1H, 13C, and 15N NMR Resonance Assignments, Secondary Structure, and Backbone Topology of a Variant of Human Interleukin-3

Feng, Yiqing; Klein, Barbara K.; Vu, Linh; Aykent, Serdar; McWherter, Charles A.

doi:10.1021/bi00019a036

Cited by 27 publications

(30 citation statements)

References 47 publications

(57 reference statements)

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“…To rule out the possibility that structural perturbation leads to the loss of activity, seven of the mutants (G42A, G42D, E43N, D44A, E50D, F113Y, and K116W), along with the parental molecule hIL-3 , were characterized using far UV circular dichroism spectroscopy. All of the variants had a helical content of ϳ40% that was indistinguishable from that of hIL-3 and was also consistent with the solution structure of SC-65369 (6). These data indicate that the mutants are properly folded with no large scale changes in global conformation.…”

Section: Resultssupporting

confidence: 76%

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The Receptor Binding Site of Human Interleukin-3 Defined by Mutagenesis and Molecular Modeling

Klein¹,

Feng²,

McWherter³

et al. 1997

Journal of Biological Chemistry

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Interleukin-3 (IL-3) is a member of the cytokine superfamily that promotes multi-potential hematopoietic cell growth by interacting with a cell surface receptor composed of ␣ and ␤ chains. The newly available threedimensional structure of a variant of human (h) IL-3 allowed us to evaluate new and existing mutagenesis data and to rationally interpret the structure-function relationship of hIL-3 on a structural basis. The amino acid residues that were identified to be important for hIL-3 activity are grouped into two classes. The first class consists of largely hydrophobic residues required for the structural integrity of the protein, including the residues in IL-3 that are largely conserved among 10 mammalian species. These residues form the core of a scaffold for the second class of more rapidly diverging solvent-exposed residues, likely to be required for interaction with the receptor. , map to one side of the protein and form a putative binding site for the ␣ subunit of the receptor. A model of the IL-3⅐IL-3 receptor complex based on the human growth hormone (hGH)⅐hGH soluble receptor complex structure suggests that the interface between IL-3 and the IL-3 receptor ␣ subunit consists of a cluster of hydrophobic residues flanked by electrostatic interactions. Although the IL-3/ IL-3 receptor ␤ subunit interface cannot be uniquely located due to the lack of sufficient experimental data, several residues of the ␤ subunit that may interact with Glu 22 of IL-3 are proposed. The role of these residues can be tested in future mutagenesis studies to define the interaction between IL-3 and IL-3 receptor ␤ subunit. Interleukin-3 (IL-3)1 is a multi-lineage hematopoietic growth factor that promotes the growth of most lineages of blood cell precursors (1, 2). It belongs to the helical cytokine superfamily and is further classified as a short chain cytokine similar to granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-5 (IL-5) (3-5). Characteristic of the short chain helical cytokines, the structural core of the hIL-3 variant SC-65369 (6, 7) consists of an up-up-down-down four-helical bundle (designated as helix A through D) with a 30 -40°packing angle. This helical bundle motif is completed by long overhand loops connecting the helices (loops AB and CD) and a type II turn (turn BC) between helices B and C. Distinct from the other short chain cytokines, the hIL-3 variant structure also revealed that loop AB contains an additional helix (helix AЈ) that is approximately parallel to helix D and interacts extensively with both helix D and loop CD. Furthermore, loop AB, which passes in front of helix D, has a threading topology that is more like that of the long chain cytokines such as growth hormone (8, 9).Binding to a cell surface receptor (IL-3R) composed of at least ␣ and ␤ chains is the initial event in the expression of IL-3's proliferative activity on a target cell. The ␣ chain of this receptor (IL-3R␣) is specific for IL-3, whereas the ␤ chain (␤ c ) is shared with the related hematopoietic cytokine...

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Section: Resultssupporting

confidence: 76%

“…As mentioned previously the ligands show little sequence homology but have substantial structural homology (6,8,9). The receptors of IL-3 and GH, however, share sequence homology in their extracellular domains consisting of a pattern of conserved Cys residues and the WSXWS box near the C terminus (3).…”

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confidence: 86%

The Receptor Binding Site of Human Interleukin-3 Defined by Mutagenesis and Molecular Modeling

Klein¹,

Feng²,

McWherter³

et al. 1997

Journal of Biological Chemistry

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“…Thomas, W. Hood, unpublished results), and (3) it is closely related to SC-55494 (Thomas et al, 1995), a similarly truncated and multiply substituted hIL-3 variant which has a 10 to 20-fold increase in its growth promoting activity and which is undergoing clinical evaluation for reducing the degree and duration of chemotherapy-induced cytopenias. Previously, the 1 H, 13 C and 15 N resonance assignments, secondary structure and chain folding topology of SC-65369 have been determined using 13 C/ 15 N-enriched samples of SC-65369 (Feng et al, 1995). In addition to the expected four-helical bundle, an unexpected observation was the small helix designated as helix A' in the loop connecting the first two helices (helices A and B).…”

Section: Resultsmentioning

confidence: 99%

“…In order to provide a more complete basis for interpreting these studies, we initiated a project to determine the three-dimensional structure of a soluble variant of hIL-3 (SC-65369) using multi-dimensional heteronuclear NMR techniques. In a previous paper, we reported the 1 H, 15 N, and 13 C NMR resonance assignments, the secondary structure, and the chain-folding topology of SC-65369 (Feng et al, 1995). In this paper, we report the three-dimensional structure and backbone 15 N dynamics of SC-65369.…”

Section: Introductionmentioning

confidence: 85%

“…The initial biophysical characterization of recombinant IL-3 and IL-3 variants by CD (Freeman et al, 1991;R. Schilling & R. McKinnie, unpublished results) and NMR spectroscopy (Feng et al, 1995) have served to confirm this designation. Stimulation of the IL-3 receptor on immature multipotential and lineage-restricted hematopoietic progenitors, either alone or in the presence of other cytokines, leads to their proliferation and thereby enhances the development of erythrocytes, platelets, macrophages, neutrophils, basophils, and eosinophils (Metcalf & Nicola, 1995).…”

Section: Introductionmentioning

confidence: 87%

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Three-dimensional Solution Structure and Backbone Dynamics of a Variant of Human Interleukin-3

Feng¹,

Klein²,

McWherter³

1996

Journal of Molecular Biology

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The three-dimensional structure and backbone dynamics of a truncated G.D. Searle and Companyand multiply substituted recombinant human interleukin-3 (IL-3) variant 700 Chesterfield (SC-65369) have been determined from multidimensional heteronuclear Parkway North, St. Louis MO 63198, USA nuclear magnetic resonance spectroscopic data. Sequential application of distance geometry and restrained molecular dynamics calculations produced a family of 25 convergent structures which satisfy a total of 1812 experimental constraints (1659 proton-proton NOEs, 75 backbone dihedral angle constraints, and 39 pairs of hydrogen bond constraints) with an average root-mean-square deviation from the mean coordinate positions of 0.88(20.15) Å and 1.37(20.13) Å for the backbone and all heavy atoms, respectively, of all residues except 28 to 39. The structure is a left-handed four-helix bundle (comprised of helices A through D) with two long overhand loops (designated as loops AB and CD). Loop AB contains a short fifth helix (helix A') which is closely packed against helix D in an approximately parallel fashion and which has multiple contacts with loop CD. The overall molecular tumbling time (6.5 ns) determined from the 15 N relaxation data was consistent with a monomeric protein under the conditions of the experiment (1 mM protein, pH 4.6, 30°C). The 15 N relaxation data indicate that the helical regions of SC-65369 are quite rigid, while portions of loop AB, loop CD, and the C terminus undergo significant internal motions. Among the structurally related four-helical bundle cytokines, the structure of SC-65369 is most similar to those of granulocyte-macrophage colony stimulating factor (GM-CSF) and the single structural domain of interleukin-5 (IL-5), all of which share a common receptor subunit required for signal transduction and activation of their hematopoietic target cells. Indeed, the C a atoms in the four-helix core of these three proteins can be superimposed to 1.71 Å (SC-65369 and GM-CSF, 62 C a atoms) and 1.96 Å (SC-65369 and IL-5 single structural domain, 58 C a atoms), respectively. When the structures of the IL-3 variant, GM-CSF, and IL-5 were aligned, the conserved and conservatively substituted residues were found to be hydrophobic and buried, with the single exception of Glu-22 (IL-3 numbering), which is strictly conserved but nonetheless fully exposed to solvent. The most remarkable differences between the SC-65369 structure and that of GM-CSF occur in loop AB. This loop in GM-CSF crosses over the top of helix D and passes underneath loop CD on its way to helix B. In contrast, loop AB of SC-65369 passes in front of helix D, similar to the first crossover loop in human growth hormone and granulocyte colony-stimulating factor. In addition, helix A', which is interdigitated into the helical bundle in a manner similar to the helices in the CD loop of interferon-b and interferon-g, exists in a region where short *Corresponding authors stretches of b-structure are found at analogous positions in GM-CSF and Abbreviations use...

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Haematopoietic Growth Factors

Thomas¹,

López²

2009

Encyclopedia of Life Sciences

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Haematopoietic growth factors regulate the production of all the cellular elements of blood by stimulating the proliferation, survival and differentiation of haematopoietic cells in various stages of maturation and also enhancing certain mature cell functions. Recombinant growth factors are used in the clinic to stimulate cell production as part of treatment for anaemia, neutropenia and thrombocytopenia and to aid in the collection of bone marrow stem cells.

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1H, 13C, and 15N NMR Resonance Assignments, Secondary Structure, and Backbone Topology of a Variant of Human Interleukin-3

Cited by 27 publications

References 47 publications

The Receptor Binding Site of Human Interleukin-3 Defined by Mutagenesis and Molecular Modeling

The Receptor Binding Site of Human Interleukin-3 Defined by Mutagenesis and Molecular Modeling

Three-dimensional Solution Structure and Backbone Dynamics of a Variant of Human Interleukin-3

Haematopoietic Growth Factors

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