Alanine-scanning mutagenesis of human interleukin-11: identification of regions important for biological activity.
We have identified functionally important regions of human interleukin-11 (hIL-11) by means of alanine-scanning mutagenesis. A total of 61 mutated forms of hIL-11 were produced in E. coli as thioredoxin fusion proteins and tested in a murine T10 plasmacytoma proliferation assay. Mutations made at several positions proximal to the hIL-11 C-terminus caused substantial reduction in biological activity. In addition a number of other mutations in this region affected either protein folding or stability. Both effects displayed a characteristic periodicity with respect to the primary sequence which suggested that residues close to the C-terminus of hIL-11 adopt a helical conformation. Mutations made proximal to the N-terminus of hIL-11 also exhibited reduced bioactivity, although no effects on protein folding or stability were observed. The N-terminal mutations with reduced activity also mapped with a periodicity suggestive of a helical conformation. We previously have proposed a four-helix bundle topology for the hIL-11 structure based on physical studies, selective chemical modifications, positions of intron/exon boundaries, limit proteolysis experiments and site-directed mutagenesis. The alanine-scanning mutagenesis data we report here provide additional support for this model.
Czupryn
M
M
Genetics Institute Inc., Cambridge, Massachusetts 02140, USA.
Bennett
F
F
Dube
J
J
Grant
K
K
Scoble
H
H
Sookdeo
H
H
McCoy
J M
JM
D000409:Alanine; D000595:Amino Acid Sequence; D001681:Biological Assay; D006801:Humans; D017370:Interleukin-11 / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship; D013879:Thioredoxins / Q000737:chemistry
D017370:Interleukin-11; D010455:Peptides; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D000409:Alanine