A potent and highly specific FN3 monobody inhibitor of the Abl SH2 domain.
Interactions between Src homology 2 (SH2) domains and phosphotyrosine sites regulate tyrosine kinase signaling networks. Selective perturbation of these interactions is challenging due to the high homology among the 120 human SH2 domains. Using an improved phage-display selection system, we generated a small antibody mimic (or 'monobody'), termed HA4, that bound to the Abelson (Abl) kinase SH2 domain with low nanomolar affinity. SH2 protein microarray analysis and MS of intracellular HA4 interactors showed HA4's specificity, and a crystal structure revealed how this specificity is achieved. HA4 disrupted intramolecular interactions of Abl involving the SH2 domain and potently activated the kinase in vitro. Within cells, HA4 inhibited processive phosphorylation activity of Abl and also inhibited STAT5 activation. This work provides a design guideline for highly specific and potent inhibitors of a protein interaction domain and shows their utility in mechanistic and cellular investigations.
Wojcik
John
J
Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA.
Hantschel
Oliver
O
Grebien
Florian
F
Kaupe
Ines
I
Bennett
Keiryn L
KL
Barkinge
John
J
Jones
Richard B
RB
Koide
Akiko
A
Superti-Furga
Giulio
G
Koide
Shohei
S
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016374:Oncogene Proteins v-abl / Q000737:chemistry / Q000276:immunology*; D010766:Phosphorylation; D017386:Sequence Homology, Amino Acid; D018909:src Homology Domains
D000911:Antibodies, Monoclonal; D016374:Oncogene Proteins v-abl