The A-chain of insulin contacts the insert domain of the insulin receptor. Photo-cross-linking and mutagenesis of a diabetes-related crevice.
The contribution of the insulin A-chain to receptor binding is investigated by photo-cross-linking and nonstandard mutagenesis. Studies focus on the role of Val(A3), which projects within a crevice between the A- and B-chains. Engineered receptor alpha-subunits containing specific protease sites ("midi-receptors") are employed to map the site of photo-cross-linking by an analog containing a photoactivable A3 side chain (para-azido-Phe (Pap)). The probe cross-links to a C-terminal peptide (residues 703-719 of the receptor A isoform, KTFEDYLHNVVFVPRPS) containing side chains critical for hormone binding (underlined); the corresponding segment of the holoreceptor was shown previously to cross-link to a Pap(B25)-insulin analog. Because Pap is larger than Val and so may protrude beyond the A3-associated crevice, we investigated analogs containing A3 substitutions comparable in size to Val as follows: Thr, allo-Thr, and alpha-aminobutyric acid (Aba). Substitutions were introduced within an engineered monomer. Whereas previous studies of smaller substitutions (Gly(A3) and Ser(A3)) encountered nonlocal conformational perturbations, NMR structures of the present analogs are similar to wild-type insulin; the variant side chains are accommodated within a native-like crevice with minimal distortion. Receptor binding activities of Aba(A3) and allo-Thr(A3) analogs are reduced at least 10-fold; the activity of Thr(A3)-DKP-insulin is reduced 5-fold. The hormone-receptor interface is presumably destabilized either by a packing defect (Aba(A3)) or by altered polarity (allo-Thr(A3) and Thr(A3)). Our results provide evidence that Val(A3), a site of mutation causing diabetes mellitus, contacts the insert domain-derived tail of the alpha-subunit in a hormone-receptor complex.
Huang
Kun
K
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Chan
Shu Jin
SJ
Hua
Qing-xin
QX
Chu
Ying-Chi
YC
Wang
Run-ying
RY
Klaproth
Birgit
B
Jia
Wenhua
W
Whittaker
Jonathan
J
De Meyts
Pierre
P
Nakagawa
Satoe H
SH
Steiner
Donald F
DF
Katsoyannis
Panayotis G
PG
Weiss
Michael A
MA
D000613:Aminobutyrates / Q000737:chemistry; D000818:Animals; D003432:Cross-Linking Reagents / Q000737:chemistry*; D003920:Diabetes Mellitus / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000737:chemistry*; D008027:Light; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D016296:Mutagenesis; D009154:Mutation; D010777:Photochemistry / Q000379:methods; D011487:Protein Conformation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011972:Receptor, Insulin / Q000737:chemistry*; D013552:Swine; D014633:Valine / Q000737:chemistry
D000613:Aminobutyrates; D003432:Cross-Linking Reagents; D007328:Insulin; D020033:Protein Isoforms; C012223:alpha-aminobutyric acid; D011972:Receptor, Insulin; D014633:Valine