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pH-Dependent Interactions of Human Islet Amyloid Polypeptide Segments with Insulin Studied by Replica Exchange Molecular Dynamics Simulations Ping Jiang, Lei Wei, Konstantin Pervushin, and Yuguang Mu* School of Biological Sciences, Nanyang Technological UniVersity, Singapore, Singapore ReceiVed: February 28, 2010; ReVised Manuscript ReceiVed: June 26, 2010
Amyloidogenesis of human islet amyloid polypeptide (hIAPP) within or surrounding secretory β-cells of pancreas has long been related to the pathology of type II diabetes. Insulin, coexpressed and cosecreted with hIAPP in vivo, has the capacity of interacting with hIAPP and further inhibiting the amyloid deposition of the peptide. On the basis of the reported experimental data, we conducted replica exchange molecular dynamics simulations on the complexes of insulin and the binding segment 9-20 of rat/human IAPP under different pH values. The deprotonation of H18 at neutral pH reduces the possibility of polar interactions at position 18 as well as the flanking positions. Moreover, it destabilizes the helical motif of insulin-bound hIAPP. From several perspectives, hIAPP with charged H18 bears more resemblance to rat IAPP (rIAPP) with a point mutation of R18 than its neutral version does. It is likely that the positively charged residue at position 18 is critical to insulin binding. At neutral pH, three loosely binding sites can be recognized on the insulin surface, while at acidic pH one dominant binding site is resolved, which is similar to the previously identified rIAPP-insulin binding site. Several critical intermolecular contacts, including salt-bridges and π-stacking interactions, identified in the current study, could be used as the starting point for devising a potent peptide inhibitor of hIAPP amyloidogenesis. Introduction Human islet amyloid polypeptide (hIAPP) is known as a 37residue hormone coexpressed and cosecreted with insulin in β-cells of the pancreas.1 In the condition of Type-II diabetes (T2D), pancreatic β-cell apoptosis synchronizes with the deposition of hIAPP amyloid fibrils in more than 90% of the patients.1 Amyloid-like deposition of intrinsically soluble proteins or peptides in tissues and organs is a common characteristic of a series of conformational diseases comprising Alzheimer’s disease, Parkinson’s disease, spongiform encephalopathy, T2D, and so forth.2 A dramatically increasing number of studies have contributed to elucidating mechanisms of hIAPP deposition and cytotoxicity, and great progression has been made in recent years. Surprisingly, it has been found that hIAPP remains soluble in secretory vesicles at a concentration range of 0.8-4 mM, whereas the critical concentration of hIAPP to convert into amyloid deposition is incredibly lower by orders of magnitude in vitro.3,4 Therefore, the in vivo environment including pH values, ionic strength (Ca2+, Zn2+), and protein components such as insulin, proinsulin, and C-peptide must play a critical role in avoidance of hIAPP misfolding into fibrillar amyloid.5,6 Among all the factors, acidic pH at ∼6.0 and insulin whose concentration is at a ratio of ∼100:1 to hIAPP in secretory vesicles are found to have significant impacts on the unnatural transition of hIAPP from soluble peptides to deposited amyloid fibrils.4 Condensed crystalline insulin as well as cellular membranes occupy the most space in secretory granules where hIAPP exists in between and interplays with both. Crystalline insulin of hexamer units is the main form of insulin stored in the pancreas and is most likely in equilibrium with soluble monomers, dimers, and hexamers at the neutral pH.7,8 Insulin monomer consists of A and B chains, which are linked by a pair of disulfide bonds. * To whom correspondence should be addressed. Phone: +65-63162885. E-mail:
[email protected].
The A chain folds into two antiparallel arranged, short helices connected by an extended segment, while the B chain adopts a longer helix in the middle together with two extended segments on the N and C termini.7 In a way of placing plenty of aromatic and hydrophobic residues into close contacts, the helical segment of B chain acts as a significant contributor to the stability of dimeric and hexameric insulin oligomers.7 A series of works performed under a mimicked secretory vesicle condition in Vitro have made progresses in clarifying the interplay between IAPP and insulin in solvated/assembled forms.5,9-11 The misfolding and subsequent amyloid regression of soluble IAPP can be inhibited by interaction with insulin in assembled array or in a soluble/monomeric form whose B chain is truncated to prevent oligomerization.9,12 Insulin is also found to be capable of associating with preformed IAPP fibrils, but with no evidence of disaggregation.9 These findings assume that insulin-IAPP interfaces may be solvent exposed, and it is likely that insulin-IAPP interaction resides in more than one site and involves distinct residue clusters from both partners. There are several valuable trials to characterize the insulinIAPP binding sites, and most have identified the insulin B chain as playing the key role in mediating binding and inhibitory ability.11,13,14 Insulin-degrading enzyme (IDE), with insulindegrading activity, can catalyze both insulin B chain and IAPP as its substrates, both of which show a similar two discrete β-sheet pattern in their bound states.15 Sequence alignment has been performed between the two peptides, assuming binding in a manner of homogeneous recognition.9 Residual interaction mapping has been performed by Gilead et al. who have successfully shortened the binding regions to two segments as residues B8-25 (B chain) and B20-30. Additionally, a segment of N-terminal IAPP consisting of residues 7-19 was identified to be the insulin-binding sequence.11 The binding sequences within insulin B8-25 and IAPP 7-19 were confirmed by our recent study on structural characterization of insulin-rat IAPP
10.1021/jp101811u 2010 American Chemical Society Published on Web 07/21/2010
Interactions of hIAPP Segments with Insulin (rIAPP), a nonamyloidogenic variant of IAPP from rodent species.14 One mutation of R18H occurs at the binding region of hIAPP, and all the others locate in a decameric region of 20-29, which is the hydrophobic core of the hIAPP amyloid fibril.16 The bound rIAPP segment was found to be locally structured, consisting of two short helices in proximity to insulin. A salt bridge well formed between rIAPP and insulin and a flanking hydrophobic patch on the insulin B chain were suggested to mediate the specific interaction. Taking advantage of the existing depiction of the binding interface and the assumption of homogeneous recognition and aromatic interactions that might be involved,11 we extend our studies to construct the binding interfaces of hIAPP-insulin complexes at acidic and neutral pH, to explore the different interaction patterns and inhibitory mechanisms of insulin on hIAPP aggregation. In the current study, a replica exchange molecule dynamics (REMD)17-19 simulation scheme was used to enhance the sampling of the huge configuration space of an insulin-IAPP complex. The binding sites and binding modes involving both electrostatic and π-stacking interactions will be presented, which can provide an atomic detailed mechanism for the inhibitory effects of insulin on hIAPP amyloid formation. The influences of varied pH, H18R mutation, and the pH dependence of IAPP-insulin interactions are also disclosed, which could be used as the starting point for devising a potent peptide inhibitor of hIAPP amyloidogenesis. Materials and Methods Sequences. The IAPP sequences for human and rodent are shown here: KCNTATCATQ10RLANFLVHSS20NNFGAILSST30NVGSNTY (hIAPP) KCNTATCATQ10RLANFLVRSS20NNLGPVLPPT30NVGSNTY (rIAPP) Six residues disparate between the two sequences are highlighted. REMD Simulations. A temperature-swapping replica exchange simulation scheme was applied to a docking r/hIAPP fragment comprising residues 9-20 onto monomeric insulin (PDB entry code: 1MSO).7 To mimic the neutral and the low pH environments, all histidine residues whose pKa values are approximately 6.0 were modeled as deprotonated or protonated states separately in two systems (referred to as hIAPP and hIAPP(His+) systems hereafter). The rIAPP system is simulated at neutral pH as a control. The temperatures of 24 replicas are listed here: 315.0, 318.1, 321.2, 324.4, 327.6, 330.8, 334.0, 337.3, 340.6, 344.0, 347.4, 350.8, 354.2, 357.7, 361.2, 364.8, 368.3, 372.0, 375.6, 379.3, 383.0, 386.8, 390.6, and 394.4 K. The two simulations of hIAPP-insulin complexes lasted for 100 ns. rIAPP simulations ended at 84 ns. N and C termini of IAPP9-20 were capped by acetyl (ACE) and N-methyl (NME) groups, respectively. At the beginning of the simulations, the IAPP peptide was modeled as an extended strand and randomly placed at a distance of approximately 15 Å away from the insulin molecule. The protein complex was solvated by simple point charge (SPC) water molecules in a dodecahedron box with a size of 50 Å. The simulations were performed by using the Gromacs simulation package with an OPLSAA (all-atom) force field.20,21 From the NMR data, the three-dimensional (3D) fold of insulin is well reserved in IAPP titration experiments.14 Thereby, throughout all simulations, the positions of backbone atoms of insulin were restrained with side chains capable of rotating freely. All bonds involving hydrogen atoms were constrained in length according to LINCS protocol.22 Electro-
J. Phys. Chem. B, Vol. 114, No. 31, 2010 10177 static interactions were treated with the particle mesh Ewald (PME) method with a cutoff of 9 Å, and a cutoff of 14 Å was used for the van der Waals interactions.23 The integration step was set as 0.002 ps, and the nonbonded pair list was updated every five integration steps. The solutes and solvents were separately coupled to the external heat bath with the relaxation time of 0.1 ps. Replica exchange was attempted every 1000 integration steps (2 ps). The structure snapshots were output every 1 ps, and an ensemble of 100 000 (or 84 000 for rIAPP) structures at 315.0 K was produced for the analysis. REMD simulations for two control systems of an isolated hIAPP9-20 segment were also performed. Residue H18 was modeled to be either protonated or deprotonated to mimic the charged state in neutral or acidic pH solution. The peptides were initially extended and placed in the center of a dodecahedron box with a dimension of 50 × 50 × 50 Å. The simulations run for 50 ns. Clustering Analysis. The REMD scheme enhances the IAPP-insulin complex configuration sampling in two aspects: recognition sites on insulin and conformations of bound IAPP. Thus, the clustering analysis of conformational ensemble relies not only upon structure-based grouping of IAPP but also upon recognition sites which may be composed of disparate groups from IAPP or insulin. A pair of intermolecular residues between IAPP and insulin is counted as a contact when the distance of any pair of atoms is not larger than a cutoff distance (4 Å in our case). The most contacts found can be grouped together on the insulin as binding sites rather than isolated distributed. Therefore, the first level of our clustering scheme is to search the correlated contacts and then group them together. Before searching the correlated contacts, the random contacts whose occurrence probability is less than 5% were filtered out. After the filtering, there were in total 44 contacts for hIAPP(His+), 79 contacts for hIAPP, and 126 contacts for rIAPP left, which were considered the “conserved” contacts. The correlation of these conserved contacts was analyzed in the following way. All contacts were ranked based on their occurrence. The first contact has the largest occurrence. The correlation degree of two contacts was defined as the ratio of the probability of the two contacts happening simultaneously to the probability of the contact with lower occurrence. The correlation degrees for all contact pairs were calculated. On the basis of the distribution of the correlation degrees, a value of 62.5% was assigned to be the threshold of correlation: if the correlation degree is larger than this value, the two contacts are considered to be correlated and are classified into one contact group. Other contacts can join the contact group only when the correlation degrees between this contact and all group members are larger than the threshold. Such first-level clustering categorized all contacts into 13 contact groups for hIAPP(His+), 20 contact groups for hIAPP, and 21 groups for rIAPP. One contact can be assigned into different contact groups. The second level of clustering relies on pairwise heavy atom root-mean-square deviations (rmsd) of all structural snapshots belonging to a single contact group. The central structure of the highest populated cluster was taken as the representative structure of the group. After the first level of clustering by which contact groups were identified, we found that majority members of the IAPP-insulin complex ensemble (62.37% and 97.58% for two hIAPP systems and 97.65% for rIAPP) have been selected, which guarantees that the representative structures obtained from the second-level structure-based clustering have significant statistical meanings.
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Figure 1. Insulin-IAPP complex simulation convergence monitored by SSP. The simulation trajectories at 315 K of hIAPP(His+) (a), hIAPP (b), and rIAPP (c) were grouped every 10 ns. The SS assessment was performed for individual residues by the DSSP package.36 (d) Propensities for the R-helix of each residue are compared. The helix propensities were calculated for every 20 ns of the last 60 ns trajectories at 315 K. The values in the figure are averaged over the three time intervals. Error bars indicate the standard deviation of three calculated values.
Binding Energy Calculation. The binding energy of the IAPP-insulin complex can be estimated by the equation
∆Ebind ) Ecomplex - Einsulin - EIAPP Ecomplex, Einsulin, and EIAPP are the total potential energies of the IAPP-insulin complex, insulin-only, and IAPP-only protein, respectively, all of which can be decomposed into two terms: intraprotein potential energy calculated by the Gromacs package and solvation energy estimated by using generalized Born (GB) model in the sander module of the AMBER 9 package.24,25 The source code of the amber leap program was modified to import solute nonbonded parameters from the OPLS-AA force field. Results In the Presence of Insulin, the Helix Proportion of the hIAPP(His+) Segment Is Converged at ∼10%. To analyze the secondary structure proportion (SSP) of IAPP and also to verify the convergence of a total of five REMD simulations, the secondary structure (SS) composition of IAPP segments in the presence and absence of insulin was assessed. The SSP averaged over an every 10 ns ensemble is plotted in Figures 1a-c and 2a,b. The convergence of REMD simulations is demonstrated by the stable SSP values. From the last 30 ns data of IAPP-insulin complexes, the SS compositions of hIAPP(His+) (Figure 1a) and rIAPP (Figure 1c) are quite similar, consisting of ∼10% helices, 15% turns,
