A deletion variant of human interleukin-3, hIL-3 15-125 , was produced in the periplasmic space of Escherichia coli and had full activity in an AML193.1.3 cell proliferation assay. Libraries of random single-amino acid substitutions were constructed at each of 105 positions in the gene for hIL-3 . Approximately eight single-site substitutions at each position were produced in osmotic shock fractions and screened for activity. 15 mutants were found with bioactivity of 5-26-fold greater than that of native hIL-3. The majority of amino acids in hIL-3 15-125 could be substituted without substantial loss of activity. Substitution of residues predicted to be in the hydrophobic core of the protein often resulted in reduced activity and/or low accumulation levels. Only five residues predicted to be on the surface of the protein were intolerant of substitution and hence are candidates for sites of interaction with the receptor. We therefore propose that the majority of residues in hIL-3 serve a structural role and permit the display of a few key residues in the correct configuration for recognition by the receptor.Human interleukin-3 (hIL-3) 1 is a multilineage hematopoietic cytokine acting in the bone marrow to promote the growth of most lineages of blood cell precursors (1). Recently, exogenously administered hIL-3 has shown promise for the clinical relief of neutropenia and thrombocytopenia induced by cancer chemotherapy (2, 3). Sequence homology comparisons of hIL-3 with other proteins indicate that it is a member of the hematopoietic cytokine family (4 -6) and that it adopts a four-␣-helix bundle topology (7-10). The protein binds to a receptor comprising at least two nonidentical subunits (11, 12). Although the precise nature of interaction between hIL-3 and its receptor is not known, studies using site-specific mutants have shed some light on which portions of the protein are important for function (8,(13)(14)(15)(16)(17). In particular, mutagenesis of the adjacent helices A and D indicate that these regions are important for interaction with the receptor. This is similar to the findings for human interleukin-5 and human granulocyte-macrophage colony stimulating factor, whose receptors share a common  subunit with the hIL-3 receptor (11,18,19). Other members of the hematopoietic cytokine family also have important residues in helices A and D (19 -23) and in helix C (20,22,24,25).In this paper we have undertaken an extensive mutagenesis of hIL-3 in order to discover mutants with enhanced proliferative activity and to define residues necessary for activity. Although alanine scanning mutagenesis has been successfully used to derive structure-activity information (20, 26 -32), we chose to perform a more extensive mutagenesis, permitting the incorporation of any of the possible 19 substitutions (33). MATERIALS AND METHODS Production of hIL-3 and Variants in the Escherichia coli Cytoplasm-General techniques for manipulation of DNA are described elsewhere (34). The hIL-3 gene (35) was obtained from British Biotechnolo...
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...
A systematic evaluation of structure-activity information led to the construction of genetically engineered interleukin 3 (IL-3) receptor agonists (synthokines) with enhanced hematopoietic potency. SC-55494, the most extensively characterized member of this series, exhibits 10-to 20-fold greater biological activity than recombinant human
Myelopoietins (MPOs) are a family of engineered dual interleukin-3 (IL-3) and granulocyte colony-stimulating factor (G-CSF) receptor agonists that are superior in comparison to the single agonists in their ability to promote the growth and maturation of hematopoietic cells of the myeloid lineage. A series of MPO molecules were created which incorporated circularly permuted G-CSF (cpG-CSF) sequences with an IL-3 receptor (IL-3R) agonist moiety attached at locations that correspond to the loops that connect the helices of the G-CSF four-helix bundle structure. The cpG-CSF linkage sites (using the original sequence numbering) were residue 39, which is at the beginning of the first loop connecting helices 1 and 2; residue 97, which is in the turn connecting helices 2 and 3; and residues 126, 133, and 142, which are at the beginning, middle, and end, respectively, of the loop connecting helices 3 and 4. The N- and C-terminal helices of each cpG-CSF domain were constrained, either by direct linkage of the termini (L0) or by replacement of the amino-terminal 10-residue segment with a seven-residue linker composed of SGGSGGS (L1). All of the MPO molecules stimulated the proliferation of both IL-3-dependent (EC50 = 13-95 pM) and G-CSF-dependent (EC50 = 35-710 pM) cell lines. MPOs with the IL-3R agonist domain linked to cpG-CSFs in the first (residue 39) or second (residue 133) long overhand loops were found by CD spectroscopy to have helical contents similar to that expected for a protein comprised of two linked four-helix bundles. The MPOs retained the ability to bind to the IL-3R with affinities similar to that of the parental MPO. Using both a cell surface competitive binding assay and surface plasmon resonance detection of binding kinetics, the MPOs were found to bind to the G-CSF receptor with low nanomolar affinities, similar to that of G-CSF(S17). In a study of isolated cpG-CSF domains [Feng, Y., et al. (1999) Biochemistry 38, 4553-4563], domains with the L1 linker had lower G-CSF receptor-mediated proliferative activities and conformational stabilities than those which had the L0 linker. A similar trend was found for the MPOs in which the G-CSFR agonist activity is mostly a property of the cpG-CSF domain. Important exceptions were found in which the linkage to the IL-3R agonist domain either restored (e.g., attachment at residue 142) or further decreased (linkage at residue 39) the G-CSFR-mediated proliferative activity. MPO in which the IL-3R agonist domain is attached to the cpG-CSF(L1)[133/132] domain was shown to be more potent than the coaddition of the IL-3R agonist and G-CSF in stimulating the production of CFU-GM colonies in a human bone marrow-derived CD34+ colony-forming unit assay. Several MPOs also had decreased proinflammatory activity in a leukotriene C4 release assay using N-formyl-Met-Leu-Phe-primed human monocytes. It was found that circular permutation of the G-CSF domain can alter the ratio of G-CSFR:IL-3R agonist activities, demonstrating that it is a useful tool in engineering chimeric protein...
The main problems to be studied in the field of phage morphogenesis are size and shape determination in head and tail, DNA condensation, and various regulative mechanisms to ensure the proper sequential order of morphogenesis steps. Concerning DNA condensation, evidence is accumulating in various systems of complex bacteriophages that DNA is packaged into a preformed precursor particle which sometimes has a smaller size and often a shape different from that of the mature phage head (1-10). The DNA substrate, on the other hand, is often of more than unit size length. Thus, immediately related to the packaging question is the one of size determination of the head and length determination of the DNA. How are pieces of unit length with (as in A) or without (as in T4) unique ends cut from concatemeric DNA? Is the head size determined by the amount of DNA packaged? What is the relationship between cutting and packaging? Finally, the packaging of the DNA into the phage head has to be explained as a reversible process: condensation during morphogenesis, decondensation during injection into the bacterial host. The protein components, however, have to assemble in a sequential, irreversible manner, each step depending upon the completion of the previous one until a stable capsid is formed to protect its DNA.In the case of morphogenesis of the head of bacteriophage h the special relationship between the formation of a normal-sized head and DNA maturation requires that the following observations be explained. Neither in vivo (1 I , 12) nor in vitro (1 3) does a substantial amount of phage DNA mature from the concatemeric precursor DNA without simultaneous production of normal-sized phage heads. Conversely, in the absence of precursor DNA, be it as a consequence of overall inhibition of DNA synthesis (14) or as a consequence of a genetic defect that blocks the production of concatemeric DNA from 302
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