Chiral mutagenesis of insulin. Foldability and function are inversely regulated by a stereospecific switch in the B chain

How insulin binds to its receptor is unknown despite decades of investigation. Here, we employ chiral mutagenesis-comparison of corresponding d and l amino acid substitutions in the hormone-to define a structural switch between folding-competent and active conformations. Our strategy is motivated by the T --> R transition, an allosteric feature of zinc-hexamer assembly in which an invariant glycine in the B chain changes conformations. In the classical T state, Gly(B8) lies within a beta-turn and exhibits a positive phi angle (like a d amino acid); in the alternative R state, Gly(B8) is part of an alpha-helix and exhibits a negative phi angle (like an l amino acid). Respective B chain libraries containing mixtures of d or l substitutions at B8 exhibit a stereospecific perturbation of insulin chain combination: l amino acids impede native disulfide pairing, whereas diverse d substitutions are well-tolerated. Strikingly, d substitutions at B8 enhance both synthetic yield and thermodynamic stability but markedly impair biological activity. The NMR structure of such an inactive analogue (as an engineered T-like monomer) is essentially identical to that of native insulin. By contrast, l analogues exhibit impaired folding and stability. Although synthetic yields are very low, such analogues can be highly active. Despite the profound differences between the foldabilities of d and l analogues, crystallization trials suggest that on protein assembly substitutions of either class can be accommodated within classical T or R states. Comparison between such diastereomeric analogues thus implies that the T state represents an inactive but folding-competent conformation. We propose that within folding intermediates the sign of the B8 phi angle exerts kinetic control in a rugged landscape to distinguish between trajectories associated with productive disulfide pairing (positive T-like values) or off-pathway events (negative R-like values). We further propose that the crystallographic T -->R transition in part recapitulates how the conformation of an insulin monomer changes on receptor binding. At the very least the ostensibly unrelated processes of disulfide pairing, allosteric assembly, and receptor binding appear to utilize the same residue as a structural switch; an "ambidextrous" glycine unhindered by the chiral restrictions of the Ramachandran plane. We speculate that this switch operates to protect insulin-and the beta-cell-from protein misfolding.     

Role of the phenylalanine B24 side chain in directing insulin interaction with its receptor. Importance of main chain conformation

We have investigated (by use of semisynthetic insulin analogs and isolated canine hepatocytes) the role of invariant residue PheB24 in determining the affinity of insulin-receptor interactions. Our results confirm that replacement of PheB24 by D-Phe is not detrimental to ligand binding to receptor, show that D-Ala is well tolerated at position B24 (whereas Ala is not), and demonstrate that [GlyB24]insulin retains as much as 78% of the receptor binding potency of native insulin. Additional findings show that replacement of PheB24 by D-Pro or by alpha-aminoisobutyric acid results in analogs with severely decreased binding potency, and that the COOH-terminal domain containing residues B26-B30 plays a positive role in determining receptor binding potency in GlyB24-substituted insulin (whereas it plays a negative role in determining the receptor binding potency of its GlyB25-substituted counterpart). We interpret our results as identifying (a) a critical role for the insulin main chain near residue B24 in determining the affinity of receptor for ligand, (b) the importance of main chain flexibility in achieving a high affinity state of receptor-bound hormone, and (c) a potential interaction of the PheB24 side chain with receptor which initiates main chain structural changes in the natural hormone, but which does not itself confer affinity to ligand-receptor interactions.     

The immunoglobulin heavy chain switch: structural features of gamma 1 recombinant switch regions

The immunoglobulin heavy chain isotype switch is mediated by a DNA rearrangement involving specific genomic segments referred to as switch regions. Switch regions are composed of tandemly repeated simple sequences. The role of the tandemly repeated structure of switch regions in the switch recombination process is not understood. We mapped eight recombination sites--six in the gamma 1 and two in the gamma 3 tandem arrays. In addition, we obtained molecular clones representing three of the six gamma 1 rearrangements, and determined the nucleotide sequences of the recombination sites in each. In general, the rearrangements are confined to the tandem repeat units, and are not clustered in a particular portion of either the gamma 3 or gamma 1 switch region. Nucleotide sequence analysis of one of the recombinant clones, gamma M35, reveals evidence for a successive switch event wherein a recombination between S mu and S gamma 3 was followed by recombination 57 bp downstream with S gamma 1. gamma 1 sequence data from the molecular clones we obtained, together with similar data from other investigators regarding the gamma 1, gamma 2b, and gamma 2a switch regions, reveals that recombinations tend to occur at homologous positions of the respective gamma-unit repeats, adjacent to the elements AGCT and GGGG found in each. This finding suggests that the cutting and religation step of the recombination process is mediated by a recombinase common to the four gamma-isotypes.     

Diabetes-associated mutations in insulin: consecutive residues in the B chain contact distinct domains of the insulin receptor

How insulin binds to and activates the insulin receptor has long been the subject of speculation. Of particular interest are invariant phenylalanine residues at consecutive positions in the B chain (residues B24 and B25). Sites of mutation causing diabetes mellitus, these residues occupy opposite structural environments: Phe(B25) projects from the surface of insulin, whereas Phe(B24) packs against the core. Despite these differences, site-specific cross-linking suggests that each contacts the insulin receptor. Photoactivatable derivatives of insulin containing respective p-azidophenylalanine substitutions at positions B24 and B25 were synthesized in an engineered monomer (DKP-insulin). On ultraviolet irradiation each derivative cross-links efficiently to the receptor. Packing of Phe(B24) at the receptor interface (rather than against the core of the hormone) may require a conformational change in the B chain. Sites of cross-linking in the receptor were mapped to domains by Western blot. Remarkably, whereas B25 cross-links to the C-terminal domain of the alpha subunit in accord with previous studies (Kurose, T., et al. (1994) J. Biol. Chem. 269, 29190-29197), the probe at B24 cross-links to its N-terminal domain (the L1 beta-helix). Our results demonstrate that consecutive residues in insulin contact widely separated sequences in the receptor and in turn suggest a revised interpretation of electron-microscopic images of the complex. By tethering the N- and C-terminal domains of the extracellular alpha subunit, insulin is proposed to stabilize an active conformation of the disulfide-linked transmembrane tyrosine kinase.     

An open conformation determined by a structural switch for 2A protease from coxsackievirus A16

Coxsackievirus A16 belongs to the family Picornaviridae, and is a major agent of hand-foot-and-mouth disease that infects mostly children, and to date no vaccines or antiviral therapies are available. 2A protease of enterovirus is a nonstructural protein and possesses both self-cleavage activity and the ability to cleave the eukaryotic translation initiation factor 4G. Here we present the crystal structure of coxsackievirus A16 2A protease, which interestingly forms hexamers in crystal as well as in solution. This structure shows an open conformation, with its active site accessible, ready for substrate binding and cleavage activity. In conjunction with a previously reported “closed” state structure of human rhinovirus 2, we were able to develop a detailed hypothesis for the conformational conversion triggered by two “switcher” residues Glu88 and Tyr89 located within the bll2-cII loop. Substrate recognition assays revealed that amino acid residues P1′, P2 and P4 are essential for substrate specificity, which was verifi ed by our substrate binding model. In addition, we compared the in vitro cleavage effi ciency of 2A proteases from coxsackievirus A16 and enterovirus 71 upon the same substrates by fl uorescence resonance energy transfer (FRET), and observed higher protease activity of enterovirus 71 compared to that of coxsackievirus A16. In conclusion, our study shows an open conformation of coxsackievirus A16 2A protease and the underlying mechanisms for conformational conversion and substrate specifi city. These new insights should facilitate the future rational design of effi cient 2A protease inhibitors.     

Periodic electric field as a biopolymer conformation switch: a possible mechanism

A theoretical model is proposed to describe the influence of a periodic electric field (PEF) upon a biopolymer. The biopolymer is treated as a classical mechanical system consisting of subsystems (molecular groups) which interact with each other through potential forces. The PEF is treated as a periodic driving force applied to a molecular group. The energy dissipation is considered using the model of fluid (viscous) friction. Arguments for the non-linear character of the friction-velocity dependence caused by the non-Newtonian rheology of a viscous medium are formulated.A forced molecular-group motion is investigated for the situation of a small driving-force period, with oscillations overdamped and a driving force consisting of more than one harmonic. As a result, it is established that the motion always gets to a terminal stage when only a small-scale vibration about some point, X ^*, takes place. The terminal motion is preceded by a transient characterized by the presence of a directional velocity component and so by a drift along a potential profile. the drift goes on until a barrier is met which has a sufficiently large steepness (the barrier height is not important). As a result, the point X ^* may happen to be remote from the conformation potential local minimum (conformational state). The physical reasons for the drift are described.If we consider the small-scale vibration about X ^* in the framework of the hierarchy of scales for intramolecular mobility, it can be regarded as an equilibrium mobility, whereas the drift can be regarded as a functionally important motion, and X ^* as a new conformational state which is realizable only in the presence of the PEF. It may be concluded, as the result of a consistent treatment and neglecting the small-scale vibration, that the conformational potential is modified by adding a linear term (in the one-dimensional case). In this connection, the set of conformational states can both deform (deviation of the positions of minima and their relative depth) and rearrange qualitatively (some minima can vanish and/or new minima can appear). In particular, the transition from one conformation to another one may happen due to rearrangement.     

Antagonism of insulin action on muscle by the albumin-bound B chain of insulin

1. The presence of a substance associated with human albumin that exerts anti-insulin activity on the isolated rat diaphragm has been confirmed. This factor has been removed from albumin, thereby providing a source of non-antagonistic carrier protein. 2. Derivatives of the polypeptide B chain of insulin obtained by chemical scission of the interchain disulphide bonds have been separated by conventional techniques. In the presence of non-antagonistic albumin, the reduced and sulpho-B chain preparations inhibited insulin action on muscle. 3. The B chain resulting from reductive cleavage of insulin by bovine-liver extracts, in association with human albumin, exhibited a comparable anti-insulin effect. 4. It is postulated that the B chain interacts with albumin to enable solubilization of the chain and that inhibition of insulin action on muscle may occur as a result of competition for cellular receptor sites by the B chain. 5. The implication of these findings in relation to a circulating insulin antagonist is discussed.     

Crystal structure of Rab9 complexed to GDP reveals a dimer with an active conformation of switch II

The small GTPase Rab9 is an essential regulator of vesicular transport from the late endosome to the trans-Golgi network, as monitored by the redirection of the mannose-6-phosphate receptors. The crystal structure of Rab9 complexed to GDP, Mg(2+), and Sr(2+) reveals a unique dimer formed by an intermolecular beta-sheet that buries the switch I regions. Surface area and shape complementarity calculations suggest that Rab9 dimers can form an inactive, membrane-bound pool of Rab9 . GDP that is independent of GDI. Mg(2+)-bound Rab9 represents an inactive state, but Sr(2+)-bound Rab9 . GDP displays activated switch region conformations, mimicking those of the GTP state. A hydrophobic tetrad is formed resembling an effector-discriminating epitope found only in GTP-bound Rab proteins.     

The conformation of the lysyl side chain of substrates at the active center of trypsin

In order to study the conformation of the side chain of lysine substrates bound to the active center of trypsin, two lysine analogs, cis- and trans-2,6-diamino-4-hexenoic acids (4,5-dehydrolysines) were synthesized and kinetic parameters for the hydrolysis of benzoyl methyl esters and phenylthiazolones of these analogs by this enzyme were compared with those of the corresponding lysine derivatives. The derivatives of cis-4,5-dehydrolysine were hydrolyzed much more slowly than those of lysine, owing largely to the small kcat values for the former. On the other hand, the derivatives of the trans-isomer were hydrolyzed at about the same rates as those of lysine and the values of both Km and kcat of the former are also similar to those of the latter. These results indicate that the conformation of the side chain of the lysine derivatives hydrolyzed by trypsin is such that the beta- and epsilon-carbons are in a trans-like conformation, as suggested by X-ray crystallographic studies of inhibitor-trypsin complex.     

Limited proteolysis patterns of the B chain of insulin.

The oxidized B chain of insulin was used as a simple model for further consideration of limited proteolysis with low substrate:enzyme ratios. With low B chain:trypsin ratios, the ordinarily slower cleavage rate of the -Lys₂₉-Ala₃₀ bond essentially equaled the cleavage saturation rate of the -Arg₂₂-Gly₂₃ bond. This led to the disappearance of octapeptide which ordinarily forms most rapidly. Heptapeptide and alanine, formed mainly by cleavage of the octapeptide, decreased somewhat at high enzyme relative levels. Trypsin added to B chain formed a single chromatographic peak.     

Protein flexibility: multiple molecular dynamics simulations of insulin chain B

Multiple molecular dynamics simulations totaling more than 100 ns were performed on chain B of insulin in explicit solvent at 300 K and 400 K. Despite some individual variations, a comparison of the protein dynamics of each simulation showed similar trends and most structures were consistent with NMR experimental values, even at the elevated temperature. The importance of packing interactions in determining the conformational transitions of the protein was observed, sometimes resulting in conformations induced by localized hydrophobic interactions. The high temperature simulation generated a more diverse range of structures with similar elements of secondary structure and populated conformations to the simulations at room temperature. A broad sampling of the conformational space of insulin chain B illustrated a wide range of conformational states with many transitions at room temperature in addition to the conformational states observed experimentally. The T-state conformation associated with insulin activity was consistently present and a possible mechanism of behavior was suggested.     

Action of human liver cathepsin B on the oxidized insulin B chain.

The lysosomal cysteine proteinase cathepsin B (from human liver) was tested for its peptide-bond specificity against the oxidized B-chain of insulin. Sixteen peptide degradation products were separated by high-pressure liquid chromatography and thin-layer chromatography and were analysed for their amino acid content and N-terminal amino acid residue. Five major and six minor cleavage sites were identified; the major cleavage sites were Gln(4)-His(5), Ser(9)-His(10), Glu(13)-Ala(14), Tyr(16)-Leu(17) and Gly(23)-Phe(24). The findings indicate that human cathepsin B has a broad specificity, with no clearly defined requirement for any particular amino acid residues in the vicinity of the cleavage sites. The enzyme did not display peptidyldipeptidase activity with this substrate, and showed a specificity different from those reported for two other cysteine proteinases, papain and rat cathepsin L.     

Evolutionary origins of insulin resistance: a behavioral switch hypothesis

Background  

Insulin resistance, which can lead to a number of diseases including type 2 diabetes and coronary heart disease, is believed to have evolved as an adaptation to periodic starvation. The "thrifty gene" and "thrifty phenotype" hypotheses constitute the dominant paradigm for over four decades. With an increasing understanding of the diverse effects of impairment of the insulin signaling pathway, the existing hypotheses are proving inadequate.     

Periodic electric field as a possible switch of biopolymer conformation

A theoretical model describing the effect of periodic electrical fields (PEF) on the conformation of biopolymers is proposed. The biopolymer is interpreted as a system of classical mechanics consisting of subsystems (molecular groups) connected with each other by potential forces. The existence of PEF manifests itself as a periodic inducing force applied to the molecular group. Dissipation of energy is considered on the basis of liquid (viscous) friction. Arguments are presented in favor of non-Newton rheology of the viscous medium and as a consequence of non-linear dependence of the friction force on velocity. It is found that if the periodic field has more than one harmonics, then due to PEF effect the conformation potential can change to the linear in co-ordinate item.     

Revisiting the structural flexibility of the complex p21(ras)-GTP: the catalytic conformation of the molecular switch II.

The hydrolysis of GTP in p21(ras) triggers conformational changes that regulate the ras/ERK signaling pathway. An important active site residue is Gln61, which has been found to be mutated in 30% of human tumors. The dynamics of the active site conformation is studied by using molecular dynamics simulation of two independent structures of the GTP-bound uncomplexed enzyme. Two distinct conformations of the enzyme are observed, in which the side-chain residue Gln61 is in different orientations. Essential dynamics analysis is used to describe the essential motions in the transition between the two conformations. Results are compared with earlier simulations of p21(ras) and its complex with GTPase activating protein p21-GAP.     

Activation of the nicotinic acetylcholine receptor involves a switch in conformation of the alpha subunits

The nicotinic acetylcholine (ACh) receptor belongs to a superfamily of synaptic ion channels that open in response to the binding of chemical transmitters. Their mechanism of activation is not known in detail, but a time-resolved electron microscopic study of the muscle-type ACh receptor had suggested that a local disturbance in the ligand-binding region and consequent rotations in the ligand-binding alpha subunits, connecting to the transmembrane portion, are involved. A more precise interpretation of this structural change is given here, based on comparison of the extracellular domain of the ACh receptor with an ACh-binding protein (AChBP) to which a putative agonist is bound. We find that, to a good approximation, there are two alternative extended conformations of the ACh receptor subunits, one characteristic of either alpha subunit before activation, and the other characteristic of all three non-alpha subunits and the protomer of AChBP. Substitution in the three-dimensional maps of alpha by non-alpha subunits mimics the changes seen on activation, suggesting that the structures of the alpha subunits are modified initially by their interactions with neighbouring subunits and switch to the non-alpha form when ACh binds. This structural change, which entails 15-16 degrees rotations of the inner pore-facing parts of the alpha subunits, most likely acts as the trigger that opens the gate in the membrane-spanning pore.     

Immunogenicity of B chain in insulin responder and nonresponder mice.

The potential immunogenicity of insulin B chain in beef insulin low-responder H-2k,a and high-responder H-2b,d mice was examined using lymph node proliferation assays. Oxidized B chain was immunogenic in H-2k,a, but not H-2b,d, mice. The T cell population recognized a determinant in OX-B chain associated with I-Ak. These cells did not respond to intact insulin, suggesting that the B chain determinant was not available to I-Ak during immunologic processing of insulin. Responses were observed in H-2k and H-2d, but not H-2b, after immunization with reduced and carboxyamidomethylated-insulin which contains equimolar A chain and B chain. These responses were I-A-restricted and heterogeneous, with reactivity to A chain and B chain determinants. In each case, little or no cross-reactivity was observed between RCAM-insulin and intact insulin. Furthermore, T cell populations induced in H-2k mice selectively recognized OX-B chain or RCAM-B chain, which differ in chemical modification of the thiols of Cys B7 and Cys B19. Similarly, RCAM-BINS-immune T cells from H-2d did not react to OX-B chain. These results indicate that derivatization of the cysteine thiols, through disulfide bonds, oxidation, or carboxyamidomethylation, radically affects T cell recognition of insulin B chain.     

Tyrocidine-induced modulation of the DNA conformation in Bacillus brevis

Using the [3H]trimethylpsoralen photobinding method [Sinden, R.R., Carlson, J.O. & Pettijohn, D.E. (1980) Cell 21, 773-783], a decrease in unrestrained torsional tension of DNA was detected in Bacillus brevis cells when they had entered the sporulation phase. This decrease in superhelicity was found in cells which synthesized the peptide antibiotic tyrocidine and which were stimulated to sporulate. Fluctuations in superhelicity probably reflect a highly complicated picture of tension-relaxing and tension-inducing activities. Addition of tyrocidine to vegetative cells reduced by one-half the torsional tension from DNA, whereas ethidium bromide relaxes DNA completely. Cross-links between DNA and tyrocidine were introduced with ultraviolet light in vitro and in vivo indicating that the modulation of the DNA conformation in the cell may in fact be due to a DNA-tyrocidine interaction. In a growing B. brevis culture exogenous [3H]tyrocidine could only be photobound to DNA after the cells had entered the sporulation phase. Our results could mean that the peptide antibiotic tyrocidine is active in B. brevis on the DNA level as one regulatory factor controlling DNA functions.     

Toward a structural understanding of arf family:effector specificity

Arf family proteins are critical regulators of intracellular trafficking and actin cytoskeleton dynamics. To carry out their cellular functions, Arf family proteins interact with various effectors that differ in nature and structure. Understanding how these proteins interact with structurally different partners and are distinguished by specific effectors while being closely related requires a structural characterization and comparison of the various Arf family:effector complexes. Recent structural reports of Arf and Arl proteins in complex with different downstream effectors shed new light on general and specific structural recognition determinants characteristic of Arf family proteins.