Scott A. Robson

Functionally Distinct NEAT (NEAr Transporter) Domains within the Staphylococcus aureus IsdH/HarA Protein Extract Heme from Methemoglobin

The pathogen Staphylococcus aureus uses iron-regulated surface determinant (Isd) proteins to scavenge the essential nutrient iron from host hemoproteins. The IsdH protein (also known as HarA) is a receptor for hemoglobin (Hb), haptoglobin (Hp), and the Hb-Hp complex. It contains three NEAT (NEAr Transporter) domains: IsdHN1, IsdHN2, and IsdHN3. Here we show that they have different functions; IsdHN1 binds Hb and Hp, whereas IsdHN3 captures heme that is released from Hb. The staphylococcal IsdB protein also functions as an Hb receptor. Primary sequence homology to IsdH indicates that it will also employ functionally distinct NEAT domains to bind heme and Hb. We have used site-directed mutagenesis and surface plasmon resonance methods to localize the Hp and Hb binding surface on IsdHN1. High affinity binding to these structurally unrelated proteins requires residues located within a conserved aromatic motif that is positioned at the end of the β-barrel structure. Interestingly, this site is quite malleable, as other NEAT domains use it to bind heme. We also demonstrate that the IsdC NEAT domain can capture heme directly from Hb, suggesting that there are multiple pathways for heme transfer across the cell wall.

The IsdC Protein from Staphylococcus aureus Uses a Flexible Binding Pocket to Capture Heme

Staphylococcus aureus scavenges heme-iron from host hemoproteins using iron-regulated surface determinant (Isd) proteins. IsdC is the central conduit through which heme is passed across the cell wall and binds this molecule using a NEAr Transporter (NEAT) domain. NMR spectroscopy was used to determine the structure of IsdC in complex with a heme analog, zinc-substituted protoporphyrin IX (ZnPPIX). The backbone coordinates of the ensemble of conformers representing the structure exhibit a root mean square deviation to the mean structure of 0.53 ± 0.11Å. IsdC partially buries protoporphyrin within a large hydrophobic pocket that is located at the end of itsβ-barrel structure. The central metal ion of the analog adopts a pentacoordinate geometry in which a highly conserved tyrosine residue serves as a proximal ligand. Consistent with the structure and its role in heme transfer across the cell wall, we show that IsdC weakly binds heme (KD = 0.34 ± 0.12 μm) and that ZnPPIX rapidly dissociates from the protein at a rate of 126 ± 30 s-1. NMR studies of the apo-form of IsdC reveal that a 310 helix within the binding pocket undergoes a flexible to rigid transition as heme is captured. This structural plasticity may increase the efficiency of heme transfer across the cell wall by facilitating protein-protein interactions between apoIsdC and upstream hemoproteins.

Perspectives in magnetic resonance: NMR in the post-FFT era.

Multi-dimensional NMR spectra have traditionally been processed with the fast Fourier transformation (FFT). The availability of high field instruments, the complexity of spectra of large proteins, the narrow signal dispersion of some unstructured proteins, and the time needed to record the necessary increments in the indirect dimensions to exploit the resolution of the highfield instruments make this traditional approach unsatisfactory. New procedures need to be developed beyond uniform sampling of the indirect dimensions and reconstruction methods other than the straight FFT are necessary. Here we discuss approaches of non-uniform sampling (NUS) and suitable reconstruction methods. We expect that such methods will become standard for multi-dimensional NMR data acquisition with complex biological macromolecules and will dramatically enhance the power of modern biological NMR.

Exploring signal-to-noise ratio and sensitivity in non-uniformly sampled multi-dimensional NMR spectra.

It is well established that non-uniform sampling (NUS) allows acquisition of multi-dimensional NMR spectra at a resolution that cannot be obtained with traditional uniform acquisition through the indirect dimensions. However, the impact of NUS on the signal-to-noise ratio (SNR) and sensitivity are less well documented. SNR and sensitivity are essential aspects of NMR experiments as they define the quality and extent of data that can be obtained. This is particularly important for spectroscopy with low concentration samples of biological macromolecules. There are different ways of defining the SNR depending on how to measure the noise, and the distinction between SNR and sensitivity is often not clear. While there are defined procedures for measuring sensitivity with high concentration NMR standards, such as sucrose, there is no clear or generally accepted definition of sensitivity when comparing different acquisition and processing methods for spectra of biological macromolecules with many weak signals close to the level of noise. Here we propose tools for estimating the SNR and sensitivity of NUS spectra with respect to sampling schedule and reconstruction method. We compare uniformly acquired spectra with NUS spectra obtained in the same total measuring time. The time saving obtained when only 1/k of the Nyquist grid points are sampled is used to measure k-fold more scans per increment. We show that judiciously chosen NUS schedules together with suitable reconstruction methods can yield a significant increase of the SNR within the same total measurement time. Furthermore, we propose to define the sensitivity as the probability to detect weak peaks and show that time-equivalent NUS can considerably increase this detection sensitivity. The sensitivity gain increases with the number of NUS indirect dimensions. Thus, well-chosen NUS schedules and reconstruction methods can significantly increase the information content of multidimensional NMR spectra of challenging biological macromolecules.

The Sortase A Enzyme That Attaches Proteins to the Cell Wall of Bacillus anthracis Contains an Unusual Active Site Architecture

The pathogen Bacillus anthracis uses the Sortase A (SrtA) enzyme to anchor proteins to its cell wall envelope during vegetative growth. To gain insight into the mechanism of protein attachment to the cell wall in B. anthracis we investigated the structure, backbone dynamics, and function of SrtA. The NMR structure of SrtA has been determined with a backbone coordinate precision of 0.40 ± 0.07 Å. SrtA possesses several novel features not previously observed in sortase enzymes including the presence of a structurally ordered amino terminus positioned within the active site and in contact with catalytically essential histidine residue (His126). We propose that this appendage, in combination with a unique flexible active site loop, mediates the recognition of lipid II, the second substrate to which proteins are attached during the anchoring reaction. pKa measurements indicate that His126 is uncharged at physiological pH compatible with the enzyme operating through a “reverse protonation” mechanism. Interestingly, NMR relaxation measurements and the results of a model building study suggest that SrtA recognizes the LPXTG sorting signal through a lock-in-key mechanism in contrast to the prototypical SrtA enzyme from Staphylococcus aureus.

Assembly of Minicellulosomes on the Surface of Bacillus subtilis

ABSTRACT To cost-efficiently produce biofuels, new methods are needed to convert lignocellulosic biomass into fermentable sugars. One promising approach is to degrade biomass using cellulosomes, which are surface-displayed multicellulase-containing complexes present in cellulolytic Clostridium and Ruminococcus species. In this study we created cellulolytic strains of Bacillus subtilis that display one or more cellulase enzymes. Proteins containing the appropriate cell wall sorting signal are covalently anchored to the peptidoglycan by coexpressing them with the Bacillus anthracis sortase A (SrtA) transpeptidase. This approach was used to covalently attach the Cel8A endoglucanase from Clostridium thermocellum to the cell wall. In addition, a Cel8A-dockerin fusion protein was anchored on the surface of B. subtilis via noncovalent interactions with a cell wall-attached cohesin module. We also demonstrate that it is possible to assemble multienzyme complexes on the cell surface. A three-enzyme-containing minicellulosome was displayed on the cell surface; it consisted of a cell wall-attached scaffoldin protein noncovalently bound to three cellulase-dockerin fusion proteins that were produced in Escherichia coli. B. subtilis has a robust genetic system and is currently used in a wide range of industrial processes. Thus, grafting larger, more elaborate minicellulosomes onto the surface of B. subtilis may yield cellulolytic bacteria with increased potency that can be used to degrade biomass.

The Bacillus subtilis cell division proteins FtsL and DivIC are intrinsically unstable and do not interact with one another in the absence of other septasomal components

The bacterial septum appears to comprise a macromolecular assembly of essential cell division proteins (the ‘septasome’) that are responsible for physically dividing the cell during cytokinesis. FtsL and DivIC are essential components of this division machinery in Bacillus subtilis. We used yeast two‐hybrid analysis as well as a variety of biochemical and biophysical methods to examine the proposed interaction between Bacillus subtilis FtsL and DivIC. We show that FtsL and DivIC are thermodynamically unstable proteins that are likely to be unfolded and therefore targeted for degradation unless stabilized by interactions with other components of the septasome. However, we show that this stabilization does not result from a direct interaction between FtsL and DivIC. We propose that the observed interdepend‐ence of DivIC and FtsL stability is a result of indirect interactions that are mediated by other septasomal proteins.

Staphylococcus aureus Uses a Novel Multidomain Receptor to Break Apart Human Hemoglobin and Steal Its Heme

Background: During infections, Staphylococcus aureus acquires heme-iron from human hemoglobin using the receptor proteins IsdH and IsdB. Results: A conserved multidomain unit in IsdH and IsdB synergistically captures heme and destabilizes the hemoglobin tetramer. Conclusion: Receptor domain synergy and hemoglobin dissociation allow efficient heme uptake by S. aureus. Significance: IsdH and IsdB may represent novel targets for antibiotics that limit microbial access to iron. Staphylococcus aureus is a leading cause of life-threatening infections in the United States. It requires iron to grow, which must be actively procured from its host to successfully mount an infection. Heme-iron within hemoglobin (Hb) is the most abundant source of iron in the human body and is captured by S. aureus using two closely related receptors, IsdH and IsdB. Here we demonstrate that each receptor captures heme using two conserved near iron transporter (NEAT) domains that function synergistically. NMR studies of the 39-kDa conserved unit from IsdH (IsdHN2N3, Ala326–Asp660) reveals that it adopts an elongated dumbbell-shaped structure in which its NEAT domains are properly positioned by a helical linker domain, whose three-dimensional structure is determined here in detail. Electrospray ionization mass spectrometry and heme transfer measurements indicate that IsdHN2N3 extracts heme from Hb via an ordered process in which the receptor promotes heme release by inducing steric strain that dissociates the Hb tetramer. Other clinically significant Gram-positive pathogens capture Hb using receptors that contain multiple NEAT domains, suggesting that they use a conserved mechanism.

NMR Solution Structure and Condition-Dependent Oligomerization of the Antimicrobial Peptide Human Defensin 5

Human defensin 5 (HD5) is a 32-residue host-defense peptide expressed in the gastrointestinal, reproductive, and urinary tracts that has antimicrobial activity. It exhibits six cysteine residues that are regiospecifically oxidized to form three disulfide bonds (Cys(3)-Cys(31), Cys(5)-Cys(20), and Cys(10)-Cys(30)) in the oxidized form (HD5(ox)). To probe the solution structure and oligomerization properties of HD5(ox), and select mutant peptides lacking one or more disulfide bonds, NMR solution studies and analytical ultracentrifugation experiments are reported in addition to in vitro peptide stability assays. The NMR solution structure of HD5(ox), solved at pH 4.0 in 90:10 H(2)O/D(2)O, is presented (PDB: 2LXZ ). Relaxation T(1)/T(2) measurements and the rotational correlation time (τ(c)) estimated from a (15)N-TRACT experiment demonstrate that HD5(ox) is dimeric under these experimental conditions. Exchange broadening of the Hα signals in the NMR spectra suggests that residues 19-21 (Val(19)-Cys(20)-Glu(21)) contribute to the dimer interface in solution. Exchange broadening is also observed for residues 7-14 comprising the loop. Sedimentation velocity and equilibrium studies conducted in buffered aqueous solution reveal that the oligomerization state of HD5(ox) is pH-dependent. Sedimentation coefficients of ca. 1.8 S and a molecular weight of 14 363 Da were determined for HD5(ox) at pH 7.0, supporting a tetrameric form ([HD5(ox)] ≥ 30 μM). At pH 2.0, a sedimentation coefficient of ca. 1.0 S and a molecular weight of 7079 Da, corresponding to a HD5(ox) dimer, were obtained. Millimolar concentrations of NaCl, CaCl(2), and MgCl(2) have a negligible effect on the HD5(ox) sedimentation coefficients in buffered aqueous solution at neutral pH. Removal of a single disulfide bond results in a loss of peptide fold and quaternary structure. These biophysical investigations highlight the dynamic and environmentally sensitive behavior of HD5(ox) in solution, and provide important insights into HD5(ox) structure/activity relationships and the requirements for antimicrobial action.

Evidence from Artificial Septal Targeting and Site-Directed Mutagenesis that Residues in the Extracytoplasmic β Domain of DivIB Mediate Its Interaction with the Divisomal Transpeptidase PBP 2B

Bacterial cytokinesis is achieved through the coordinated action of a multiprotein complex known as the divisome. The Escherichia coli divisome is comprised of at least 10 essential proteins whose individual functions are mostly unknown. Most divisomal proteins have multiple binding partners, making it difficult to pinpoint epitopes that mediate pairwise interactions between these proteins. We recently introduced an artificial septal targeting approach that allows the interaction between pairs of proteins to be studied in vivo without the complications introduced by other interacting proteins (C. Robichon, G. F. King, N. W. Goehring, and J. Beckwith, J. Bacteriol. 190:6048-6059, 2008). We have used this approach to perform a molecular dissection of the interaction between Bacillus subtilis DivIB and the divisomal transpeptidase PBP 2B, and we demonstrate that this interaction is mediated exclusively through the extracytoplasmic domains of these proteins. Artificial septal targeting in combination with mutagenesis experiments revealed that the C-terminal region of the β domain of DivIB is critical for its interaction with PBP 2B. These findings are consistent with previously defined loss-of-function point mutations in DivIB as well as the recent demonstration that the β domain of DivIB mediates its interaction with the FtsL-DivIC heterodimer. These new results have allowed us to construct a model of the DivIB/PBP 2B/FtsL/DivIC quaternary complex that strongly implicates DivIB, FtsL, and DivIC in modulating the transpeptidase activity of PBP 2B.