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Streptococcus pyogenes causes a wide range of human infections. Currently, antibiotics are the main treatment for S. pyogenes infection, but serious anti-microbial resistance requires alternative treatment options. To develop a novel strategy for treatment, we physicochemically characterized SPs0871, a putative maltose/maltodextrin-binding protein that is thought to have important roles in the pathogenesis of invasive streptococci. We obtained a variable domain of heavy chain of heavy-chain antibody, the smallest unit of an antibody, which specifically binds to SPs0871. Although the VHH completely inhibited the binding of maltodextrins to SPs0871, the inhibition did not lead to growth suppression of the bacteria. Our results provide important insights for development of VHH as an anti-streptococcal therapeutic., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Hemes (iron-porphyrins) are critical for biological processes in all organisms. Hemolytic bacteria survive by acquiring b-type heme from hemoglobin in red blood cells from their animal hosts. These bacteria avoid the cytotoxicity of excess heme during hemolysis by expressing heme-responsive sensor proteins that act as transcriptional factors to regulate the heme efflux system in response to the cellular heme concentration. Here, the underlying regulatory mechanisms were investigated using crystallographic, spectroscopic, and biochemical studies to understand the structural basis of the heme-responsive sensor protein PefR from Streptococcus agalactiae, a causative agent of neonatal life-threatening infections. Structural comparison of heme-free PefR, its complex with a target DNA, and heme-bound PefR revealed that unique heme coordination controls a >20 Å structural rearrangement of the DNA binding domains to dissociate PefR from the target DNA. We also found heme-bound PefR stably binds exogenous ligands, including carbon monoxide, a by-product of the heme degradation reaction.

Staphylococcus aureus uses IsdG and IsdI to convert heme into a mixture of staphylobilin isomers, 15-oxo-β-bilirubin and 5-oxo-δ-bilirubin, formaldehyde, and iron. The highly ruffled heme found in the heme-IsdI and IsdG complexes has been proposed to be responsible for the unique heme degradation products. We employed resonance Raman (RR) and electron paramagnetic resonance (EPR) spectroscopies to examine the coordination and electronic structures of heme bound to IsdG and IsdI. Heme complexed to IsdG and IsdI is coordinated by a neutral histidine. The trans ligand is hydroxide in the ferric alkaline form of both proteins. In the ferric neutral form at pH 6.0, heme is six-coordinated with water as the sixth ligand for IsdG and is in the mixture of the five-coordinated and six-coordinated species for IsdI. In the ferrous CO-bound form, CO is strongly hydrogen bonded with a distal residue. The marker lines, ν 2 and ν 3 , appear at frequencies that are distinct from other proteins having planar hemes. The EPR spectra for the ferric hydroxide and cyanide states might be explained by assuming the thermal mixing of the d -electron configurations, ( d xy ) 2 ( d xz , d yz ) 3 and ( d xz , d yz ) 4 ( d xy ) 1 . The fraction for the latter becomes larger for the ferric cyanide form. In the ferric neutral state at pH 6.0, the quantum mechanical mixing of the high and intermediate spin configurations might explain the peculiar frequencies of ν 2 and ν 3 in the RR spectra. The heme ruffling imposed by IsdG and IsdI gives rise to unique electronic structures of heme, which are expected to modulate the first and subsequent steps of the heme oxygenation.

Streptococcus pyogenes, an important pathogen that causes a wide range of diseases, possesses the sia gene cluster, which encodes proteins involved in the heme acquisition system. Although this system was previously described, the molecular mechanism of effective heme transfer remains to be elucidated. Here, we have characterized the interactions between heme and each domain of Streptococcal hemoprotein receptor (Shr) and Streptococcal heme-binding protein (Shp). Our kinetic and thermodynamic analyses suggested that effective heme transfer within this system is achieved not only by affinity-based transfer but also by the difference of the binding driving force. The biophysical characterization of the above-mentioned interaction will lead to an indication for the selection of the target for a chemical screening of inhibitors as novel antibacterial agents based on biophysical approaches., (Copyright © 2017 Elsevier Inc. All rights reserved.)

In vertebrates, most iron is present as heme or is chelated by proteins. Thus, Gram-positive pathogens such as Staphylococcus aureus have evolved an iron-regulated surface determinant (Isd) system that transports heme across thick cell walls into the cytoplasm. Recent studies have demonstrated that heme is rapidly transferred between the NEAr Transporter (NEAT) domains of the Isd system, despite its high affinity toward each domain, suggesting the presence of an intermediate NEAT•heme•NEAT complex. In the present study, we performed short restrained molecular dynamics (MD) simulations to dock the acceptor NEAT domain to the donor NEAT•heme complex and obtained models where the two NEAT domains were arranged with two-fold pseudo symmetry around the heme molecule. After turning off the restraints, complex structures were stably maintained during subsequent unrestrained MD simulations, except for the hydrogen bond between the propionate group of the heme molecule and the donor NEAT domain, potentially facilitating the transition of heme from the donor to the acceptor. Subsequent structural optimization using the quantum mechanics/molecular mechanics (QM/MM) method showed that two tyrosine residues, one from each NEAT domain, were simultaneously coordinated to the ferric heme iron in the intermediate complex only if they were deprotonated. Based on these results, we propose a reaction scheme for heme transfer between NEAT domains.

The rapid spread of antibiotic-resistance among pathogenic bacteria poses a serious risk for public health. The search for novel therapeutic strategies and antimicrobial compounds is needed to ameliorate this menace. The bifunctional metalloenzyme CapF is an antibacterial target produced by certain pathogenic bacteria essential in the biosynthetic route of capsular polysaccharide, a mucous layer on the surface of bacterium that facilitates immune evasion and infection. We report the first inhibitor of CapF from Staphylococcus aureus, which was identified by employing fragment-based methodologies. The hit compound 3-isopropenyl-tropolone inhibits the first reaction catalyzed by CapF, disrupting the synthesis of a key precursor of capsular polysaccharide. Isothermal titration calorimetry demonstrates that 3-isopropenyl-tropolone binds tightly (KD = 27 ± 7 μM) to the cupin domain of CapF. In addition, the crystal structure of the enzyme-inhibitor complex shows that the compound engages the essential Zn(2+) ion necessary for the first reaction catalyzed by the enzyme, explaining its inhibitory effect. Moreover, the tropolone compound alters the coordination sphere of the metal, leading to the overall destabilization of the enzyme. We propose 3-isopropenyl-tropolone as a precursor to develop stronger inhibitors for this family of enzymes to impair the synthesis of capsular polysaccharide in Staphylococcus aureus.

The chemical modification of proteins to provide desirable functions and/or structures broadens their possibilities for use in various applications. Usually, proteins can acquire new functions and characteristics, in addition to their original ones, via the introduction of synthetic functional moieties. Here, we adopted a more radical approach to protein modification, i.e., the replacement of a functional domain of proteins with alternative chemical compounds to build "cyborg proteins." As a proof of concept model, we chose staphylococcal α-hemolysin (Hla), which is a well-studied, pore-forming toxin. The hemolytic activity of Hla mutants was dramatically decreased by truncation of the stem domain, which forms a β-barrel pore in the membrane. However, the impaired hemolytic activity was significantly restored by attaching a pyrenyl-maleimide unit to the cysteine residue that was introduced in the remaining stem domain. In contrast, negatively charged fluorescein-maleimide completely abolished the remaining activity of the mutants.

Staphylococcus aureus is one of the most important human pathogens because it is a common cause of nosocomial infections. The elastin-binding protein of Staphylococcus aureus (EbpS) is an adhesin that is responsible for attachment to host cells via its binding to elastin. Despite its relatively weak contribution to adhesion, the ebpS gene is highly conserved among S. aureus isolates, suggesting that EbpS may have other crucial functions. Here, we found that EbpS binds Zn(2+) with its N-terminal region, which leads to local conformational changes that result in the assembly of the EbpS protein. The growth rate of the EbpS-deficient strain was considerably decreased. Zn(2+) chelation decreased the growth rate of the wild-type strain but did not alter that of the EbpS-deficient strain. Furthermore, biofilm formation by the EbpS-deficient strain was abnormally enhanced in the Zn(2+) concentration-dependent manner. All the results suggest that ebpS deficiency led to a zinc concentration-dependent inability to modulate the growth/biofilm maturation phase appropriately. Given the high conservation of ebpS and that appropriate regulation of biofilm formation is thought to be essential for effective staphylococcal infection, inhibition of EbpS binding to Zn(2+) could lead to the development of novel therapeutic strategies for controlling S. aureus infections., (© The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Near transporter (NEAT) domains of the iron-regulated surface determinant (Isd) proteins are essential for the import of nutritional heme from host animals to Gram-positive pathogens such as Staphylococcus aureus. The order of transfer of heme between NEAT domains occurs from IsdH to IsdA to IsdC, without any energy input despite the similarity of their three-dimensional structures. We measured the free energy of binding of heme and various metalloporphyrins to each NEAT domain and found that the affinity of heme and non-iron porphyrins for NEAT domains increased gradually in the same order as that for heme transfer. To gain insight into the atomistic mechanism for the differential affinities, we performed in silico molecular dynamics simulation and in vitro site-directed mutagenesis. The simulations revealed that the negatively charged residues that are abundant in the loop between strand β1b and the 310 helix of IsdH-NEAT3 destabilize the interaction with the propionate group of heme. The higher affinity of IsdC was in part attributed to the formation of a salt bridge between its unique residue, Glu88, and the conserved Arg100 upon binding to heme. In addition, we found that Phe130 of IsdC makes the β7-β8 hairpin less flexible in the ligand-free form, which serves to reduce the magnitude of the entropy loss on binding to heme. We confirmed that substitution of these key residues of IsdC decreased its affinity for heme. Furthermore, IsdC mutants, whose affinities for heme were lower than those of IsdA, transferred heme back to IsdA. Thus, NEAT domains have evolved the characteristic residues on the common structural scaffold such that they exhibit different affinities for heme, thus promoting the efficient transfer of heme.

CapE is an essential enzyme for the synthesis of capsular polysaccharide (CP) of pathogenic strains of Staphylococcus aureus. Herein we demonstrate that CapE is a 5-inverting 4,6-dehydratase enzyme. However, in the absence of downstream enzymes, CapE catalyzes an additional reaction (5-back-epimerization) affording a by-product under thermodynamic control. Single-crystal X-ray crystallography was employed to identify the structure of the by-product. The structural analysis reveals a network of coordinated motions away from the active site governing the enzymatic activity of CapE. A second dynamic element (the latch) regulates the enzymatic chemoselectivity. The validity of these mechanisms was evaluated by site-directed mutagenesis., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Enzymes synthesizing the bacterial CP (capsular polysaccharide) are attractive antimicrobial targets. However, we lack critical information about the structure and mechanism of many of them. In an effort to reduce that gap, we have determined three different crystal structures of the enzyme CapE of the human pathogen Staphylococcus aureus. The structure reveals that CapE is a member of the SDR (short-chain dehydrogenase/reductase) super-family of proteins. CapE assembles in a hexameric complex stabilized by three major contact surfaces between protein subunits. Turnover of substrate and/or coenzyme induces major conformational changes at the contact interface between protein subunits, and a displacement of the substrate-binding domain with respect to the Rossmann domain. A novel dynamic element that we called the latch is essential for remodelling of the protein-protein interface. Structural and primary sequence alignment identifies a group of SDR proteins involved in polysaccharide synthesis that share the two salient features of CapE: the mobile loop (latch) and a distinctive catalytic site (MxxxK). The relevance of these structural elements was evaluated by site-directed mutagenesis.

IsdG and IsdI from Staphylococcus aureus are novel heme-degrading enzymes containing unusually nonplanar (ruffled) heme. While canonical heme-degrading enzymes, heme oxygenases, catalyze heme degradation coupled with the release of CO, in this study we demonstrate that the primary C1 product of the S. aureus enzymes is formaldehyde. This finding clearly reveals that both IsdG and IsdI degrade heme by an unusual mechanism distinct from the well-characterized heme oxygenase mechanism as recently proposed for MhuD from Mycobacterium tuberculosis. We conclude that heme ruffling is critical for the drastic mechanistic change for these novel bacterial enzymes.

A chimeric protein (N-PYP-Hla), consisting of staphylococcal pore-forming toxin α-hemolysin (Hla) and photoactive yellow protein (PYP), exhibited photoresponsive hemolytic activities, where visible light irradiation gave rise to retardation of hemolysis at 25 °C., (This journal is © The Royal Society of Chemistry 2012)

CP (capsular polysaccharide) is an important virulence factor during infections by the bacterium Staphylococcus aureus. The enzyme CapF is an attractive therapeutic candidate belonging to the biosynthetic route of CP of pathogenic strains of S. aureus. In the present study, we report two independent crystal structures of CapF in an open form of the apoenzyme. CapF is a homodimer displaying a characteristic dumb-bell-shaped architecture composed of two domains. The N-terminal domain (residues 1-252) adopts a Rossmann fold belonging to the short-chain dehydrogenase/reductase family of proteins. The C-terminal domain (residues 252-369) displays a standard cupin fold with a Zn2+ ion bound deep in the binding pocket of the β-barrel. Functional and thermodynamic analyses indicated that each domain catalyses separate enzymatic reactions. The cupin domain is necessary for the C3-epimerization of UDP-4-hexulose. Meanwhile, the N-terminal domain catalyses the NADPH-dependent reduction of the intermediate species generated by the cupin domain. Analysis by ITC (isothermal titration calorimetry) revealed a fascinating thermodynamic switch governing the attachment and release of the coenzyme NADPH during each catalytic cycle. These observations suggested that the binding of coenzyme to CapF facilitates a disorder-to-order transition in the catalytic loop of the reductase (N-terminal) domain. We anticipate that the present study will improve the general understanding of the synthesis of CP in S. aureus and will aid in the design of new therapeutic agents against this pathogenic bacterium.

ATP-binding cassette (ABC) transporters couple hydrolysis of ATP with vectorial transport across the cell membrane. We have reconstituted ABC transporter MsbA in nanodiscs of various sizes and lipid compositions to test whether ATPase activity is modulated by the properties of the bilayer. ATP hydrolysis rates, Michaelis-Menten parameters, and dissociation constants of substrate analog ATP-γ-S demonstrated that physicochemical properties of the bilayer modulated binding and ATPase activity. This is remarkable when considering that the catalytic unit is located ~50Å from the transmembrane region. Our results validated the use of nanodiscs as an effective tool to reconstitute MsbA in an active catalytic state, and highlighted the close relationship between otherwise distant transmembrane and ATPase modules., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Ebh, a giant protein found in staphylococci, contains several domains, including a large central region with 52 imperfect repeats of a domain composed of 126 amino acids. We used electron microscopy to observe the rod-like structure of a partial Ebh protein containing 10 repeating units. This is the first report of the direct observation of an Ebh structure containing a large number of repeating units, although structures containing one, two, or four repeating units have been reported. The observed structure of the partial Ebh protein was distorted and had a length of ca. 520A and a width of ca. 21A. The observed structures were consistent with those deduced from crystal structure analysis, suggesting that the Ebh domains are connected to form a rod-like structure. The crystal structure data revealed distorted, string-like features in the simulated structure of the whole-length Ebh protein. Superposition of fragments of the simulated whole-length structure of the Ebh protein onto each electron micrograph showed a high level of correlation between the observed and calculated structures. These results suggest that Ebh is composed of highly flexible filate molecules. The highly repetitive structure and the associated unique structural flexibility of Ebh support the proposed function of this protein, i.e. binding to sugars in the cell wall. This binding might result in intra-cell-wall cross-linking that contributes to the rigidity of bacterial cells.

Staphylococcus aureus is well known to colonize on human skin where the physiological condition is characterized by hypervariable water activity, i.e., repeated dehydration or rehydration. To determine the facilitating factors for the colonization under hypervariable water activity, we studied the giant protein Ebh (extracellular matrix (ECM)-binding protein homologue). The ebh mutant RAM8 showed invaginated vacuoles along the septum, similar to that found in partial plasmolysis, and the cells burst under osmotic upshift. RAM8 was also relatively susceptible to abrupt hyperosmotic upshift, teicoplanin, and Triton X-100. By using the green fluorescent protein (GFP) as a reporter, Ebh was localized over the entire cell surface. This suggests that Ebh might contribute to structural homeostasis by forming a bridge between the cell-wall and cytoplasmic membrane to avoid plasmolysis under hyperosmotic condition.

Green fluorescent protein (GFP) is often misfolded into nonfluorescent states when an aggregatable sequence is attached to its N-terminus. However, GFP fusions with highly aggregatable, prion-determining, and highly charged sequences from yeast prions, such as Sup35 and Ure2p, form green fibrils with properly folded GFP. To gain further insight into the general effect of an aggregatable sequence attached to fluorescent protein, we designed eight fusion proteins of a yellow variant of GFP (YFP) containing an aggregation-prone amyloidogenic sequence derived from human medin, attached via different lengths of linker sequence. Seven fusion proteins formed white fibrils lacking native YFP function. However, the fusion with an 18-residue medin sequence and a 50 amino acid linker formed fibrils with yellow color of folded YFP. Deconvolution analysis of infrared spectra also supports the presence of properly folded YFP in the fibrils formed by this protein. These results suggest that, the presence of an amyloidogenic sequence to a folded protein can promote the formation of fibrils and disrupt the native structures whereas the structure of the folded region is retained by optimizing sequences of amyloidogenic and linker regions.

The 1.1 MDa cell-wall-associated adhesion protein of staphylococci, Ebh, consists of several distinct regions, including a large central region with 52 imperfect repeats of 126 amino acid residues. We investigated the structure of this giant molecule by X-ray crystallography, circular dichroism (CD) spectrometry, and small-angle X-ray scattering (SAXS). The crystal structure of two repeats showed that each repeat consists of two distinct three-helix bundles, and two such repeats are connected along the long axis, resulting in a rod-like structure that is 120 A in length. CD and SAXS analyses of the samples with longer repeats suggested that each repeat has an identical structure, and that such repeats are connected tandemly to form a rod-like structure in solution, the length of which increased proportionately with the number of repeating units. On the basis of these results, it was proposed that Ebh is a 320 nm rod-like molecule with some plasticity at module junctions.

Elastin-binding protein of Staphylococcus aureus (EbpS) has been identified as an adhesin that can bind to soluble elastin or tropoelastin. However, the structure and exact function of EbpS remain to be elucidated. To gain insight into the molecular characteristics of EbpS, we investigated the physical properties of its N-terminal extracellular domain in various environments. CD spectroscopy showed that this protein was soluble and unstructured under aqueous conditions. Non-native secondary structures, however, were induced by several alcohols that provided membrane-mimetic environments. These changes may have some correlation with the function of this protein.

Drp35 is a protein induced by cell wall-affecting antibiotics or detergents; it possesses calcium-dependent lactonase activity. To determine the molecular basis of the lactonase activity, we first solved the crystal structures of Drp35 with and without Ca(2+); these showed that the molecule has a six-bladed beta-propeller structure with two calcium ions bound at the center of the beta-propeller and surface region. Mutational analyses of evolutionarily conserved residues revealed that the central calcium-binding site is essential for the enzymatic activity of Drp35. Substitution of some other amino acid residues for the calcium-binding residues demonstrated the critical contributions of Glu(48), Asp(138), and Asp(236) to the enzymatic activity. Differential scanning calorimetric analysis revealed that the loss of activity of E48Q and D236N, but not D138N, was attributed to their inability to hold the calcium ion. Further structural analysis of the D138N mutant indicates that it lacks a water molecule bound to the calcium ion rather than the calcium ion itself. Based on these observations and structural information, a possible catalytic mechanism in which the calcium ion and its binding residues play direct roles was proposed for the lactonase activity of Drp35.

Acidic pH is commonly used to elute antibodies from Protein-A affinity column, although low pH may result in aggregation of the proteins. As an alternative, here arginine was tested as an eluent and compared with a more conventional eluent of citrate. Using purified monoclonal antibodies, recovery of antibodies with 0.1M citrate, pH 3.8, was less than 50% and decreased further as the pH was increased to 4.3. At the same pH, the recovery of antibodies was greatly increased with 0.5M arginine and more so with 2M arginine. Even at pH 5.0, 2M arginine resulted in 31% recovery, although the elution under such condition showed extensive tailing. Such tailing was observed at pH 3.8 when 0.1M citrate was used. Size exclusion analysis indicated that the eluted antibodies were mostly monomeric whether eluted with citrate or arginine. This demonstrates the usefulness of arginine as an efficient eluent for Protein-A chromatography.