Structural Lessons From the Mutant Proinsulin Syndrome.

Insight into folding mechanisms of proinsulin has been provided by analysis of dominant diabetes-associated mutations in the human insulin gene (INS). Such mutations cause pancreatic ß-cell dysfunction due to toxic misfolding of a mutant proinsulin and impairment in trans of wild-type insulin secretion. Anticipated by the "Akita" mouse (a classical model of monogenic diabetes mellitus; DM), this syndrome illustrates the paradigm endoreticulum (ER) stress leading to intracellular proteotoxicity. Diverse clinical mutations directly or indirectly perturb native disulfide pairing leading to protein misfolding and aberrant aggregation. Although most introduce or remove a cysteine (Cys; leading in either case to an unpaired thiol group), non-Cys-related mutations identify key determinants of folding efficiency. Studies of such mutations suggest that the hormone's evolution has been constrained not only by structure-function relationships, but also by the susceptibility of its single-chain precursor to impaired foldability. An intriguing hypothesis posits that INS overexpression in response to peripheral insulin resistance likewise leads to chronic ER stress and ß-cell dysfunction in the natural history of non-syndromic Type 2 DM. Cryptic contributions of conserved residues to folding efficiency, as uncovered by rare genetic variants, define molecular links between biophysical principles and the emerging paradigm of Darwinian medicine: Biosynthesis of proinsulin at the edge of non-foldability provides a key determinant of "diabesity" as a pandemic disease of civilization.

Structural principles of insulin formulation and analog design: A century of innovation.

BACKGROUND: The discovery of insulin in 1921 and its near-immediate clinical use initiated a century of innovation. Advances extended across a broad front, from the stabilization of animal insulin formulations to the frontiers of synthetic peptide chemistry, and in turn, from the advent of recombinant DNA manufacturing to structure-based protein analog design. In each case, a creative interplay was observed between pharmaceutical applications and then-emerging principles of protein science; indeed, translational objectives contributed to a growing molecular understanding of protein structure, aggregation and misfolding. SCOPE OF REVIEW: Pioneering crystallographic analyses-beginning with Hodgkin's solving of the 2-Zn insulin hexamer-elucidated general features of protein self-assembly, including zinc coordination and the allosteric transmission of conformational change. Crystallization of insulin was exploited both as a step in manufacturing and as a means of obtaining protracted action. Forty years ago, the confluence of recombinant human insulin with techniques for site-directed mutagenesis initiated the present era of insulin analogs. Variant or modified insulins were developed that exhibit improved prandial or basal pharmacokinetic (PK) properties. Encouraged by clinical trials demonstrating the long-term importance of glycemic control, regimens based on such analogs sought to resemble daily patterns of endogenous ß-cell secretion more closely, ideally with reduced risk of hypoglycemia. MAJOR CONCLUSIONS: Next-generation insulin analog design seeks to explore new frontiers, including glucose-responsive insulins, organ-selective analogs and biased agonists tailored to address yet-unmet clinical needs. In the coming decade, we envision ever more powerful scientific synergies at the interface of structural biology, molecular physiology and therapeutics.

Insertion of a synthetic switch into insulin provides metabolite-dependent regulation of hormone-receptor activation.

Insulin-signaling requires conformational change: whereas the free hormone and its receptor each adopt autoinhibited conformations, their binding leads to structural reorganization. To test the functional coupling between insulin's "hinge opening" and receptor activation, we inserted an artificial ligand-dependent switch into the insulin molecule. Ligand-binding disrupts an internal tether designed to stabilize the hormone's native closed and inactive conformation, thereby enabling productive receptor engagement. This scheme exploited a diol sensor (meta-fluoro-phenylboronic acid at GlyA1) and internal diol (3,4-dihydroxybenzoate at LysB28). The sensor recognizes monosaccharides (fructose > glucose). Studies of insulin-signaling in human hepatoma-derived cells (HepG2) demonstrated fructose-dependent receptor autophosphorylation leading to appropriate downstream signaling events, including a specific kinase cascade and metabolic gene regulation (gluconeogenesis and lipogenesis). Addition of glucose (an isomeric ligand with negligible sensor affinity) did not activate the hormone. Similarly, metabolite-regulated signaling was not observed in control studies of 1) an unmodified insulin analog or 2) an analog containing a diol sensor without internal tethering. Although secondary structure (as probed by circular dichroism) was unaffected by ligand-binding, heteronuclear NMR studies revealed subtle local and nonlocal monosaccharide-dependent changes in structure. Insertion of a synthetic switch into insulin has thus demonstrated coupling between hinge-opening and allosteric holoreceptor signaling. In addition to this foundational finding, our results provide proof of principle for design of a mechanism-based metabolite-responsive insulin. In particular, replacement of the present fructose sensor by an analogous glucose sensor may enable translational development of a "smart" insulin analog to mitigate hypoglycemic risk in diabetes therapy.

Diabetes mellitus due to toxic misfolding of proinsulin variants.

BACKGROUND: Dominant mutations in the human insulin gene (INS) lead to pancreatic ß-cell dysfunction and diabetes mellitus (DM) due to toxic misfolding of a mutant proinsulin. Analogous to a classical mouse model of monogenic DM ("Akita"), this syndrome highlights the susceptibility of ß-cells to endoreticulum (ER) stress due to protein misfolding and aberrant aggregation. SCOPE OF REVIEW: Diverse clinical mutations directly or indirectly perturb native disulfide pairing. Whereas most introduce or remove a cysteine (Cys; leading in either case to an unpaired thiol group), non-Cys-related mutations identify key determinants of folding efficiency. Studies of such mutations suggest that the hormone's evolution has been constrained not only by structure-function relationships but also by the susceptibility of its single-chain precursor to impaired foldability. An intriguing hypothesis posits that INS overexpression in response to peripheral insulin resistance likewise leads to chronic ER stress and ß-cell dysfunction in the natural history of nonsyndromic Type 2 DM. MAJOR CONCLUSIONS: Cryptic contributions of conserved residues to folding efficiency, as uncovered by rare genetic variants, define molecular links between biophysical principles and the emerging paradigm of Darwinian medicine: Biosynthesis of proinsulin at the edge of nonfoldability provides a key determinant of "diabesity" as a pandemic disease of civilization.

NMR structure and localization of a large fragment of the SARS-CoV fusion protein: Implications in viral cell fusion.

The lethal Coronaviruses (CoVs), Severe Acute Respiratory Syndrome-associated Coronavirus (SARS-CoV) and most recently Middle East Respiratory Syndrome Coronavirus, (MERS-CoV) are serious human health hazard. A successful viral infection requires fusion between virus and host cells carried out by the surface spike glycoprotein or S protein of CoV. Current models propose that the S2 subunit of S protein assembled into a hexameric helical bundle exposing hydrophobic fusogenic peptides or fusion peptides (FPs) for membrane insertion. The N-terminus of S2 subunit of SARS-CoV reported to be active in cell fusion whereby FPs have been identified. Atomic-resolution structure of FPs derived either in model membranes or in membrane mimic environment would glean insights toward viral cell fusion mechanism. Here, we have solved 3D structure, dynamics and micelle localization of a 64-residue long fusion peptide or LFP in DPC detergent micelles by NMR methods. Micelle bound structure of LFP is elucidated by the presence of discretely folded helical and intervening loops. The C-terminus region, residues F42-Y62, displays a long hydrophobic helix, whereas the N-terminus is defined by a short amphipathic helix, residues R4-Q12. The intervening residues of LFP assume stretches of loops and helical turns. The N-terminal helix is sustained by close aromatic and aliphatic sidechain packing interactions at the non-polar face. 15N{1H}NOE studies indicated dynamical motion, at ps-ns timescale, of the helices of LFP in DPC micelles. PRE NMR showed that insertion of several regions of LFP into DPC micelle core. Together, the current study provides insights toward fusion mechanism of SARS-CoV.

Synthesis of a new ß-amino acid with a 3-deoxy-L-ara furnaoside side chain: the influence of the side chain on the conformation of α/ß-peptides.

The important role of side chains in the stabilization of helical folds in peptidic foldamers containing C-linked carbo-ß-amino acids (ß-Caa), an interesting class of ß-amino acids, with carbohydrate side chains has been extensively elaborated. As a pragmatic approach to alleviate the interference of substituents in the side chains on the folding propensities of the peptides, they are often modified or removed. The present study reports the synthesis of a new ß-Caa with a 3-deoxy-L-ara furanoside side chain, [(R)-ß-Caa(da)], from D-glucose, and its use in the synthesis of α/ß-peptides in 1 : 1 alternation with D-Ala. The synthesis of peptides using (R)-ß-Caa(da), was facile unlike those from (R)-ß-Caa(a) having the L-ara furanoside side chain. The detailed NMR, molecular dynamics (MD) and CD studies on the new α/ß-peptides showed the presence of robust left-handed 11/9-mixed helices. The study demonstrates that the new (R)-ß-Caa(da), behaves differently compared to the other two related monomers, (R)-ß-Caa(x) with the D-xylo furanoside side chain and (R)-ß-Caa(a).

Synthesis of C-linked carbo-ß2-amino acids and ß2-peptides: design of new motifs for left-handed 12/10- and 10/12-mixed helices.

C-linked carbo-ß(2)-amino acids (ß(2)-Caa), a new class of ß-amino acid with a carbohydrate side chain having d-xylo configuration, were prepared from d-glucose. The main idea behind the design of the new ß-amino acids was to move the steric strain of the bulky carbohydrate side chain from the Cß- to the Cα-carbon atom and to explore its influence on the folding propensities in peptides with alternating (R)- and (S)-ß(2)-Caas. The tetra- and hexapeptides derived were studied employing NMR (in CDCl(3)), CD, and molecular dynamics simulations. The ß(2)-peptides of the present study form left-handed 12/10- and 10/12-mixed helices independent of the order of the alternating chiral amino acids in the sequence and result in a new motif. These results differ from earlier findings on ß(3)-peptides of the same design, containing a carbohydrate side chain with d-xylo configuration, which form exclusively right-handed 12/10-mixed helices. Quantum chemical calculations employing ab initio MO theory suggest the side chain chirality as an important factor for the observed definite left- or right-handedness of the helices in the ß(2)- and ß(3)-peptides.

Synthesis and structural studies of homooligomers of geminally disubstituted ß(2,2)-amino acids with carbohydrate side chain.

A new class of geminally disubstituted C-linked carbo-ß(2,2)-amino acids (ß(2,2)-Caa) were prepared from d-glucose. The structures of homooligomeric di-, tetra-, and hexapeptides prepared from (S)-ß(2,2)-Caa were studied with NMR (in CDCl(3)), CD, and Molecular Dynamics calculations. These ß(2,2)-peptides have shown the presence of stable 6-membered (6-mr) NH(i)···CO(i) intra-residue H-bonded (C(6)) strands. It was found that the strand structures realized in these systems were additionally stabilized by the electrostatic interaction arising due to the proximity of amide proton (NH(i)) to the oxygen of the preceding methoxy group (O(Me)(i-1)) at the C3 carbon of the carbohydrate ring. The new ß(2,2)-Caa residues with additional support to H-bonding considerably expand the domain of foldamers.

Theoretical and experimental studies on alpha/epsilon-hybrid peptides: design of a 14/12-helix from peptides with alternating (S)-C-linked carbo-epsilon-amino acid [(S)-epsilon-Caa((x))] and L-ala.

An (S)-C-linked carbo-epsilon-amino acid [(S)-epsilon-Caa((x))] was prepared from the known (S)-delta-Caa. This monomer was utilized together with l-Ala to give novel alpha/epsilon-hybrid peptides in 1:1 alternation. Conformational analysis on penta- and hexapeptides by NMR (in CDCl(3)), CD, and MD studies led to the identification of robust 14/12-mixed helices. This is in agreement with the data from a theoretical conformational analysis on the basis of ab initio MO theory providing a complete overview on all formally possible hydrogen-bonded helix patterns of alpha/epsilon-hybrid peptides with 1:1 backbone alternation. The "new motif" of a mixed 14/12-helix was predicted as most stable in vacuum. Obviously, the formation of ordered secondary structures is also possible in peptide foldamers with amino acid constituents of considerable backbone lengths. Thus, alpha/epsilon-hybrid peptides expand the domain of foldamers and allow the introduction of desired functionalities via the alpha-amino acid constituents.