Bernard Clantin

Secretion signal of the filamentous haemagglutinin, a model two‐partner secretion substrate

The sorting of proteins to their proper subcellular compartment requires specific addressing signals that mediate interactions with ad hoc transport machineries. In Gram‐negative bacteria, the widespread two‐partner secretion (TPS) pathway is dedicated to the secretion of large, mostly virulence‐related proteins. The secreted TpsA proteins carry a characteristic 250‐residue‐long N‐terminal ‘TPS domain’ essential for secretion, while their TpsB transporters are pore‐forming proteins that specifically recognize their respective TpsA partners and mediate their translocation across the outer membrane. However, the nature of the secretion signal has not been elucidated yet. The whooping cough agent Bordetella pertussis secretes its major adhesin filamentous haemagglutinin (FHA) via the TpsB transporter FhaC. In this work, we show specific interactions between an N‐terminal fragment of FHA containing the TPS domain and FhaC by using two different techniques, an overlay assay and a pull‐down of the complex. FhaC recognizes only non‐native conformations of the TPS domain, corroborating the model that in vivo, periplasmic FHA is not yet folded. By generating single amino acid substitutions, we have identified interaction determinants forming the secretion signal. They are found unexpectedly far into the TPS domain and include both conserved and variable residues, which most likely explains the specificity of the TpsA–TpsB interaction. The N‐terminal domain of FhaC is involved in the FHA–FhaC interaction, in agreement with its proposed function and periplasmic localization.

Crystal structure of isopentenyl diphosphate:dimethylallyl diphosphate isomerase

Isopentenyl diphosphate:dimethylallyl diphosphate (IPP:DMAPP) isomerase catalyses a crucial activation step in the isoprenoid biosynthesis pathway. This enzyme is responsible for the isomerization of the carbon–carbon double bond of IPP to create the potent electrophile DMAPP. DMAPP then alkylates other molecules, including IPP, to initiate the extraordinary variety of isoprenoid compounds found in nature. The crystal structures of free and metal‐bound Escherichia coli IPP isomerase reveal critical active site features underlying its catalytic mechanism. The enzyme requires one Mn2+ or Mg2+ ion to fold in its active conformation, forming a distorted octahedral metal coordination site composed of three histidines and two glutamates and located in the active site. Two critical residues, C67 and E116, face each other within the active site, close to the metal‐binding site. The structures are compatible with a mechanism in which the cysteine initiates the reaction by protonating the carbon–carbon double bond, with the antarafacial rearrangement ultimately achieved by one of the glutamates involved in the metal coordination sphere. W161 may stabilize the highly reactive carbocation generated during the reaction through quadrupole– charge interaction.

First structural insights into the TpsB/Omp85 superfamily

Abstract Proteins of the TpsB/Omp85 superfamily are involved in protein transport across, or assembly into, the outer membrane of Gram-negative bacteria, and their distant eukaryotic relatives exert similar functions in chloroplasts and mitochondria. The X-ray structure of one TpsB transporter, FhaC, provides the bases to decipher the mechanisms of action of these proteins. With two POTRA domains in the periplasm, a transmembrane β barrel and a large loop harboring a functionally important motif, FhaC epitomizes the conserved features of the super-family.

X-ray Structure of Papaya Chitinase Reveals the Substrate Binding Mode of Glycosyl Hydrolase Family 19 Chitinases

The crystal structure of a chitinase from Carica papaya has been solved by the molecular replacement method and is reported to a resolution of 1.5 A. This enzyme belongs to family 19 of the glycosyl hydrolases. Crystals have been obtained in the presence of N-acetyl- d-glucosamine (GlcNAc) in the crystallization solution and two well-defined GlcNAc molecules have been identified in the catalytic cleft of the enzyme, at subsites -2 and +1. These GlcNAc moieties bind to the protein via an extensive network of interactions which also involves many hydrogen bonds mediated by water molecules, underlying their role in the catalytic mechanism. A complex of the enzyme with a tetra-GlcNAc molecule has been elaborated, using the experimental interactions observed for the bound GlcNAc saccharides. This model allows to define four major substrate interacting regions in the enzyme, comprising residues located around the catalytic Glu67 (His66 and Thr69), the short segment E89-R90 containing the second catalytic residue Glu89, the region 120-124 (residues Ser120, Trp121, Tyr123, and Asn124), and the alpha-helical segment 198-202 (residues Ile198, Asn199, Gly201, and Leu202). Water molecules from the crystal structure were introduced during the modeling procedure, allowing to pinpoint several additional residues involved in ligand binding that were not previously reported in studies of poly-GlcNAc/family 19 chitinase complexes. This work underlines the role played by water-mediated hydrogen bonding in substrate binding as well as in the catalytic mechanism of the GH family 19 chitinases. Finally, a new sequence motif for family 19 chitinases has been identified between residues Tyr111 and Tyr125.

Channel Properties of TpsB Transporter FhaC Point to Two Functional Domains with a C-terminal Protein-conducting Pore

Integral outer membrane transporters of the Omp85/TpsB superfamily mediate the translocation of proteins across, or their integration into, the outer membranes of Gram-negative bacteria, chloroplasts, and mitochondria. The Bordetella pertussis FhaC/FHA couple serves as a model for the two-partner secretion pathway in Gram-negative bacteria, with the TpsB protein, FhaC, being the specific transporter of its TpsA partner, FHA, across the outer membrane. In this work, we have investigated the structure/function relationship of FhaC by analyzing the ion channel properties of the wild type protein and a collection of mutants with varied FHA secretion activities. We demonstrated that the channel is formed by the C-terminal two-thirds of FhaC most likely folding into a β-barrel domain predicted to be conserved throughout the family. A C-proximal motif that represents the family signature appears essential for pore function. The N-terminal 200 residues of FhaC constitute a functionally distinct domain that modulates the pore properties and may participate in FHA recognition.

Substrate recognition by the POTRA domains of TpsB transporter FhaC

Widespread in Gram‐negative bacteria, the two‐partner secretion (TPS) pathway mediates the secretion of large, β‐helical ‘TpsA’ proteins with various functions. TpsA proteins harbour a conserved, N‐proximal TPS domain essential for secretion. TpsB transporters specifically recognize their TpsA partners in the periplasm and mediate their translocation across the outer membrane through a hydrophilic channel. The FHA/FhaC pair of Bordetella pertussis represents a model TPS system. FhaC is composed of a β barrel preceded by two periplasmic POTRA domains in tandem. Here we show that both POTRAs are involved in FHA recognition. Surface plasmon resonance analyses indicated an interaction of micromolar affinity between the POTRAs and the TPS domain with fast association and dissociation steps, consistent with the transient character of this interaction in vivo. Major interaction sites in POTRAs correspond to hydrophobic grooves formed by a β sheet edge and the flanking α helix, well‐suited to accommodate extended, amphipathic strands of the substrate and consistent with β augmentation. The initial recruitment of the TPS domain to POTRAs appears to be facilitated by electrostatic attractions. A domain corresponding to the first part of the repeat‐rich central region of FHA is also recognized by the POTRAs, suggesting successive interactions in the course of secretion.

Functional importance of a conserved sequence motif in FhaC, a prototypic member of the TpsB/Omp85 superfamily.

In Gram‐negative bacteria, the two‐partner secretion pathway mediates the secretion of TpsA proteins with various functions. TpsB transporters specifically recognize their TpsA partners in the periplasm and mediate their transport through a hydrophilic channel. The filamentous haemagglutinin adhesin (FHA)/FhaC pair represents a model two‐partner secretion system, with the structure of the TpsB transporter FhaC providing the bases to decipher the mechanism of action of these proteins. FhaC is composed of a β‐barrel preceded by two periplasmic polypeptide‐transport‐associated (POTRA) domains in tandem. The barrel is occluded by an N‐terminal helix and an extracellular loop, L6, folded back into the FhaC channel. In this article, we describe a functionally important motif of FhaC. The VRGY tetrad is highly conserved in the TpsB family and, in FhaC, it is located at the tip of L6 reaching the periplasm. Replacement by Ala of the invariant Arg dramatically affects the secretion efficiency, although the structure of FhaC and its channel properties remain unaffected. This substitution affects the secretion mechanism at a step beyond the initial TpsA–TpsB interaction. Replacement of the conserved Tyr affects the channel properties, but not the secretion activity, suggesting that this residue stabilizes the loop in the resting conformation of FhaC. Thus, the conserved motif at the tip of L6 represents an important piece of two‐partner secretion machinery. This motif is conserved in a predicted loop between two β‐barrel strands in more distant relatives of FhaC involved in protein transport across or assembly into the outer membranes of bacteria and organelles, suggesting a conserved function in the molecular mechanism of transport.

Structural basis for haem piracy from host haemopexin by Haemophilus influenzae.

Haemophilus influenzae is an obligate human commensal/pathogen that requires haem for survival and can acquire it from several host haemoproteins, including haemopexin. The haem transport system from haem-haemopexin consists of HxuC, a haem receptor, and the two-partner-secretion system HxuB/HxuA. HxuA, which is exposed at the cell surface, is strictly required for haem acquisition from haemopexin. HxuA forms complexes with haem-haemopexin, leading to haem release and its capture by HxuC. The key question is how HxuA liberates haem from haemopexin. Here, we solve crystal structures of HxuA alone, and HxuA in complex with the N-terminal domain of haemopexin. A rational basis for the release of haem from haem-haemopexin is derived from both in vivo and in vitro studies. HxuA acts as a wedge that destabilizes the two-domains structure of haemopexin with a mobile loop on HxuA that favours haem ejection by redirecting key residues in the haem-binding pocket of haemopexin.

Structure of the secretion domain of HxuA from Haemophilus influenzae.

Haemophilus influenzae HxuA is a cell-surface protein with haem-haemopexin binding activity which is key to haem acquisition from haemopexin and thus is one of the potential sources of haem for this microorganism. HxuA is secreted by its specific transporter HxuB. HxuA/HxuB belongs to the so-called two-partner secretion systems (TPSs) that are characterized by a conserved N-terminal domain in the secreted protein which is essential for secretion. Here, the 1.5 Å resolution structure of the secretion domain of HxuA, HxuA301, is reported. The structure reveals that HxuA301 folds into a β-helix domain with two extra-helical motifs, a four-stranded β-sheet and an N-terminal cap. Comparisons with other structures of TpsA secretion domains are reported. They reveal that despite limited sequence identity, strong structural similarities are found between the β-helix motifs, consistent with the idea that the TPS domain plays a role not only in the interaction with the specific TpsB partners but also as the scaffold initiating progressive folding of the TpsA proteins at the bacterial surface.

Probing the conformation of FhaC with small-angle neutron scattering and molecular modeling.

Probing the solution structure of membrane proteins represents a formidable challenge, particularly when using small-angle scattering. Detergent molecules often present residual scattering contributions even at their match point in small-angle neutron scattering (SANS) measurements. Here, we studied the conformation of FhaC, the outer-membrane, β-barrel transporter of the Bordetella pertussis filamentous hemagglutinin adhesin. SANS measurements were performed on homogeneous solutions of FhaC solubilized in n-octyl-d17-βD-glucoside and on a variant devoid of the α helix H1, which critically obstructs the FhaC pore, in two solvent conditions corresponding to the match points of the protein and the detergent, respectively. Protein-bound detergent amounted to 142 ± 10 mol/mol as determined by analytical ultracentrifugation. By using molecular modeling and starting from three distinct conformations of FhaC and its variant embedded in lipid bilayers, we generated ensembles of protein-detergent arrangement models with 120-160 detergent molecules. The scattered curves were back-calculated for each model and compared with experimental data. Good fits were obtained for relatively compact, connected detergent belts, which occasionally displayed small detergent-free patches on the outer surface of the β barrel. The combination of SANS and modeling clearly enabled us to infer the solution structure of FhaC, with H1 inside the pore as in the crystal structure. We believe that our strategy of combining explicit atomic detergent modeling with SANS measurements has significant potential for structural studies of other detergent-solubilized membrane proteins.