Subramanian Vivekanandan

A partially folded structure of amyloid-beta(1-40) in an aqueous environment

Aggregation of the Aβ(1-40) peptide is linked to the development of extracellular plaques characteristic of Alzheimers disease. While previous studies commonly show the Aβ(1-40) is largely unstructured in solution, we show that Aβ(1-40) can adopt a compact, partially folded structure. In this structure (PDB ID: 2LFM), the central hydrophobic region of the peptide forms a 3(10) helix from H13 to D23 and the N- and C-termini collapse against the helix due to the clustering of hydrophobic residues. Helical intermediates have been predicted to be crucial on-pathway intermediates in amyloid fibrillogenesis, and the structure presented here presents a new target for investigation of early events in Aβ(1-40) fibrillogenesis.

Small molecule modulators of copper-induced Aβ aggregation

Our design of bifunctional metal chelators as chemical probes and potential therapeutics for Alzheimers disease (AD) is based on the incorporation of a metal binding moiety into structural frameworks of Abeta aggregate-imaging agents. Using this strategy, two compounds 2-[4-(dimethylamino)phenyl]imidazo[1,2-a]pyridine-8-ol (1) and N(1),N(1)-dimethyl-N(4)-(pyridin-2-ylmethylene)benzene-1,4-diamine (2) were prepared and characterized. The bifunctionality for metal chelation and Abeta interaction of 1 and 2 was verified by spectroscopic methods. Furthermore, the reactivity of 1 and 2 with Cu(II)-associated Abeta aggregates was investigated. The modulation of Cu(II)-triggered Abeta aggregation by 1 and 2 was found to be more effective than that by the known metal chelating agents CQ, EDTA, and phen. These studies suggest a new class of multifunctional molecules for the development of chemical tools to unravel metal-associated events in AD and potential therapeutic agents for metal-ion chelation therapy.

Structure and membrane orientation of IAPP in its natively amidated form at physiological pH in a membrane environment.

Human islet amyloid polypeptide is a hormone coexpressed with insulin by pancreatic beta-cells. For reasons not clearly understood, hIAPP aggregates in type II diabetics to form oligomers that interfere with beta-cell function, eventually leading to the loss of insulin production. The cellular membrane catalyzes the formation of amyloid deposits and is a target of amyloid toxicity through disruption of the membranes structural integrity. Therefore, there is considerable current interest in solving the 3D structure of this peptide in a membrane environment. NMR experiments could not be directly utilized in lipid bilayers due to the rapid aggregation of the peptide. To overcome this difficulty, we have solved the structure of the naturally occurring peptide in detergent micelles at a neutral pH. The structure has an overall kinked helix motif, with residues 7-17 and 21-28 in a helical conformation, and with a 3(10) helix from Gly 33-Asn 35. In addition, the angle between the N- and C-terminal helices is constrained to 85°. The greater helical content of human IAPP in the amidated versus free acid form is likely to play a role in its aggregation and membrane disruptive activity.

Role of zinc in human islet amyloid polypeptide aggregation

Human Islet Amyloid Polypeptide (hIAPP) is a highly amyloidogenic protein found in islet cells of patients with type II diabetes. Because hIAPP is highly toxic to beta-cells under certain conditions, it has been proposed that hIAPP is linked to the loss of beta-cells and insulin secretion in type II diabetics. One of the interesting questions surrounding this peptide is how the toxic and aggregation prone hIAPP peptide can be maintained in a safe state at the high concentrations that are found in the secretory granule where it is stored. We show here zinc, which is found at millimolar concentrations in the secretory granule, significantly inhibits hIAPP amyloid fibrillogenesis at concentrations similar to those found in the extracellular environment. Zinc has a dual effect on hIAPP fibrillogenesis: it increases the lag-time for fiber formation and decreases the rate of addition of hIAPP to existing fibers at lower concentrations, while having the opposite effect at higher concentrations. Experiments at an acidic pH which partially neutralizes the change in charge upon zinc binding show inhibition is largely due to an electrostatic effect at His18. High-resolution structures of hIAPP determined from NMR experiments confirm zinc binding to His18 and indicate zinc induces localized disruption of the secondary structure of IAPP in the vicinity of His18 of a putative helical intermediate of IAPP. The inhibition of the formation of aggregated and toxic forms of hIAPP by zinc provides a possible mechanism between the recent discovery of linkage between deleterious mutations in the SLC30A8 zinc transporter, which transports zinc into the secretory granule, and type II diabetes.

Insights into antiamyloidogenic properties of the green tea extract (−)-epigallocatechin-3-gallate toward metal-associated amyloid-β species

Despite the significance of Alzheimer’s disease, the link between metal-associated amyloid-β (metal–Aβ) and disease etiology remains unclear. To elucidate this relationship, chemical tools capable of specifically targeting and modulating metal–Aβ species are necessary, along with a fundamental understanding of their mechanism at the molecular level. Herein, we investigated and compared the interactions and reactivities of the green tea extract, (−)-epigallocatechin-3-gallate [(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro-2H-1-benzopyran-3-yl 3,4,5-trihydroxybenzoate; EGCG], with metal [Cu(II) and Zn(II)]–Aβ and metal-free Aβ species. We found that EGCG interacted with metal–Aβ species and formed small, unstructured Aβ aggregates more noticeably than in metal-free conditions in vitro. In addition, upon incubation with EGCG, the toxicity presented by metal-free Aβ and metal–Aβ was mitigated in living cells. To understand this reactivity at the molecular level, structural insights were obtained by ion mobility-mass spectrometry (IM-MS), 2D NMR spectroscopy, and computational methods. These studies indicated that (i) EGCG was bound to Aβ monomers and dimers, generating more compact peptide conformations than those from EGCG-untreated Aβ species; and (ii) ternary EGCG–metal–Aβ complexes were produced. Thus, we demonstrate the distinct antiamyloidogenic reactivity of EGCG toward metal–Aβ species with a structure-based mechanism.

A Two Site Mechanism for the Inhibition of IAPP Amyloidogenesis by Zinc

Human islet amyloid polypeptide (hIAPP) is a highly amyloidogenic protein co-secreted with insulin in response to glucose levels. The formation of hIAPP amyloid plaques near islet cells has been linked to the death of insulin-secreting β-cells in humans and the progression of type II diabetes. Since both healthy individuals and those with type II diabetes produce and secrete hIAPP, it is reasonable to look for factors involved in storing hIAPP and preventing amyloidosis. We have previously shown that zinc inhibits the formation of insoluble amyloid plaques of hIAPP; however, there remains significant ambiguity in the underlying mechanisms. In this study, we show that zinc binds unaggregated hIAPP at micromolar concentrations similar to those found in the extracellular environment. By contrast, the fibrillar amyloid form of hIAPP has low affinity for zinc. The binding stoichiometry obtained from isothermal titration calorimetry experiments indicates that zinc favors the formation of hIAPP hexamers. High-resolution NMR structures of hIAPP bound to zinc reveal changes in the electron environment along residues that would be located along one face of the amphipathic hIAPP α-helix proposed as an intermediate for amyloid formation. Results from electrospray ionization mass spectroscopy investigations showed that a single zinc atom is predominantly bound to hIAPP and revealed that zinc inhibits the formation of the dimer. At higher concentrations of zinc, a second zinc atom binds to hIAPP, suggesting the presence of a low-affinity secondary binding site. Combined, these results suggest that zinc promotes the formation of oligomers while creating an energetic barrier for the formation of amyloid fibers.

A Model of the Membrane-bound Cytochrome b5-Cytochrome P450 Complex from NMR and Mutagenesis Data

Background: cytb5 modulates catalysis performed by cytsP450, in vivo and in vitro. Results: The structure of full-length cytb5 was solved by NMR, and the cytP450-binding site on cytb5 was identified by mutagenesis and NMR. Conclusion: A model of the cytb5-cytP450 complex is presented. Addition of a substrate strengthens the cytb5-cytP450 interaction. Significance: The cytb5-cytP450 complex structure will help unravel the mechanism by which cytb5 regulates catalysis by cytP450. Microsomal cytochrome b5 (cytb5) is a membrane-bound protein that modulates the catalytic activity of its redox partner, cytochrome P4502B4 (cytP450). Here, we report the first structure of full-length rabbit ferric microsomal cytb5 (16 kDa), incorporated in two different membrane mimetics (detergent micelles and lipid bicelles). Differential line broadening of the cytb5 NMR resonances and site-directed mutagenesis data were used to characterize the cytb5 interaction epitope recognized by ferric microsomal cytP450 (56 kDa). Subsequently, a data-driven docking algorithm, HADDOCK (high ambiguity driven biomolecular docking), was used to generate the structure of the complex between cytP4502B4 and cytb5 using experimentally derived restraints from NMR, mutagenesis, and the double mutant cycle data obtained on the full-length proteins. Our docking and experimental results point to the formation of a dynamic electron transfer complex between the acidic convex surface of cytb5 and the concave basic proximal surface of cytP4502B4. The majority of the binding energy for the complex is provided by interactions between residues on the C-helix and β-bulge of cytP450 and residues at the end of helix α4 of cytb5. The structure of the complex allows us to propose an interprotein electron transfer pathway involving the highly conserved Arg-125 on cytP450 serving as a salt bridge between the heme propionates of cytP450 and cytb5. We have also shown that the addition of a substrate to cytP450 likely strengthens the cytb5-cytP450 interaction. This study paves the way to obtaining valuable structural, functional, and dynamic information on membrane-bound complexes.

Site specific interaction of the polyphenol EGCG with the SEVI amyloid precursor peptide PAP(248-286).

Recently, a 39 amino acid peptide fragment from prostatic acid phosphatase has been isolated from seminal fluid that can enhance infectivity of the HIV virus by up to 4-5 orders of magnitude. PAP(248-286) is effective in enhancing HIV infectivity only when it is aggregated into amyloid fibers termed SEVI. The polyphenol EGCG (epigallocatechin-3-gallate) has been shown to disrupt both SEVI formation and HIV promotion by SEVI, but the mechanism by which it accomplishes this task is unknown. Here, we show that EGCG interacts specifically with the side chains of monomeric PAP(248-286) in two regions (K251-R257 and N269-I277) of primarily charged residues, particularly lysine. The specificity of interaction to these two sites is contrary to previous studies on the interaction of EGCG with other amyloidogenic proteins, which showed the nonspecific interaction of EGCG with exposed backbone sites of unfolded amyloidogenic proteins. This interaction is specific to EGCG as the related gallocatechin (GC) molecule, which shows greatly decreased antiamyloid activity, exhibits minimal interaction with monomeric PAP(248-286). The EGCG binding was shown to occur in two steps, with the initial formation of a weakly bound complex followed by a pH dependent formation of a tightly bound complex. Experiments in which the lysine residues of PAP(248-286) have been chemically modified suggest the tightly bound complex is created by Schiff-base formation with lysine residues. The results of this study could aid in the development of small molecule inhibitors of SEVI and other amyloid proteins.

NMR Structure in a Membrane Environment Reveals Putative Amyloidogenic Regions of the SEVI Precursor Peptide PAP248−286

Semen is the main vector for HIV transmission worldwide. Recently, a peptide fragment (PAP(248-286)) has been isolated from seminal fluid that dramatically enhances HIV infectivity by up to 4-5 orders of magnitude. PAP(248-286) appears to enhance HIV infection by forming amyloid fibers known as SEVI, which are believed to enhance the attachment of the virus by bridging interactions between virion and host-cell membranes. We have solved the atomic-level resolution structure of the SEVI precursor PAP(248-286) using NMR spectroscopy in SDS micelles, which serve as a model membrane system. PAP(248-286), which does not disrupt membranes like most amyloid proteins, binds superficially to the surface of the micelle, in contrast to other membrane-disruptive amyloid peptides that generally penetrate into the core of the membrane. The structure of PAP(248-286) is unlike most amyloid peptides in that PAP(248-286) is mostly disordered when bound to the surface of the micelle, as opposed to the alpha-helical structures typically found of most amyloid proteins. The highly disordered nature of the SEVI peptide may explain the unique ability of SEVI amyloid fibers to enhance HIV infection as partially disordered amyloid fibers will have a greater capture radius for the virus than compact amyloid fibers. Two regions of nascent structure (an alpha-helix from V262-H270 and a dynamic alpha/3(10) helix from S279-L283) match the prediction of highly amyloidogenic sequences and may serve as nuclei for aggregation and amyloid fibril formation. The structure presented here can be used for the rational design of mutagenesis studies on SEVI amyloid formation and viral infection enhancement.

Zinc stabilization of prefibrillar oligomers of human islet amyloid polypeptide

The aggregation of human islet amyloid polypeptide (hIAPP) has been linked to beta-cell death in type II diabetes. Zinc present in secretory granules has been shown to affect this aggregation. A combination of EXAFS, NMR, and AFM experiments shows that the influence of zinc is most likely due to the stabilization of prefibrillar aggregates of hIAPP.