Crystal structure of destripeptide (B28-B30) insulin: implications for insulin dissociation

Destripeptide (B28-B30) insulin (DTRI) is an insulin analogue that has much weaker association ability than native insulin but keeps most of its biological activity. It can be crystallized from a solution containing zinc ions at near-neutral pH. Its crystal structure has been determined by molecular replacement and refined at 1.9 A resolution. DTRI in the crystal exists as a loose hexamer compared with 2Zn insulin. The hexamer only contains one zinc ion that coordinates to the B10 His residues of three monomers. Although residues B28-B30 are located in the monomer-monomer interface within a dimer, the removal of them can simultaneously weaken both the interactions between monomers within the dimer and the interactions between dimers. Because the B-chain C-terminus of insulin is very flexible, we take the DTRI hexamer as a transition state in the native insulin dissociation process and suggest a possible dissociation process of the insulin hexamer based on the DTRI structure.     

The solution structure of a monomeric insulin. A two-dimensional 1H-NMR study of des-(B26-B30)-insulin in combination with distance geometry and restrained molecular dynamics.

The solution conformation of des-(B26-B30)-insulin (DPI) has been investigated by 1H-NMR spectroscopy. A set of 250 approximate interproton distance restraints, derived from two-dimensional nuclear Overhauser enhancement spectra, were used as the basis of a structure determination using distance geometry (DG) and distance-bound driven dynamics (DDD). Sixteen DG structures were optimized using energy minimization (EM) and submitted to short 5-ps restrained molecular dynamics (RMD) simulations. A further refinement of the DDD structure with the lowest distance errors was done by energy minimization, a prolonged RMD simulation in vacuo and a time-averaged RMD simulation. An average structure was obtained from a trajectory generated during 20-ps RMD. The final structure was compared with the des-(B26-B30)-insulin crystal structure refined by molecular dynamics and the 2-Zn crystal structure of porcine insulin. This comparison shows that the overall structure of des-(B26-B30)-insulin is retained in solution with respect to the crystal structures with a high flexibility at the N-terminal part of the A chain and at the N-terminal and C-terminal parts of the B chain. In the RMD run a high mobility of Gly A1, Asn A21 and of the side chain of Phe B25 is noticed. One of the conformations adopted by des-(B26-B30)-insulin in solution is similar to that of molecule 1 (Chinese nomenclature) in the crystal structure of porcine insulin.     

A comparison of the dynamic behavior of monomeric and dimeric insulin shows structural rearrangements in the active monomer

Molecular dynamics (MD) simulations (5-10ns in length) and normal mode analyses were performed for the monomer and dimer of native porcine insulin in aqueous solution; both starting structures were obtained from an insulin hexamer. Several simulations were done to confirm that the results obtained are meaningful. The insulin dimer is very stable during the simulation and remains very close to the starting X-ray structure; the RMS fluctuations calculated from the MD simulation agree with the experimental B-factors. Correlated motions were found within each of the two monomers; they can be explained by persistent non-bonded interactions and disulfide bridges. The correlated motions between residues B24 and B26 of the two monomers are due to non-bonded interactions between the side-chains and backbone atoms. For the isolated monomer in solution, the A chain and the helix of the B chain are found to be stable during 5ns and 10ns MD simulations. However, the N-terminal and the C-terminal parts of the B chain are very flexible. The C-terminal part of the B chain moves away from the X-ray conformation after 0.5-2.5ns and exposes the N-terminal residues of the A chain that are thought to be important for the binding of insulin to its receptor. Our results thus support the hypothesis that, when monomeric insulin is released from the hexamer (or the dimer in our study), the C-terminal end of the monomer (residues B25-B30) is rearranged to allow binding to the insulin receptor. The greater flexibility of the C-terminal part of the beta chain in the B24 (Phe-->Gly) mutant is in accord with the NMR results. The details of the backbone and side-chain motions are presented. The transition between the starting conformation and the more dynamic structure of the monomers is characterized by displacements of the backbone of Phe B25 and Tyr B26; of these, Phe B25 has been implicated in insulin activation.     

Conformational flexibility of aqueous monomeric and dimeric insulin: a molecular dynamics study

A series of molecular dynamics simulations have been used to investigate the nature of monomeric and dimeric insulin in aqueous solution. It is shown that in the absence of crystal contacts both monomeric and dimeric insulin have a high degree of intrinsic flexibility. Neither of the two monomer conformations of 2Zn crystalline insulin appears to be favored in solution nor is the asymmetry of the crystal dimer reduced in the absence of crystal contacts. A shift is observed in the relative positions of molecules 1 and 2 in the dimer compared with that found in the crystal, which may have consequences for the prediction of the effects of mutants in the monomer-monomer interface designed to alter the self-association properties of insulin.     

Thermal dissociation and unfolding of insulin

The thermal stability of human insulin was studied by differential scanning microcalorimetry and near-UV circular dichroism as a function of zinc/protein ratio, to elucidate the dissociation and unfolding processes of insulin in different association states. Zinc-free insulin, which is primarily dimeric at room temperature, unfolded at approximately 70 degrees C. The two monomeric insulin mutants Asp(B28) and Asp(B9),Glu(B27) unfolded at higher temperatures, but with enthalpies of unfolding that were approximately 30% smaller. Small amounts of zinc caused a biphasic thermal denaturation pattern of insulin. The biphasic denaturation is caused by a redistribution of zinc ions during the heating process and results in two distinct transitions with T(m)'s of approximately 70 and approximately 87 degrees C corresponding to monomer/dimer and hexamer, respectively. At high zinc concentrations (>or=5 Zn(2+) ions/hexamer), only the hexamer transition is observed. The results of this study show that the thermal stability of insulin is closely linked to the association state and that the zinc hexamer remains stable at much higher temperatures than the monomer. This is in contrast to studies with chemical denaturants where it has been shown that monomer unfolding takes place at much higher denaturant concentrations than the dissociation of higher oligomers [Ahmad, A., et al. (2004) J. Biol. Chem. 279, 14999-15013].     

Novel covalently linked insulin dimer engineered to investigate the function of insulin dimerization

An ingenious system evolved to facilitate insulin binding to the insulin receptor as a monomer and at the same time ensure sufficient stability of insulin during storage. Insulin dimer is the cornerstone of this system. Insulin dimer is relatively weak, which ensures dissociation into monomers in the circulation, and it is stabilized by hexamer formation in the presence of zinc ions during storage in the pancreatic β-cell. Due to the transient nature of insulin dimer, direct investigation of this important form is inherently difficult. To address the relationship between insulin oligomerization and insulin stability and function, we engineered a covalently linked insulin dimer in which two monomers were linked by a disulfide bond. The structure of this covalent dimer was identical to the self-association dimer of human insulin. Importantly, this covalent dimer was capable of further oligomerization to form the structural equivalent of the classical hexamer. The covalently linked dimer neither bound to the insulin receptor, nor induced a metabolic response in vitro. However, it was extremely thermodynamically stable and did not form amyloid fibrils when subjected to mechanical stress, underlining the importance of oligomerization for insulin stability.     

Structural relationships in the two-zinc insulin hexamer.

The refinement of the crystal structure of two-Zn pig insulin using 1.5-A (1A = 0.1 nm) resolution data by Fourier and fast Fourier least-squares methods allows us to make detailed comparisons between the two independent molecules present in the two-Zn insulin dimer and to describe their interactions in the monomer, dimer, and hexamer. The main chain structures for the two molecules agree well except at the N terminus of the A chain and the C terminus of the B chain. The residues along the line of the local two-fold axes, apart from the B25 side chain, conform extremely closely to the two-fold symmetry, although the discrepancies are much more apparent away from this axis. The ability of the insulin molecule to adopt different conformations may be an important factor in the expression of its biological activity.     

Partially folded intermediates in insulin fibrillation

Native zinc-bound insulin exists as a hexamer at neutral pH. Under destabilizing conditions, the hexamer dissociates, and is very prone to forming fibrils. Insulin fibrils exhibit the typical properties of amyloid fibrils, and pose a problem in the purification, storage, and delivery of therapeutic insulin solutions. We have carried out a systematic investigation of the effect of guanidine hydrochloride (Gdn.HCl)-induced structural perturbations on the mechanism of fibrillation of insulin. At pH 7.4, the addition of as little as 0.25 M Gdn.HCl leads to dissociation of insulin hexamers into dimers. Moderate concentrations of Gdn.HCl lead to formation of a novel partially unfolded dimer state, which dissociates into a partially unfolded monomer state. High concentrations of Gdn.HCl resulted in unfolded monomers with some residual structure. The addition of even very low concentrations of Gdn.HCl resulted in substantially accelerated fibrillation, although the yield of fibrils decreased at high concentrations. Accelerated fibrillation correlated with the population of the expanded (partially folded) monomer, which existed up to >6 M Gdn.HCl, accounting for the formation of substantial amounts of fibrils under such conditions. In the presence of 20% acetic acid, where insulin exists as the monomer, fibrillation was also accelerated by Gdn.HCl. The enhanced fibrillation of the monomer was due to the increased ionic strength at low denaturant concentrations, and due to the presence of the partially unfolded, expanded conformation at Gdn.HCl concentrations above 1 M. The data suggest that under physiological conditions, the fibrillation of insulin involves both changes in the association state (with rate-limiting hexamer dissociation) and conformational changes, leading to formation of the amyloidogenic expanded monomer intermediate.     

Cryo-EM structure of monomeric photosystem II at 2.78 Å resolution reveals factors important for the formation of dimer

Photosystem II (PSII) functions mainly as a dimer to catalyze the light energy conversion and water oxidation reactions. However, monomeric PSII also exists and functions in vivo in some cases. The crystal structure of monomeric PSII has been solved at 3.6 Å resolution, but it is still not clear which factors contribute to the formation of the dimer. Here, we solved the structure of PSII monomer at a resolution of 2.78 Å using cryo-electron microscopy (cryo-EM). From our cryo-EM density map, we observed apparent differences in pigments and lipids in the monomer-monomer interface between the PSII monomer and dimer. One β-carotene and two sulfoquinovosyl diacylglycerol (SQDG) molecules are found in the monomer-monomer interface of the dimer structure but not in the present monomer structure, although some SQDG and other lipid molecules are found in the analogous region of the low-resolution crystal structure of the monomer, or cryo-EM structure of an apo-PSII monomer lacking the extrinsic proteins from Synechocystis sp. PCC 6803. In the current monomer structure, a large part of the PsbO subunit was also found to be disordered. These results indicate the importance of the β-carotene, SQDG and PsbO in formation of the PSII dimer.     

Sulfolobus acidocaldarius inorganic pyrophosphatase: structure, thermostability, and effect of metal ion in an archael pyrophosphatase.

The first crystal structure of an inorganic pyrophosphatase (S-PPase) from an archaebacterium, the thermophile Sulfolobus acidocaldarius, has been solved by molecular replacement and refined to an R-factor of 19.7% at 2.7 A. S-PPase is a D3 homohexameric protein with one Mg2+ per active site in a position similar to, but not identical with, the first activating metal in mesophilic pyrophosphatases (PPase). In mesophilic PPases, Asp65, Asp70, and Asp102 coordinate the Mg2+, while only Asp65 and Asp102 do in S-PPase, and the Mg2+ moves by 0.7 A. S-PPase may therefore be deactivated at low temperature by mispositioning a key metal ion. The monomer S-PPase structure is very similar to that of Thermus thermophilus (T-PPase) and Escherichia coli (E-PPase), root-mean-square deviations around 1 A/Calpha. But the hexamer structures of S- and T-PPase are more tightly packed and more similar to each other than they are to that of E-PPase, as shown by the increase in surface area buried upon oligomerization. In T-PPase, Arg116 creates an interlocking ionic network to both twofold and threefold related monomers; S-PPase has hydrophilic interactions to threefold related monomers absent in both E- and T-PPase. In addition, the thermostable PPases have about 7% more hydrogen bonds per monomer than E-PPase, and, especially in S-PPase, additional ionic interactions anchor the C-terminus to the rest of the protein. Thermostability in PPases is thus due to subtle improvements in both monomer and oligomer interactions.     

Solution structure of a polypeptide dimer comprising the fourth Ca(2+)-binding site of troponin C by nuclear magnetic resonance spectroscopy

The structure of a 39 amino acid proteolytic fragment of rabbit skeletal troponin C containing the fourth Ca(2+)-binding site has been determined by an approach involving nuclear magnetic resonance (NMR) spectroscopy combined with hybrid distance geometry-dynamical simulated annealing calculations. Hydrodynamic and NMR evidence establishes unambiguously that the fragment forms a stable dimer in solution in the presence of excess Ca2+. The calculation of the dimeric structure is based on a total of 1056 experimental restraints comprising 422 interproton distances, 35 phi, 28 psi, and 28 chi 1 torsion angle restraints within each subunit, 30 intermonomer distance restraints, and 6 Ca2+ restraints per subunit. A total of 48 final structures were calculated having an rms deviation about the mean atomic backbone coordinate positions of 1.0 A for residues Asp128-Glu156. The solution structure consists of a dimer of helix-loop-helix motifs related by a 2-fold axis of symmetry. The overall architecture of the dimer is very similar to the C-terminal domain in the crystal structure of chicken skeletal troponin C.     

Physicochemical basis for the rapid time-action of LysB28ProB29-insulin: dissociation of a protein-ligand complex.

The rate-limiting step for the absorption of insulin solutions after subcutaneous injection is considered to be the dissociation of self-associated hexamers to monomers. To accelerate this absorption process, insulin analogues have been designed that possess full biological activity and yet have greatly diminished tendencies to self-associate. Sedimentation velocity and static light scattering results show that the presence of zinc and phenolic ligands (m-cresol and/or phenol) cause one such insulin analogue, LysB28ProB29-human insulin (LysPro), to associate into a hexameric complex. Most importantly, this ligand-bound hexamer retains its rapid-acting pharmacokinetics and pharmacodynamics. The dissociation of the stabilized hexameric analogue has been studied in vitro using static light scattering as well as in vivo using a female pig pharmacodynamic model. Retention of rapid time-action is hypothesized to be due to altered subunit packing within the hexamer. Evidence for modified monomer-monomer interactions has been observed in the X-ray crystal structure of a zinc LysPro hexamer (Ciszak E et al., 1995, Structure 3:615-622). The solution state behavior of LysPro, reported here, has been interpreted with respect to the crystal structure results. In addition, the phenolic ligand binding differences between LysPro and insulin have been compared using isothermal titrating calorimetry and visible absorption spectroscopy of cobalt-containing hexamers. These studies establish that rapid-acting insulin analogues of this type can be stabilized in solution via the formation of hexamer complexes with altered dissociation properties.     

1H n.m.r. studies of insulin. Reversible transformation of 2-zinc to 4-zinc insulin hexamer

1H n.m.r. studies at 270 MHz were made of the transformation of 2 Zn insulin hexamer to 4 Zn hexamer produced by the addition of anions (thiocyanate ion). Four separate H2 histidine resonances were observed for the B5 and B10 histidines in 2 Zn hexamer at pH 7 and 9 and four separate resonances also occurred in the 4 Zn hexamer. The observation of these resonances and others from phenylalanine, tyrosine and leucine residues showed that the 2 Zn to 4 Zn transformation probably occurred in solution in a similar manner to that observed in the crystal. Furthermore as occurred in the crystal, it was found that in solution the transformation was reversible (on removal of thiocyanate) and that 2 Cd insulin was unable to undergo the transformation. Des-Phe-Bl-insulin did not undergo the transformation. Addition of SCN- to Zn-free insulin (mainly dimer) produced only a small transformation, consistent with the idea that Zn2+ promotes formation of hexamer from dimer but probably does not otherwise affect the transformation.     

Structural analysis of proinsulin hexamer assembly by hydroxyl radical footprinting and computational modeling

Mutations in the insulin gene can impair proinsulin folding and cause diabetes mellitus. Although crystal structures of insulin dimers and hexamers are well established, proinsulin is refractory to crystallization. Although an NMR structure of an engineered proinsulin monomer has been reported, structures of the wild-type monomer and hexamer remain undetermined. We have utilized hydroxyl radical footprinting and molecular modeling to characterize these structures. Differences between the footprints of insulin and proinsulin, defining a "shadow" of the connecting (C) domain, were employed to refine the model. Our results demonstrate that in its monomeric form, (i) proinsulin contains a native-like insulin moiety and (ii) the C-domain footprint resides within an adjoining segment (residues B23-B29) that is accessible to modification in insulin but not proinsulin. Corresponding oxidation rates were observed within core insulin moieties of insulin and proinsulin hexamers, suggesting that the proinsulin hexamer retains an A/B structure similar to that of insulin. Further similarities in rates of oxidation between the respective C-domains of proinsulin monomers and hexamers suggest that this loop in each case flexibly projects from an outer surface. Although dimerization or hexamer assembly would not be impaired, an ensemble of predicted C-domain positions would block hexamer-hexamer stacking as visualized in classical crystal lattices. We anticipate that protein footprinting in combination with modeling, as illustrated here, will enable comparative studies of diabetes-associated mutant proinsulins and their aberrant modes of aggregation.     

Metal induced conformational changes in human insulin: crystal structures of Sr2+, Ni2+ and Cu2+ complexes of human insulin

Crystal structures of Sr(2+), Ni(2+) and Cu(2+) of human insulin complexes have been determined. The structures of Sr(2+) and Ni(2+) complexes are similar to Zn(2+) insulin and are in T6 conformation. (All the six monomers in the insulin hexamer are in Tensed conformation (T), which means the first eight residues of B-chain are in an extended conformation). Cu(2+) complex, though it assumes T6 conformation, has more structural differences due to lowering of crystal symmetry and space group shift from H3 (Hexagonal crystal system) to P3 (Trigonal crystal system) and a doubling of the c axis. 2Ni(2+) human insulin when compared to 4Ni(2+) Arg insulin suggests that terminal modifications may be responsible for additional metal binding. All the three metals have been shown to have a role in diabetes and hence may be therapeutically useful.     

Role of B13 Glu in insulin assembly. The hexamer structure of recombinant mutant (B13 Glu-->Gln) insulin.

The assembly of the insulin hexamer brings the six B13 glutamate side-chains at the centre into close proximity. Their mutual repulsion is unfavourable and zinc co-ordination to B10 histidine is necessary to stabilize the well known zinc-containing hexamers. Since B13 is always a carboxylic acid in all known sequences of hexamer forming insulins, it is likely to be important in the hormone's biology. The mutation of B13 Glu-->Gln leads to a stable zinc-free hexamer with somewhat reduced potency. The structures of the zinc-free B13 Gln hexamer and the 2Zn B13 insulin hexamer have been determined by X-ray analysis and refined with 2.5 A and 2.0 A diffraction data, respectively. Comparisons show that in 2Zn B13 Gln insulin, the hexamer structure (T6) is very like that of the native hormone. On the other hand, the zinc-free hexamer assumes a quaternary structure (T3/R3) seen in the native 4Zn insulin hexamer, and normally associated only with high chloride ion concentrations in the medium. The crystal structures show the B13 Gln side-chains only contact water in contrast to the B13 glutamate in 2Zn insulin. The solvation of the B13 Gln may be associated with this residue favouring helix at B1 to B8. The low potency of the B13 Gln insulin also suggests the residue influences the hormone's conformation.     

Changes in secondary structure follow the dissociation of human insulin hexamers: a circular dichroism study

Vacuum UV circular dichroism spectra measured down to 178 nm for hexameric 2-zinc human insulin, zinc-free human insulin, and the two engineered and biologically active monomeric mutants, [B/S9D] and [B/S9D,T27E] human insulin, show significant differences. The secondary structure analysis of the 2-zinc human insulin (T6) in neutral solution was determined: 57% helix, 1% beta-strand, 18% turn, and 24% random coil. This is very close to the corresponding crystal structure showing that the solution and solid structures are similar. The secondary structure of the monomer shows a 10-15% increase in antiparallel beta-structure and a corresponding reduction in random coil structure. These structural changes are consistent with an independent analysis of the corresponding difference spectra. The advantage of secondary structure analyses of difference spectra is that the contribution of odd spectral features stemming mainly from side chain chromophores is minimized and the sensitivity of the analyses improved. Analysis of the CD spectra of T6 2-zinc, zinc-free human insulin and monomeric mutant insulin by singular value decomposition indicates that the secondary structure changes following the dissociation of hexamers into dimers and monomers are two-state processes.     

Simulation of conformational changes in 2 Zn insulin

Isolated insulin monomers, the dimer and higher aggregates from the 2 Zn crystal structure are subjected to convergent energy minimization in Cartesian co-ordinates using a force-field that includes the position of all hydrogen atoms. The minimizations are found, for the first time, to produce conformational changes of appreciable magnitude, which agree well with observed structural differences between monomers in the 2 Zn crystal and with the mechanism proposed previously for the coupling between deformations in different parts of the molecule. Our results also suggest that insulin would tend to adopt a molecule 1-like conformation in the absence of crystal packing forces, and that dimer formation is not at the origin of the observed asymmetry in the 2 Zn crystal.     

Stimulation of insulin fibrillation by urea-induced intermediates

Fibrillar deposits of insulin cause serious problems in implantable insulin pumps, commercial production of insulin, and for some diabetics. We performed a systematic investigation of the effect of urea-induced structural perturbations on the mechanism of fibrillation of insulin. The addition of as little as 0.5 m urea to zinc-bound hexameric insulin led to dissociation into dimers. Moderate concentrations of urea led to accumulation of a partially unfolded dimer state, which dissociates into an expanded, partially folded monomeric state. Very high concentrations of urea resulted in an unfolded monomer with some residual structure. The addition of even very low concentrations of urea resulted in increased fibrillation. Accelerated fibrillation correlated with population of the partially folded intermediates, which existed at up to 8 m urea, accounting for the formation of substantial amounts of fibrils under such conditions. Under monomeric conditions the addition of low concentrations of urea slowed down the rate of fibrillation, e.g. 5-fold at 0.75 m urea. The decreased fibrillation of the monomer was due to an induced non-native conformation with significantly increased alpha-helical content compared with the native conformation. The data indicate a close-knit relationship between insulin conformation and propensity to fibrillate. The correlation between fibrillation and the partially unfolded monomer indicates that the latter is a critical amyloidogenic intermediate in insulin fibrillation.     

Crystal structure of cyanovirin-N, a potent HIV-inactivating protein, shows unexpected domain swapping.

The crystal structure of cyanovirin-N (CV-N), a protein with potent antiviral activity, was solved at 1.5 A resolution by molecular replacement using as the search model the solution structure previously determined by NMR. The crystals belong to the space group P3221 with one monomer of CV-N in each asymmetric unit. The primary structure of CV-N contains 101 residues organized in two domains, A (residues 1 to 50) and B (residues 51 to 101), with a high degree of internal sequence and structural similarity. We found that under the conditions of the crystallographic experiments (low pH and 26 % isopropanol), two symmetrically related monomers form a dimer by domain swapping, such that domain A of one monomer interacts with domain B' of its crystallographic symmetry mate and vice versa. Because the two swapped domains are distant from each other, domain swapping does not result in additional intramolecular interactions. Even though one of the protein sample solutions that was used for crystallization clearly contained 100 % monomeric CV-N molecules, as judged by various methods, we were only able to obtain crystals containing domain-swapped dimers. With the exception of the unexpected phenomenon of domain swapping, the crystal structure of CV-N is very similar to the NMR structure, with a root-mean-square deviation of 0.55 A for the main-chain atoms, the best agreement reported to date for structures solved using both techniques.