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...

Mice lacking expression of the fragile X mental retardation 1 (Fmr1) gene have deficits in types of learning that are dependent on the hippocampus. Here, we report that long-term potentiation (LTP) elicited by threshold levels of theta burst afferent stimulation (TBS) is severely impaired in hippocampal field CA1 of young adult Fmr1 knock-out mice. The deficit was not associated with changes in postsynaptic responses to TBS, NMDA receptor activation, or levels of punctate glutamic acid decarboxylase-65/67 immunoreactivity. TBS-induced actin polymerization within dendritic spines was also normal. The LTP impairment was evident within 5 min of induction and, thus, may not be secondary to defects in activity-initiated protein synthesis. Protein levels for both brain-derived neurotrophic factor (BDNF), a neurotrophin that activates pathways involved in spine cytoskeletal reorganization, and its TrkB receptor were comparable between genotypes. BDNF infusion had no effect on baseline transmission or on postsynaptic responses to theta burst stimulation, but nonetheless fully restored LTP in slices from fragile X mice. These results indicate that the fragile X mutation produces a highly selective impairment to LTP, possibly at a step downstream of actin filament assembly, and suggest a means for overcoming this deficit. The possibility of a pharmacological therapy based on these results is discussed.

Cognitive problems occur in asymptomatic gene carriers of Huntington's disease (HD), and mouse models of the disease exhibit impaired learning and substantial deficits in the cytoskeletal changes that stabilize long-term potentiation (LTP). The latter effects may be related to the decreased production of brainderived neurotrophic factor (BDNF) associated with the HD mutation. This study asked whether up-regulating endogenous BDNF levels with an ampakine, a positive modulator of AMPA-type glutamate receptors, rescues plasticity and reduces learning problems in HD (CAG140) mice. Twice-daily injections of a short half-life ampakine normalized BDNF levels, activity-driven actin polymerization in dendritic spines, and LTP stabilization in 8-week-old mutants. Comparable results were obtained in 16-week-old HD mice with more severe LTP deficits. Ampakine treatments had no measurable effect on the decreased locomotor activity observed in the mutants but offset their impairments in long-term memory. Given that ampakines are well tolerated in clinical trials and were effective in this study after brief exposures, these results suggest a novel strategy for chronic treatment of the cognitive difficulties that occur in the early stages of HD.actin polymerization ͉ CAG140 ͉ long-term potentiation ͉ theta burst stimulation ͉ unsupervised learning H untington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a mutation involving the trinucleotide CAG in the huntingtin gene (1, 2). Although severe motor impairments characterize the disease, cognitive and memory deficits are also present and often appear in advance of other symptoms (3-6). Impaired learning that occurs before or concurrent with motor dysfunction or neuron loss has been described for HD mouse models (7-9) as have deficits in hippocampal long-term potentiation (LTP) (10-13), a form of synaptic plasticity widely regarded as a neurobiological substrate for memory. Understanding why LTP is severely impaired in HD mice could explain the cognitive dysfunction seen in patients with the disease.Our recent investigations into HD-associated plasticity deficits established that actin polymerization in dendritic spines, which normally stabilizes LTP (14, 15), is defective in HD knockin mice and most likely explains the rapid decay of potentiation (11). Pertinent to this finding, both HD mice and patients have reduced forebrain levels of brain-derived neurotrophic factor (BDNF) and its TrkB receptor (16,17). BDNF is a releasable neurotrophin that promotes activity-driven actin polymerization in dendritic spines (15, 18) and potently facilitates LTP induction by theta burst stimulation (TBS; refs. 19 and 20). Thus, an HD-related failure in BDNF signaling could remove an essential element of the system that modifies the spine cytoskeleton, thereby disrupting the stable synaptic changes needed to encode memory. Accordingly, applying low concentrations of BDNF fully restored TBS-induced actin polymerization and LTP in hippocampal slices prepared from HD ...

Background: Therapeutic engineering of insulin analogs is ordinarily limited by a trade-off between pharmacokinetics and stability. Results: Substitution of Tyr B26 in a rapid-acting insulin analog by 3-iodo-Tyr B26 enhances its biophysical and pharmaceutical properties. Conclusion: An unnatural amino acid substitution circumvents insulin pharmacokinetic/stability trade-off. Significance: Nonstandard mutagenesis can optimize the molecular properties of therapeutic proteins.

Structure-based protein design has enabled the engineering of insulin analogs with improved pharmacokinetic and pharmacodynamic properties. Exploiting classical structures of zinc insulin hexamers, the first insulin analog products focused on destabilization of subunit interfaces to obtain rapid-acting (prandial) formulations. Complementary efforts sought to stabilize the insulin hexamer or promote higher-order self-assembly within the subcutaneous depot toward the goal of enhanced basal glycemic control with reduced risk of hypoglycemia. Current products either operate through isoelectric precipitation (insulin glargine, the active component of Lantus®; Sanofi-Aventis) or employ an albumin-binding acyl tether (insulin detemir, the active component of Levemir®; Novo-Nordisk). In the past year second-generation basal insulin analogs have entered clinical trials in an effort to obtain ideal flat 24-hour pharmacodynamic profiles. The strategies employ non-standard protein modifications. One candidate (insulin degludec; Novo-Nordisk a/s) undergoes extensive subcutaneous supramolecular assembly coupled to a large-scale allosteric reorganization of the insulin hexamer (the TR transition). Another candidate (LY2605541; Eli Lilly and Co.) utilizes coupling to polyethylene glycol to delay absorption and clearance. On the other end of the spectrum, advances in delivery technologies (such as microneedles and micropatches) and excipients (such as the citrate/zinc-ion chelator combination employed by Biodel, Inc.) suggest strategies to accelerate PK/PD toward ultra-rapid-acting insulin formulations. Next-generation insulin analogs may also address the feasibility of hepatoselective signaling. Although not in clinical trials, early-stage technologies provide a long-range vision of “smart insulins” and glucose-responsive polymers for regulated hormone release.