope complex that translocates fully folded proteins through the outer membrane of Gram‐negative bacteria. Although T2SS is extensively studied in several bacteria pathogenic for humans, animals and plants, the molecular basis for exoprotein recruitment by this secretion machine as well as the underlying targeting motifs remain unknown. To address this question, we used bacterial two‐hybrid, surface plasmon resonance, in vivo site‐specific photo‐cross‐linking approaches and functional analyses. We showed that the fibronectin‐like Fn3 domain of exoprotein PelI from Dickeya dadantii interacts with four periplasmic domains of the T2SS components GspD and GspC. The interaction between exoprotein and the GspC PDZ domain is positively modulated by the GspD N1 domain, suggesting that exoprotein secretion is driven by a succession of synergistic interactions. We found that an exposed 9‐residue‐long loop region of PelI interacts with the GspC PDZ domain. This loop acts as a specific secretion signal that controls exoprotein recruitment by the T2SS. Concerted in silico and in vivo approaches reveal the occurrence of equivalent secretion motifs in other exoproteins, suggesting a plausible general mechanism of exoprotein recruitment by the T2SS.

Contrasting tectonically driven exhumation and incision patterns, western versus central Nepal Himalaya

Although the Himalayan range is classically presented as cylindrical along strike, segmentation of the range in terms of structure, topography, precipitation, and erosion patterns is becoming widely recognized. The potential climatic or tectonic controls on these lateral variations remain controversial. Thermokinematic models predict that the geometry of the main Himalayan detachment controls the kinematics, exhumation, and topography of the orogen: where the detachment includes a major crustal ramp, the topography shows a steep gradient that focuses orographic precipitation and exhumation, whereas the topography is gentler and exhumation less focused above a flatter detachment. We test this prediction by comparing the patterns of river incision (specific stream power) and long-term exhumation (from apatite fission track thermochronology) in central Nepal, where a major crustal ramp has been imaged by geophysical methods, with new exploratory data from the remote Karnali River transect in western Nepal, where a ramp is predicted to be absent or minor. Our results show that both exhumation rates and river incision capacity are significantly higher and focused on the crustal ramp in central Nepal, whereas they are lower and the pattern is more diffuse in western Nepal. These differences support a model in which lateral variations in topography and exhumation are controlled by variations in the geometry of the detachment, and imply that along-strike climatic variations in the Himalaya respond to tectonics rather than driving it.

C-terminal Residues Regulate Localization and Function of the Antiapoptotic Protein Bfl-1

Unlike other antiapoptotic members of the Bcl-2 family, Bfl-1 does not contain a well defined C-terminal transmembrane domain, and whether the C-terminal tail of Bfl-1 functions as a membrane anchor is not yet clearly established. The molecular modeling study of the full-length Bfl-1 performed within this work suggests that Bfl-1 may co-exist in two distinct conformational states: one in which its C-terminal helix α9 is inserted in the hydrophobic groove formed by the BH1–3 domains of Bfl-1 and one with its C terminus. Parallel analysis of the subcellular localization of Bfl-1 indicates that even if Bfl-1 may co-exist in two distinct conformational states, most of the endogenous protein is tightly associated with the mitochondria by its C terminus in both healthy and apoptotic peripheral blood lymphocytes as well as in malignant B cell lines. However, the helix α9 of Bfl-1, and therefore the binding of Bfl-1 to mitochondria, is not absolutely required for the antiapoptotic activity of Bfl-1. A particular feature of Bfl-1 is the amphipathic character of its C-terminal helix α9. Our data clearly indicate that this property of helix α9 is required for the anchorage of Bfl-1 to the mitochondria but also regulates the antiapoptotic function Bfl-1.

Dynamic Interplay between the Periplasmic and Transmembrane Domains of GspL and GspM in the Type II Secretion System

The type II secretion system (T2SS) is a multiprotein nanomachine that transports folded proteins across the outer membrane of gram-negative bacteria. The molecular mechanisms that govern the secretion process remain poorly understood. The inner membrane components GspC, GspL and GspM possess a single transmembrane segment (TMS) and a large periplasmic region and they are thought to form a platform of unknown function. Here, using two-hybrid and pull-down assays we performed a systematic mapping of the GspC/GspL/GspM interaction regions in the plant pathogen Dickeya dadantii. We found that the TMS of these components interact with each other, implying a complex interaction network within the inner membrane. We also showed that the periplasmic, ferredoxin-like, domains of GspL and GspM drive homo- and heterodimerizations of these proteins. Disulfide bonding analyses revealed that the respective domain interfaces include the equivalent secondary-structure elements, suggesting alternating interactions of the periplasmic domains, L/L and M/M versus L/M. Finally, we found that displacements of the periplasmic GspM domain mediate coordinated shifts or rotations of the cognate TMS. These data suggest a plausible mechanism for signal transmission between the periplasmic and the cytoplasmic portions of the T2SS machine.

Expression, purification and preliminary crystallographic studies of α-amylase isozyme 1 from barley seeds

The germinating barley seed contains two major alpha-amylase isozyme families, AMY1 and AMY2, involved in starch degradation to provide energy used by the plant embryo for growth. Many years of difficulty in growing three-dimensional crystals of natural AMY1 have now been overcome by a nonapeptide truncation of the enzyme C-terminus. The truncated enzyme was overexpressed in Pichia pastoris, purified and crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 8000 as precipitant and 2-propanol as an additive. Crystals belong to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 88.36, b = 72.82, c = 61.74 A and one molecule per asymmetric unit.