Structural Integrity of the B24 Site in Human Insulin Is Important for Hormone Functionality

Žáková, Lenka; Kletvíková, Emília; Veverka, Václav; Lepšı́k, Martin; Watson, Christopher J.; Turkenburg, J.P.; Jiráček, Jiřı́; Brzozowski, A.M.

doi:10.1074/jbc.m112.448050

Cited by 40 publications

(59 citation statements)

References 62 publications

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“…Class II contained Ile and Leu. Class III contained Val as well as the aromatic residues Tyr and Trp; the low affinity of [Tyr B24 ,Orn B29 ]insulin is in accordance with prior studies (11,12). Class IV (very low affinity) contained His (in accordance with Ref.…”

Section: Resultssupporting

confidence: 70%

“…Structure-Activity Relationships-The long standing view that an aromatic side chain is required at B24 (10), recently reinforced by the studies of Brzozowski and co-workers (12), is broadly consistent with possible weakly polar interactions at the receptor 11 Initial rates of fall of the blood glucose concentration were not statistically significant (Fig. 9C).…”

Section: Discussionsupporting

confidence: 50%

“…Such small differences are consistent with natural variation among mammalian insulins (species variants (52)). Aside from the special case of Gly B24 (which may reflect a shift in binding mode (6,12,53)), 8 these findings suggest that the hormone-receptor interface selects for nonpolar side chains at B24 of suitable shape and size, irrespective of aromaticity. Avoidance of packing defects within the B24-related pocket presumably underlies the affinity order Phe, Met Ͼ Ile, Leu Ͼ Val Ͼ Ala.…”

Section: Resultsmentioning

confidence: 97%

“…2D; for clarity B16 is omitted). Although past studies of selected insulin analogs suggested that aromaticity is required for such anchoring (12), the structure of the B24-related binding pocket in the IR would seem compatible with aliphatic side chains of appropriate size and shape. To resolve this apparent discrepancy, we undertook systematic mutagenesis of the B24 position in human insulin.…”

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confidence: 99%

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Aromatic Anchor at an Invariant Hormone-Receptor Interface

Pandyarajan¹,

Smith

Phillips³

et al. 2014

Journal of Biological Chemistry

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Background: Invariant insulin residue Phe B24 (a site of diabetes-associated mutation) contacts the insulin receptor. Results: Hormonal function requires hydrophobicity rather than aromaticity at this site. Conclusion: The B24 side chain provides a nonpolar anchor at the receptor interface. Significance: Nonstandard aliphatic modification of residue B24 may enhance therapeutic properties of insulin analogs.

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Section: Resultssupporting

confidence: 70%

Section: Discussionsupporting

confidence: 50%

Section: Resultsmentioning

confidence: 97%

mentioning

confidence: 99%

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Aromatic Anchor at an Invariant Hormone-Receptor Interface

Pandyarajan¹,

Smith

Phillips³

et al. 2014

Journal of Biological Chemistry

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“…Its high activity (like that of related analogs) (4, 5, 31, 42) poses a seeming paradox: how can the hormone-receptor interface tolerate the absence of an invariant side chain-especially an aromatic ring anchored within a canonical binding pocket? This question is made more pointed by the enhanced receptor-binding affinity conferred by D-Ala B24 and other D substitutions at B24 (6,42,43). On the one hand, destabilization of the B20-B30 segment (Gly B24 ) or its predetachment (D-Ala B24 ) might mitigate the cost of induced fit and so enhance binding affinity.…”

Section: Discussionmentioning

confidence: 99%

Protective hinge in insulin opens to enable its receptor engagement

Menting

Yang

Chan

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

147

257

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Insulin provides a classical model of a globular protein, yet how the hormone changes conformation to engage its receptor has long been enigmatic. Interest has focused on the C-terminal Bchain segment, critical for protective self-assembly in β cells and receptor binding at target tissues. Insight may be obtained from truncated "microreceptors" that reconstitute the primary hormone-binding site (α-subunit domains L1 and αCT). We demonstrate that, on microreceptor binding, this segment undergoes concerted hinge-like rotation at its B20-B23 β-turn, coupling reorientation of Phe B24 to a 60°rotation of the B25-B28 β-strand away from the hormone core to lie antiparallel to the receptor's L1-β 2 sheet. Opening of this hinge enables conserved nonpolar side chains (Ile A2 , Val A3 , Val B12 , Phe B24 , and Phe B25 ) to engage the receptor. Restraining the hinge by nonstandard mutagenesis preserves native folding but blocks receptor binding, whereas its engineered opening maintains activity at the price of protein instability and nonnative aggregation. Our findings rationalize properties of clinical mutations in the insulin family and provide a previously unidentified foundation for designing therapeutic analogs. We envisage that a switch between free and receptorbound conformations of insulin evolved as a solution to conflicting structural determinants of biosynthesis and function.diabetes mellitus | signal transduction | receptor tyrosine kinase | metabolism | protein structure H ow insulin engages the insulin receptor has inspired speculation ever since the structure of the free hormone was determined by Hodgkin and colleagues in 1969 (1, 2). Over the ensuing decades, anomalies encountered in studies of analogs have suggested that the hormone undergoes a conformational change on receptor binding: in particular, that the C-terminal β-strand of the B chain (residues B24-B30) releases from the helical core to expose otherwise-buried nonpolar surfaces (the detachment model) (3-6). Interest in the B-chain β-strand was further motivated by the discovery of clinical mutations within it associated with diabetes mellitus (DM) (7). Analysis of residuespecific photo-cross-linking provided evidence that both the detached strand and underlying nonpolar surfaces engage the receptor (8).The relevant structural biology is as follows. The insulin receptor is a disulfide-linked (αβ) 2 receptor tyrosine kinase (Fig. 1A), the extracellular α-subunits together binding a single insulin molecule with high affinity (9). Involvement of the two α-subunits is asymmetric: the primary insulin-binding site (site 1*) comprises the central β-sheet (L1-β 2 ) of the first leucine-rich repeat domain (L1) of one α-subunit and the partially helical Cterminal segment (αCT) of the other α-subunit (Fig. 1A) (10). Such binding initiates conformational changes leading to transphosphorylation of the β-subunits' intracellular tyrosine kinase (TK) domains. Structures of wild-type (WT) insulin (or analogs) bound to extracellular receptor fragments were recently...

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Insulin/receptor binding: The last piece of the puzzle?

Meyts

2015

BioEssays

110

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Progress in solving the structure of insulin bound to its receptor has been slow and stepwise, but a milestone has now been reached with a refined structure of a complex of insulin with a "microreceptor" that contains the primary binding site. The insulin receptor is a dimeric allosteric enzyme that belongs to the family of receptor tyrosine kinases. The insulin binding process is complex and exhibits negative cooperativity. Biochemical evidence suggested that insulin, through two distinct binding sites, crosslinks two receptor sites located on each α subunit. The structure of the unliganded receptor ectodomain showed a symmetrical folded-over conformation with an antiparallel disposition. Further work resolved the detailed structure of receptor site 1, both without and with insulin. Recently, a missing piece in the puzzle was added: the C-terminal portion of insulin's B-chain known to be critical for binding and negative cooperativity. Here I discuss these findings and their implications.

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Structural Integrity of the B24 Site in Human Insulin Is Important for Hormone Functionality

Cited by 40 publications

References 62 publications

Aromatic Anchor at an Invariant Hormone-Receptor Interface

Aromatic Anchor at an Invariant Hormone-Receptor Interface

Protective hinge in insulin opens to enable its receptor engagement

Insulin/receptor binding: The last piece of the puzzle?

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