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ABSTRACT: The Isd (iron-regulated surface determinant) system of the human pathogen Staphylococcus aureus is responsible for the adquisition of heme from the host organism. We recently reported that the extracellular heme receptor IsdH-NEAT3 captures and transfers non-iron antimicrobial porphyrins containing metals in oxidation state (III). However, it is unclear if geometric factors such as the size of the metal (ionic radius) affect binding and transfer of metalloporphyrins. We carried out an ample structural, functional, and thermodynamic analysis of the binding properties of antimicrobial indium(III)-porphyrin, which bears a much larger metal ion than the iron(III) of the natural ligand heme. The results demonstrate that the NEAT3 receptor recognizes the In(III)-containing PPIX in a manner very similar to that of heme. Site-directed mutagenesis identifies Tyr642 as the central element in the recognition mechanism as suggested from the crystal structures. Importantly, the NEAT3 receptor possesses the remarkable ability to capture dimers of metalloporphyrin. Molecular dynamics simulations reveal that IsdH-NEAT3 does not require conformational changes, or large rearrangements of the residues within its binding site, to accommodate the much larger (heme)2 ligand. We discuss the implications of these findings for the design of potent inhibitors against this family of key receptors of S. aureus.
Protein Science 05/2013; · 2.80 Impact Factor
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ABSTRACT: Enzymes synthesizing the bacterial capsular polysaccharide (CP) 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 short-chain dehydrogenase/reductase (SDR) 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 remodeling 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.
Bioscience Reports 04/2013; · 2.38 Impact Factor
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ABSTRACT: Iron is an essential nutrient for the proliferation of Staphylococcus aureus during bacterial infections. The iron-regulated surface determinant (Isd) system of S. aureus transports and metabolizes iron porphyrin (heme) captured from the host organism. Transportation of heme across the thick cell wall of this bacterium requires multiple relay points. The mechanism by which heme is physically transferred between Isd transporters is largely unknown because of the transient nature of the interactions involved. Herein, we show that the IsdC transporter not only passes heme ligand to another class of Isd transporter, as previously known, but can also perform self-transfer reactions. IsdA shows a similar ability. A genetically encoded photoreactive probe was used to survey the regions of IsdC involved in self-dimerization. We propose an updated model that explicitly considers self-transfer reactions to explain heme delivery across the cell wall. An analogous photo-cross-linking strategy was employed to map transient interactions between IsdC and IsdE transporters. These experiments identified a key structural element involved in the rapid and specific transfer of heme from IsdC to IsdE. The resulting structural model was validated with a chimeric version of the homologous transporter IsdA. Overall, our results show that the ultra-weak interactions between Isd transporters are governed by bona fide protein structural motifs.
Journal of Biological Chemistry 03/2012; 287(20):16477-87. · 4.77 Impact Factor
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ABSTRACT: 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.
Biochemical Journal 02/2012; 443(3):671-80. · 4.90 Impact Factor
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ABSTRACT: 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.
FEBS letters 11/2011; 585(22):3533-7. · 3.54 Impact Factor
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ABSTRACT: Antibiotic resistance is increasingly seen as a serious problem that threatens public health and erodes our capacity to effectively combat disease. So-called non-iron metalloporhyrins have shown promising antibacterial properties against a number of pathogenic bacteria including Staphylococcus aureus. However, little is known about the molecular mechanism(s) of action of these compounds and in particular how they reach the interior of the bacterial cells. A popular hypothesis indicates that non-iron metalloporphyrins infiltrate into bacterial cells like a "Trojan horse" using heme transport systems. Iron-regulated surface determinant (Isd) is the best characterized heme transport system of S. aureus. Herein we studied the molecular mechanism by which the extracellular heme-receptor IsdH-NEAT3 of Isd recognizes antimicrobial metalloporphyrins. We found that potent antibacterial porphyrins Ga(III)-protoporphyrin IX (PPIX) and Mn(III)-PPIX closely mimicked the properties of the natural ligand heme, namely (i) stable binding to IsdH-NEAT3 with comparable affinities for the receptor, (ii) nearly undistinghuishable three-dimensional structure when complexed with IsdH-NEAT3, and (iii) similar transfer properties to a second receptor IsdA. On the contrary, weaker antibacterial porphyrins Mg(II)-PPIX, Zn(II)-PPIX, and Cu(II)-PPIX were not captured effectively by IsdH-NEAT3 under our experimental conditions and displayed lower affinities. Moreover, reduction of Fe(III)-PPIX to Fe(II)-PPIX with dithionite abrogated stable binding to receptor. These data revealed a clear connection between oxidation state of metal and effective attachment to IsdH-NEAT3. Also, the strong correlation between binding affinity and reported antimicrobial potency suggested that the Isd system may be used by these antibacterial compounds to gain access to the interior of the cells. We hope these results will increase our understanding of Isd system of S. aureus and highlight its biomedical potential to deliver new and more efficient antibacterial treatments.
Biochemistry 08/2011; 50(34):7311-20. · 3.42 Impact Factor
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ABSTRACT: Human interleukin-11 (hIL-11) is a pleiotropic cytokine administered to patients with low platelet counts. From a structural
point of view hIL-11 belongs to the long-helix cytokine superfamily, which is characterized by a conserved core motif consisting
of four α-helices. We have investigated the region of hIL-11 that does not belong to the α-helical bundle motif, and that
for the purpose of brevity we have termed “non-core region.” The primary sequence of the interleukin was altered at various
locations within the non-core region by introducing glycosylation sites. Functional consequences of these modifications were
examined in cell-based as well as biophysical assays. Overall, the data indicated that the non-core region modulates the function
of hIL-11 in two ways. First, the majority of muteins displayed enhanced cell-stimulatory properties (superagonist behavior)
in a glycosylation-dependent manner, suggesting that the non-core region is biologically designed to limit the full potential
of hIL-11. Second, specific modification of a predicted mini α-helix led to cytokine inactivation, demonstrating that this
putative structural element belongs to site III engaging a second copy of cell-receptor gp130. These findings have unveiled
new and unexpected elements modulating the biological activity of hIL-11, which may be exploited to develop more versatile
medications based on this important cytokine.
Journal of Biological Chemistry 03/2011; 286(10):8085-8093. · 4.77 Impact Factor
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ABSTRACT: Human interleukin-11 (hIL-11) is a pleiotropic cytokine administered to patients with low platelet counts. From a structural point of view hIL-11 belongs to the long-helix cytokine superfamily, which is characterized by a conserved core motif consisting of four α-helices. We have investigated the region of hIL-11 that does not belong to the α-helical bundle motif, and that for the purpose of brevity we have termed "non-core region." The primary sequence of the interleukin was altered at various locations within the non-core region by introducing glycosylation sites. Functional consequences of these modifications were examined in cell-based as well as biophysical assays. Overall, the data indicated that the non-core region modulates the function of hIL-11 in two ways. First, the majority of muteins displayed enhanced cell-stimulatory properties (superagonist behavior) in a glycosylation-dependent manner, suggesting that the non-core region is biologically designed to limit the full potential of hIL-11. Second, specific modification of a predicted mini α-helix led to cytokine inactivation, demonstrating that this putative structural element belongs to site III engaging a second copy of cell-receptor gp130. These findings have unveiled new and unexpected elements modulating the biological activity of hIL-11, which may be exploited to develop more versatile medications based on this important cytokine.
Journal of Biological Chemistry 12/2010; 286(10):8085-93. · 4.77 Impact Factor
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ABSTRACT: N-Acylamino acids are a new family of versatile biological surfactants capable of extracting integral membrane proteins of various topologies from the biological membrane, in many instances surpassing the efficiency of commercial detergents.
Molecular BioSystems 04/2010; 6(4):677-9. · 3.53 Impact Factor
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ABSTRACT: Human interleukin 15 (hIL15) is a soluble cytokine that plays a key role in the maintenance of long-lasting responses against pathogens and a valuable target for the treatment of autoimmune diseases. In this study, we sought to elucidate the thermodynamic basis of the recognition mechanism for its private alpha-receptor (hIL15Ralpha), considered the first step of the interleukin's activation pathway. Binding of wild-type hIL15 to its alpha-receptor is characterized by a very slow dissociation rate constant and driven by a favorable enthalpy change. We further studied the kinetic and energetic consequences of substituting residues of hIL15 located at the contact interface by means of the surface plasmon resonance technique. Replacement of negatively charged residues with Ala indicates that the energetics of interaction is primarily driven by electrostatic forces, manifested by a dramatic acceleration of the dissociation step and a reduction of favorable binding enthalpy. Our analyses also unveiled a novel and critical role for residue Tyr26 in the interaction, which facilitates desolvation of key charged residues during the assembly of the complex. These results were rationalized in terms of a previously reported structure of hIL15.hIL15alpha, demonstrating that the binding energetics is dominated by interactions occurring at three hot spots whose spatial locations coincide with a previously proposed structural division of the contact interface in three regions. Specifically, Region 1 is the main contributor to the binding energy of the complex by establishing very favorable electrostatic interactions with the receptor; Region 2 is also dominated by electrostatic forces, although of a lesser intensity; and Region 3 confers specificity to the association by means of high shape complementarity and by bringing additional stabilization energy to the complex. The biological impact of hIL15 mutations with the most effect on alpha-receptor binding was evaluated in a cell-based proliferation assay, validating the conclusions of our thermodynamic analyses and highlighting the functional importance of molecular contacts that promote prolonged binding of the interleukin to the alpha-receptor. In closing, the thermodynamics and physicochemical nature of the interactions observed in IL15h.IL15Ralpha complex, together with interactions in Region 3 of the interleukin, poses a stark contrast with the structurally related and sometimes functionally redundant interleukin 2.
Journal of Molecular Biology 06/2009; 389(5):880-94. · 4.00 Impact Factor
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ABSTRACT: Human interleukin 15 (hIL15) is a soluble cytokine that plays a key role in the maintenance of long-lasting responses against pathogens and a valuable target for the treatment of autoimmune diseases. In this study, we sought to elucidate the thermodynamic basis of the recognition mechanism for its private α-receptor (hIL15Rα), considered the first step of the interleukin's activation pathway. Binding of wild-type hIL15 to its α-receptor is characterized by a very slow dissociation rate constant and driven by a favorable enthalpy change. We further studied the kinetic and energetic consequences of substituting residues of hIL15 located at the contact interface by means of the surface plasmon resonance technique. Replacement of negatively charged residues with Ala indicates that the energetics of interaction is primarily driven by electrostatic forces, manifested by a dramatic acceleration of the dissociation step and a reduction of favorable binding enthalpy. Our analyses also unveiled a novel and critical role for residue Tyr26 in the interaction, which facilitates desolvation of key charged residues during the assembly of the complex. These results were rationalized in terms of a previously reported structure of hIL15·hIL15α, demonstrating that the binding energetics is dominated by interactions occurring at three hot spots whose spatial locations coincide with a previously proposed structural division of the contact interface in three regions. Specifically, Region 1 is the main contributor to the binding energy of the complex by establishing very favorable electrostatic interactions with the receptor; Region 2 is also dominated by electrostatic forces, although of a lesser intensity; and Region 3 confers specificity to the association by means of high shape complementarity and by bringing additional stabilization energy to the complex. The biological impact of hIL15 mutations with the most effect on α-receptor binding was evaluated in a cell-based proliferation assay, validating the conclusions of our thermodynamic analyses and highlighting the functional importance of molecular contacts that promote prolonged binding of the interleukin to the α-receptor. In closing, the thermodynamics and physicochemical nature of the interactions observed in IL15h·IL15Rα complex, together with interactions in Region 3 of the interleukin, poses a stark contrast with the structurally related and sometimes functionally redundant interleukin 2.
Journal of Molecular Biology.
