Circular Permutation of Granulocyte Colony-Stimulating Factor

Abstract

The sequence of granulocyte colony-stimulating factor (G-CSF) has been circularly permuted by introducing new chain termini into interhelical loops and by constraining the N- and C-terminal helices, either by direct linkage of the termini (L0) or by substitution of the amino-terminal 10-residue segment with a seven-residue linker composed of glycines and serines (L1). All the circularly permuted G-CSFs (cpG-CSFs) were able to fold into biologically active structures that could recognize the G-CSF receptor. CD and NMR spectroscopy demonstrated that all of the cpG-CSFs adopted a fold similar to that of the native molecule, except for one [cpG-CSF(L1)[142/141]] which has the new termini at the end of loop 34 with the shorter L1 linker. All of the cpG-CSFs underwent cooperative unfolding by urea, and a systematically lower free energy change (DeltaGurea) was observed for molecules with the shorter L1 linker than for those molecules in which the original termini were directly linked (the L0 linker). The thermodynamic stability of the cpG-CSFs toward urea was found to correlate with their relative ability to stimulate proliferation of G-CSF responsive cells. Taken together, these results indicate that the G-CSF sequence is robust in its ability to undergo linear rearrangement and adopt a biologically active conformation. The choice of linker, with its effect on stability, seems to be important for realizing the full biological activity of the three-dimensional structure. The breakpoint and linker together are the ultimate determinants of the structural and biological profiles of these circularly permuted cytokines. In the following paper [McWherter, C. A., et al. (1999) Biochemistry 38, 4564-4571], McWherter and co-workers have used circularly permuted G-CSF sequences to engineer chimeric dual IL-3 and G-CSF receptor agonists in which the relative spatial orientation of the receptor agonist domains is varied. Interpreting the differences in activity for the chimeric molecules in terms of the connectivity between domains depends critically on the results reported here for the isolated cpG-CSF domains.

Testing the role of chain connectivity on the stability and structure of dihydrofolate reductase fromE. coli: Fragment complementation and circular permutation reveal stable, alternatively folded forms

“…For example, the structures of noncovalent complexes formed between pairs of fragments from barnase (Kippen and Fersht 1995), tuna cytochrome c (Yokota et al 1998), and thioredoxin (Chaffotte et al 1997) resemble their corresponding full‐length forms and retain a significant fraction of the biological activity of the full‐length protein. Similarly, circular permutants, proteins in which an internal peptide bond is broken and a peptide linker connects the natural N‐ and C‐termini, tend to retain their structure (Viguera et al 1996; Ay et al 1998), activity (Ritco‐Vonsovici et al 1995; Ay et al 1998; Feng et al 1999), oligomeric state (Vignais et al 1995; Wieligmann et al 1998), stability (Zhang et al 1993; Vignais et al 1995), and in some cases even their kinetic and equilibrium (Otzen and Fersht 1998) folding properties. Therefore, the folding reactions for a number of proteins are sufficiently robust that variations in connectivity do not alter the outcomes.…”

The enhanced in vitro hematopoietic activity of leridistim, a chimeric dual G-CSF and IL-3 receptor agonist

“…Responses to leridistim in these assays were greater than those achieved by concurrent administration of the individual agonists. 32,39 The pharmacologic rationale for generating a chimeric molecule with synergistic action was based on the observation that combining an 'early-acting' growth factor such as IL-3 with a lineage-specific growth factor such as G-CSF led to proliferative and differentiative effects. The increased potency of leridistim above the combination of equimolar concentrations of an IL-3 receptor agonist and rhG-CSF raises interesting questions relating to the mechanism of enhancement of the chimeric molecule.…”

“…32,39 IL-3R-dependent proliferative activity was measured using TF-1 cells while AML-193.1.3 cells were utilized to measure dual IL-3R and G-CSFR proliferative activity, as previously described. 27 …”

Non-core Region Modulates Interleukin-11 Signaling Activity

“…Interestingly, cytokine GSGF (whose three-dimensional structure was used to build the hIL-11 model depicted in Fig. 1) also tolerates large rearrangements of its primary sequence while keeping a biologically active conformation (42). The high propensity (Ͼ50%) of hIL-11 muteins exhibiting superagonist behavior suggests that biological evolution has not optimized this interleukin to achieve maximum cell stimulatory strength.…”