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. Study holds ProTherm entries: 16284, 16285, 16286, 16287, 16288, 16289, 16290, 16291, 16292, 16293 Extra Details: The N- and C-terminal of the original protein were connected with linker and in stead, new N- and C-terminal were created by breaking the peptide bond at the indicated position. G-CSF(S17) The unpaired Cys-17 in the wild-type G-CSF was replaced with Ser. circular permutation, colony-stimulating factor, chimeric molecules

Submission Details

ID: RbbwYxBF4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:47 p.m.

Version: 1

Publication Details
Feng Y;Minnerly JC;Zurfluh LL;Joy WD;Hood WF;Abegg AL;Grabbe ES;Shieh JJ;Thurman TL;McKearn JP;McWherter CA,Biochemistry (1999) Circular permutation of granulocyte colony-stimulating factor. PMID:10194377
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1GNC 1994-07-31 STRUCTURE AND DYNAMICS OF THE HUMAN GRANULOCYTE COLONY-STIMULATING FACTOR DETERMINED BY NMR SPECTROSCOPY. LOOP MOBILITY IN A FOUR-HELIX-BUNDLE PROTEIN
5GW9 2017-09-13 1.65 Crystal structure of C163, a backbone circularized G-CSF
5ZO6 2019-04-17 1.7 Crystal structure of C166, a backbone circularized G-CSF
6BFS 2018-09-12 2.0 The mechanism of GM-CSF inhibition by human GM-CSF auto-antibodies
5D70 2015-10-14 2.06 Crystal structure of MOR03929, a neutralizing anti-human GM-CSF antibody Fab fragment in complex with human GM-CSF
1RHG 1994-01-31 2.2 THE STRUCTURE OF GRANULOCYTE-COLONY-STIMULATING FACTOR AND ITS RELATIONSHIP TO THOSE OF OTHER GROWTH FACTORS
5D71 2015-10-14 2.25 Crystal structure of MOR04302, a neutralizing anti-human GM-CSF antibody Fab fragment in complex with human GM-CSF
2GMF 1996-11-08 2.4 HUMAN GRANULOCYTE MACROPHAGE COLONY STIMULATING FACTOR
5D72 2015-10-14 2.6 Crystal structure of MOR04252, a neutralizing anti-human GM-CSF antibody Fab fragment in complex with human GM-CSF
6BFQ 2018-09-12 2.6 The mechanism of GM-CSF inhibition by human GM-CSF auto-antibodies
4RS1 2015-11-11 2.68 Crystal structure of receptor-cytokine complex
1CSG 1993-10-31 2.7 Three-dimensional structure of recombinant human granulocyte-macrophage colony-stimulating factor
2D9Q 2006-02-07 2.8 Crystal Structure of the Human GCSF-Receptor Signaling Complex
1CD9 2000-03-08 2.8 2:2 COMPLEX OF G-CSF WITH ITS RECEPTOR
5C7X 2015-10-14 2.95 Crystal structure of MOR04357, a neutralizing anti-human GM-CSF antibody Fab fragment in complex with human GM-CSF
4NKQ 2015-09-23 3.3 Structure of a Cytokine Receptor Complex
1PGR 2000-03-08 3.5 2:2 COMPLEX OF G-CSF WITH ITS RECEPTOR

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
98.3 Granulocyte colony-stimulating factor P09919 CSF3_HUMAN
94.4 Granulocyte-macrophage colony-stimulating factor Q0MUT8 CSF2_CHLAE
100.0 Granulocyte-macrophage colony-stimulating factor P04141 CSF2_HUMAN