Abstract
Despite the recent first structural insight into the insulin-insulin receptor complex, the role of the C terminus of the B-chain of insulin in this assembly remains unresolved. Previous studies have suggested that this part of insulin must rearrange to reveal amino acids crucial for interaction with the receptor. The role of the invariant Phe(B24), one of the key residues of the hormone, in this process remains unclear. For example, the B24 site functionally tolerates substitutions to D-amino acids but not to L-amino acids. Here, we prepared and characterized a series of B24-modified insulin analogues, also determining the structures of [D-HisB24]-insulin and [HisB24]-insulin. The inactive [HisB24]-insulin molecule is remarkably rigid due to a tight accommodation of the L-His side chain in the B24 binding pocket that results in the stronger tethering of B25-B28 residues to the protein core. In contrast, the highly active [D-HisB24]-insulin is more flexible, and the reverse chirality of the B24C(α) atom swayed the D-His(B24) side chain into the solvent. Furthermore, the pocket vacated by Phe(B24) is filled by Phe(B25), which mimics the Phe(B24) side and main chains. The B25→B24 downshift results in a subsequent downshift of Tyr(B26) into the B25 site and the departure of B26-B30 residues away from the insulin core. Our data indicate the importance of the aromatic L-amino acid at the B24 site and the structural invariance/integrity of this position for an effective binding of insulin to its receptor. Moreover, they also suggest limited, B25-B30 only, unfolding of the C terminus of the B-chain upon insulin activation.
Highlights
The structure of the C-terminal B21-B30 chain of insulin bound to the insulin receptor remains undetermined
Our recent studies on highly active (ϳ200 – 450%) insulin analogues led to the identification of their structural and functional signatures, which suggest that less dramatic rearrangements of the B25-B30 residues may be required for an “active” form of the hormone [19]
The only exception was [ProB24]-insulin, which was tested for binding to the insulin receptor in membranes in IM-9 lymphocytes according to the procedure that we described recently in Morcavallo et al [36]
Summary
The structure of the C-terminal B21-B30 chain of insulin bound to the insulin receptor remains undetermined. Our recent studies on highly active (ϳ200 – 450%) insulin analogues led to the identification of their structural and functional signatures, which suggest that less dramatic rearrangements of the B25-B30 residues may be required for an “active” (i.e. as in IR complex) form of the hormone [19]. The small size of this hormone, the proximity of its key side chains, and the compactness of the long range (allosteric) intramolecular interactions combined with the complex behavior of this protein (challenging bio-synthesis, processing, storage, activation of the monomer, “induced fit” etc.), gives each of its amino acids a multifaceted and convoluted function. The possible cooperative character of B24 and B26 sites in insulin-IR interactions and their impact on insulin affinity was investigated in three double (D-HisB24, GluB26) insulin mutants
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