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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: 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.
Biochemical and Biophysical Research Communications 10/2008; 376(2):261-6. · 2.48 Impact Factor
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Yoshikazu Tanaka, Sou Sakamoto,
Makoto Kuroda,
Shuichiro Goda,
Yong-Gui Gao,
Kouhei Tsumoto,
Yuzuru Hiragi,
Min Yao,
Nobuhisa Watanabe,
Toshiko Ohta,
Isao Tanaka
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ABSTRACT: 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.
Structure 04/2008; 16(3):488-96. · 6.35 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.
