NMR assignments of the N-terminal domain of Nephila clavipes spidroin 1

The building blocks of spider dragline silk are two fibrous proteins secreted from the major ampullate gland named spidroins 1 and 2 (MaSp1, MaSp2). These proteins consist of a large central domain composed of approximately 100 tandem copies of a 35–40 amino acid repeat sequence. Non-repetitive N and C-terminal domains, of which the C-terminal domain has been implicated to transition from soluble and insoluble states during spinning, flank the repetitive core. The N-terminal domain until recently has been largely unknown due to difficulties in cloning and expression. Here, we report nearly complete assignment for all ¹H, ¹³C, and ¹⁵N resonances in the 14 kDa N-terminal domain of major ampullate spidroin 1 (MaSp1-N) of the golden orb-web spider Nephila clavipes.     

1H, 13C and 15N resonance assignments of the periplasmic signalling domain of HasR, a TonB-dependent outer membrane heme transporter

TonB-dependent transporters (TBDTs) are bacterial outer membrane proteins that internalize nutrients such as vitamin B12, metal complexes, heme, some carbohydrates, etc. In addition to their transport activity, several TBDTs are also involved in a signalling cascade from the cell surface into the cytoplasm, via their periplasmic signalling domain. Here we report the backbone and side chain resonance assignments of the signalling domain of HasR, a TonB-dependent outer membrane heme transporter from Serratia marcescens as a first step towards its structural study.     

Mechanics of force propagation in TonB-dependent outer membrane transport

For the uptake of scarce yet essential organometallic compounds, outer membrane transporters of Gram-negative bacteria work in concert with an energy-generating inner membrane complex, thus spanning the periplasmic space to drive active transport. Here, we examine the interaction of TonB, an inner membrane protein, with an outer membrane transporter based upon a recent crystal structure of a TonB-transporter complex to characterize two largely unknown steps of the transport cycle: how energy is transmitted from TonB to the transporter and how energy transduction initiates transport. Simulations of TonB in complex with BtuB reveal that force applied to TonB is transmitted to BtuB without disruption of the very small connection between the two, supporting a mechanical mode of coupling. Based on the results of different pulling simulations, we propose that the force transduction instigates a partial unfolding of the pore-occluding luminal domain of the transporter, a potential step in the transport cycle. Furthermore, analysis of the electrostatic potentials and salt bridge interactions between the two proteins during the simulations hints at involvement of electrostatic forces in long-range interaction and binding of TonB and BtuB.     

From the periplasmic signaling domain to the extracellular face of an outer membrane signal transducer of Pseudomonas aeruginosa: crystal structure of the ferric pyoverdine outer membrane receptor

The pyoverdine outer membrane receptor, FpvA, from Pseudomonas aeruginosa translocates ferric pyoverdine across the outer membrane through an energy consuming mechanism using the proton motive force and the TonB-ExbB-ExbD energy transducing complex from the inner membrane. We solved the crystal structure of the full-length FpvA bound to iron-pyoverdine at 2.7 A resolution. Signal transduction to an anti-sigma protein of the inner membrane and to TonB-ExbB-ExbD involves the periplasmic domain, which displays a beta-alpha-beta fold composed of two alpha-helices sandwiched by two beta-sheets. One iron-pyoverdine conformer is bound at the extracellular face of FpvA, revealing the conformer selectivity of the binding site. The loop that contains the TonB box, involved in interactions with TonB, and connects the signaling domain to the plug domain of FpvA is not defined in the electron density following the binding of ferric pyoverdine. The high flexibility of this loop is probably necessary for signal transduction through the outer membrane.     

Structure of outer membrane protein A transmembrane domain by NMR spectroscopy

We have determined the three-dimensional fold of the 19 kDa (177 residues) transmembrane domain of the outer membrane protein A of Escherichia coli in dodecylphosphocholine (DPC) micelles in solution using heteronuclear NMR. The structure consists of an eight-stranded beta-barrel connected by tight turns on the periplasmic side and larger mobile loops on the extracellular side. The solution structure of the barrel in DPC micelles is similar to that in n-octyltetraoxyethylene (C(8)E(4)) micelles determined by X-ray diffraction. Moreover, data from NMR dynamic experiments reveal a gradient of conformational flexibility in the structure that may contribute to the membrane channel function of this protein.     

Nuclear magnetic resonance solution structure of the periplasmic signalling domain of the TonB-dependent outer membrane transporter FecA from Escherichia coli

Gram-negative bacteria possess outer membrane receptors that utilize energy provided by the TonB system to take up iron. Several of these receptors participate in extracytoplasmic factor (ECF) signalling through an N-terminal signalling domain that interacts with a periplasmic transmembrane anti-sigma factor protein and a cytoplasmic sigma factor protein. The structures of the intact TonB-dependent outer membrane receptor FecA from Escherichia coli and FpvA from Pseudomonas aeruginosa have recently been solved by protein crystallography; however, no electron density was detected for their periplasmic signalling domains, suggesting that it was either unfolded or flexible with respect to the remainder of the protein. Here we describe the well-defined solution structure of this domain solved by multidimensional nuclear magnetic resonance (NMR) spectroscopy. The monomeric protein construct contains the 79-residue N-terminal domain as well as the next 17 residues that are part of the receptor's plug domain. These form two clearly distinct regions: a highly structured domain at the N-terminal end followed by an extended flexible tail at the C-terminal end, which includes the 'TonB-box' region, and connects it to the plug domain of the receptor. The structured region consists of two alpha-helices that are positioned side by side and are sandwiched in between two small beta-sheets. This is a novel protein fold which appears to be preserved in all the periplasmic signalling domains of bacterial TonB-dependent outer membrane receptors that are involved in ECF signalling, because the hydrophobic residues that make up the core of the protein domain are highly conserved.     

The N-terminal domain of a TonB-dependent transporter undergoes a reversible stepwise denaturation

Gram-negative bacteria contain a family of outer membrane transport proteins that function in the uptake of rare nutrients, such as iron and vitamin B(12). These proteins are termed TonB-dependent because transport requires an interaction with the inner-membrane protein TonB. Using a combination of site-directed spin labeling and chemical denaturation, we examined the site-specific unfolding of regions of the Escherichia coli vitamin B(12) transporter, BtuB. The data indicate that a portion of the N-terminal region of the protein, which occupies the lumen of the BtuB barrel, denatures prior to the unfolding of the barrel and that the free energy of folding for the N-terminus is smaller than that typically seen for globular proteins. Moreover, the data indicate that the N-terminal domain does not unfold in a single event but unfolds in a series of independent steps. The unfolding of the N-terminus is reversible, and removal of denaturant restores the native fold of the protein. These data are consistent with proposed transport mechanisms that involve a transient rearrangement or unfolding of the N-terminus of the protein, and they provide evidence of a specific protein conformation that might be an intermediate accessed during transport.     

A TonB-dependent outer membrane protein as a Bacteroides fragilis fibronectin-binding molecule

The binding of Bacteroides fragilis to plasmatic fibronectin was investigated using strains isolated from healthy subjects and from patients with bacteremia. They were cultivated in a synthetic media in which variations in cysteine concentrations determined alterations in the oxidation–reduction potential (Eh). All the strains assayed were capable of adhering to plasmatic fibronectin when cultivated under oxidizing and reducing conditions. Bacteroides fragilis 1405 showed the greatest difference when the results under these conditions were compared and it was selected for further investigations. Chemical treatments suggested the involvement of a protein in the interaction between B. fragilis and plasmatic fibronectin. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of outer membrane proteins (OMPs) revealed differences between the extracts obtained from cultures grown under the two conditions. Protein bands of c . 102, 100, 77, 73, 50 and 40 kDa were more highly expressed under oxidizing than reducing conditions. Dot blot analysis showed a stronger recognition of plasmatic fibronectin by OMPs obtained from cultures grown under higher Eh, and Western blot assays confirmed a band of c . 102 kDa as fibronectin-binding protein. This protein was sequenced and revealed to be a putative TonB-dependent OMPs. PCR analysis confirmed the presence of this gene in all the studied strains.     

Comparative structural analysis of TonB-dependent outer membrane transporters: implications for the transport cycle

TonB-dependent outer membrane transporters (TBDTs) transport organometallic substrates across the outer membranes of Gram-negative bacteria. Currently, structures of four different TBDTs have been determined by X-ray crystallography. TBDT structures consist of a 22-stranded beta-barrel enclosing a hatch domain. Structure-based sequence alignment of these four TBDTs indicates the presence of highly conserved motifs in both the hatch and barrel domains. The conserved motifs of the two domains are always in close proximity to each other and interact. We analyzed the very large interfaces between the barrel and hatch domains of TBDTs and compared their properties to those of other protein-protein interfaces. These interfaces are extensively hydrated. Most of the interfacial waters form hydrogen bonds to either the barrel or the hatch domain, with the remainder functioning as bridging waters in the interface. The hatch/barrel interfacial properties most resemble those of obligate transient protein complexes, suggesting that the interface is conducive to conformational change and/or movement of the hatch within the barrel. These results indicate that TBDTs can readily accommodate substantial conformational change and movement of their hatch domains during the active transport cycle. Also, these structural changes may require only modest forces exerted by the energy-coupling TonB protein upon the transporter.     

NMR assignments of the C-terminal domain of human galectin-8

Galectins recognize β-galectosides to promote a variety of cellular functions. Despite their sequence variations, all galectins share the same carbohydrate recognition domains (CRD) and their modes of ligand recognition at a structural level are essentially identical. Human galectin 8 plays an important role in numerous cancer and immune responses. It consists of two CRDs that are connected via a flexible linker. The substrate affinities and specificities of the N- and C-terminal domains are quite different. In order to investigate the structural basis of their substrate specificities, we complete the NMR ¹H, ¹³C, and ¹⁵N chemical shift assignments of C-terminal domain of human galectin-8 (hG8C).     

NMR assignments of the yellow fever virus envelope protein domain III

Nearly complete backbone and sidechain resonance assignments have been obtained for the third domain, residues S288–K398, of the envelope protein from the Asibi strain of yellow fever virus using double- and triple-resonance spectroscopy.     

NMR assignments of PI3-SH3 domain aided by protonless NMR spectroscopy

We report here the near complete assignments of native bovine PI3-SH3 domain, which has been a model system for protein folding, misfolding and amyloid fibril formation. The use of ¹³C-detected protonless NMR spectroscopy is instrumental in assigning the spin system of the proline residue at the C-terminus in addition to the missing resonances in proton-based NMR spectra due to rapid solvent exchange. It also helps assign the resonances of all three proline amine nitrogen nuclei, which are underrepresented in the database. Comparison of the backbone ¹³C resonances of PI3-SH3 in its native and amyloid fibril states shows that the aggregation of PI3-SH3 is accompanied by major conformational rearrangements.     

Improved membrane protein topology prediction by domain assignments

Topology predictions for integral membrane proteins can be substantially improved if parts of the protein can be constrained to a given in/out location relative to the membrane using experimental data or other information. Here, we have identified a set of 367 domains in the SMART database that, when found in soluble proteins, have compartment-specific localization of a kind relevant for membrane protein topology prediction. Using these domains as prediction constraints, we are able to provide high-quality topology models for 11% of the membrane proteins extracted from 38 eukaryotic genomes. Two-thirds of these proteins are single spanning, a group of proteins for which current topology prediction methods perform particularly poorly.     

Backbone resonance assignments of the outer membrane lipoprotein FrpD from Neisseria meningitidis

The iron-regulated FrpD protein is a unique lipoprotein embedded into the outer membrane of the Gram-negative bacterium Neisseria meningitidis. The biological function of FrpD remains unknown but might consist in anchoring to the bacterial cell surface the Type I-secreted FrpC protein, which belongs to a Repeat in ToXins (RTX) protein family and binds FrpD with very high affinity (K _d  = 0.2 nM). Here, we report the backbone ¹H, ¹³C, and ¹⁵N chemical shift assignments for the FrpD_43–271 protein that allow us to characterize the intimate interaction between FrpD and the N-terminal domain of FrpC.