Alexey B. Mantsyzov

Interplay of histidine residues of the Alzheimer's disease Aβ peptide governs its Zn-induced oligomerization.

Conformational changes of Aβ peptide result in its transformation from native monomeric state to the toxic soluble dimers, oligomers and insoluble aggregates that are hallmarks of Alzheimer’s disease (AD). Interactions of zinc ions with Aβ are mediated by the N-terminal Aβ1–16 domain and appear to play a key role in AD progression. There is a range of results indicating that these interactions trigger the Aβ plaque formation. We have determined structure and functional characteristics of the metal binding domains derived from several Aβ variants and found that their zinc-induced oligomerization is governed by conformational changes in the minimal zinc binding site 6HDSGYEVHH14. The residue H6 and segment 11EVHH14, which are part of this site are crucial for formation of the two zinc-mediated interaction interfaces in Aβ. These structural determinants can be considered as promising targets for rational design of the AD-modifying drugs aimed at blocking pathological Aβ aggregation.

NMR solution structure and function of the C-terminal domain of eukaryotic class 1 polypeptide chain release factor

Termination of translation in eukaryotes is triggered by two polypeptide chain release factors, eukaryotic class 1 polypeptide chain release factor (eRF1) and eukaryotic class 2 polypeptide chain release factor 3. eRF1 is a three‐domain protein that interacts with eukaryotic class 2 polypeptide chain release factor 3 via its C‐terminal domain (C‐domain). The high‐resolution NMR structure of the human C‐domain (residues 277–437) has been determined in solution. The overall fold and the structure of the β‐strand core of the protein in solution are similar to those found in the crystal structure. The structure of the minidomain (residues 329–372), which was ill‐defined in the crystal structure, has been determined in solution. The protein backbone dynamics, studied using 15N‐relaxation experiments, showed that the C‐terminal tail 414–437 and the minidomain are the most flexible parts of the human C‐domain. The minidomain exists in solution in two conformational states, slowly interconverting on the NMR timescale. Superposition of this NMR solution structure of the human C‐domain onto the available crystal structure of full‐length human eRF1 shows that the minidomain is close to the stop codon‐recognizing N‐terminal domain. Mutations in the tip of the minidomain were found to affect the stop codon specificity of the factor. The results provide new insights into the possible role of the C‐domain in the process of translation termination.

Zinc-induced heterodimer formation between metal-binding domains of intact and naturally modified amyloid-beta species: implication to amyloid seeding in Alzheimer’s disease?

Zinc ions and modified amyloid-beta peptides (Aβ) play a critical role in the pathological aggregation of endogenous Aβ in Alzheimer’s disease (AD). Zinc-induced Aβ oligomerization is mediated by the metal-binding domain (MBD) which includes N-terminal residues 1–16 (Aβ1–16). Earlier, it has been shown that Aβ1–16 as well as some of its naturally occurring variants undergoes zinc-induced homodimerization via the interface in which zinc ion is coordinated by Glu11 and His14 of the interacting subunits. In this study using surface plasmon resonance technique, we have found that in the presence of zinc ions Aβ1–16 forms heterodimers with MBDs of two Aβ species linked to AD: Aβ containing isoAsp7 (isoAβ) and Aβ containing phosphorylated Ser8 (pS8-Aβ). The heterodimers appear to possess the same interface as the homodimers. Simulation of 200 ns molecular dynamic trajectories in two constructed models of dimers ([Aβ1–16/Zn/Aβ1–16] and [isoAβ1–16/Zn/Aβ1–16]), has shown that conformational flexibility of the N-terminal fragments of the dimer subunits is controlled by the structure of corresponding sites 6–8. The data suggest that isoAβ and pS8-Aβ can be involved in the AD pathogenesis by means of their zinc-dependent interactions with endogenous Aβ resulting in the formation of heterodimeric seeds for amyloid aggregation.

Optimization of the Methods for Small Peptide Solution Structure Determination by NMR Spectroscopy

NMR spectroscopy was recognized as a method of protein structure determination in solution. However, determination of the conformation of small peptides, which undergo fast molecular motions, remains a challenge. This is mainly caused by the impossibility to collect the required quantity of the distance and dihedral angle restraints from NMR spectra. At the same time, short charged peptides play an important role in a number of biological processes, in particular in pathogenesis of neurodegenerative diseases including Alzheimer’s disease. Therefore, development of a method for structure simulation of small peptides in aqueous environment using the most realistic force fields seems to be of current importance. Such algorithm has been developed using the Amber-03 force field and Gromacs program after modification of its code. Calculation algorithm has been verified on a model peptide with a known solution structure and a metal-binding fragment of rat β-amyloid, whose structure has been determined by alternative methods. The developed algorithm substantially increases structure quality, in particular Ramachandran plot statistics, and decreases RMSD of atomic coordinates inside the calculated family. The described protocol can be used for determination of conformation of short peptides, and also for optimization of structure of larger proteins containing poorly structured fragments.

Structure and function of the N-terminal domain of the yeast telomerase reverse transcriptase

Abstract The elongation of single-stranded DNA repeats at the 3′-ends of chromosomes by telomerase is a key process in maintaining genome integrity in eukaryotes. Abnormal activation of telomerase leads to uncontrolled cell division, whereas its down-regulation is attributed to ageing and several pathologies related to early cell death. Telomerase function is based on the dynamic interactions of its catalytic subunit (TERT) with nucleic acids—telomerase RNA, telomeric DNA and the DNA/RNA heteroduplex. Here, we present the crystallographic and NMR structures of the N-terminal (TEN) domain of TERT from the thermotolerant yeast Hansenula polymorpha and demonstrate the structural conservation of the core motif in evolutionarily divergent organisms. We identify the TEN residues that are involved in interactions with the telomerase RNA and in the recognition of the ‘fork’ at the distal end of the DNA product/RNA template heteroduplex. We propose that the TEN domain assists telomerase biological function and is involved in restricting the size of the heteroduplex during telomere repeat synthesis.

N-domain of angiotensin-converting enzyme hydrolyzes human and rat amyloid-β(1-16) peptides as arginine specific endopeptidase potentially enhancing risk of Alzheimer’s disease

Alzheimer’s disease (AD) is a multifactorial neurodegenerative disorder. Amyloid-β (Aβ) aggregation is likely to be the major cause of AD. In contrast to humans and other mammals, that share the same Aβ sequence, rats and mice are invulnerable to AD-like neurodegenerative pathologies, and Aβ of these rodents (ratAβ) has three amino acid substitutions in the metal-binding domain 1-16 (MBD). Angiotensin-converting enzyme (ACE) cleaves Aβ-derived peptide substrates, however, there are contradictions concerning the localization of the cleavage sites within Aβ and the roles of each of the two ACE catalytically active domains in the hydrolysis. In the current study by using mass spectrometry and molecular modelling we have tested a set of peptides corresponding to MBDs of Aβ and ratAβ to get insights on the interactions between ACE and these Aβ species. It has been shown that the N-domain of ACE (N-ACE) acts as an arginine specific endopeptidase on the Aβ and ratAβ MBDs with C-amidated termini, thus assuming that full-length Aβ and ratAβ can be hydrolyzed by N-ACE in the same endopeptidase mode. Taken together with the recent data on the molecular mechanism of zinc-dependent oligomerization of Aβ, our results suggest a modulating role of N-ACE in AD pathogenesis.

Control of Azomethine Cycloaddition Stereochemistry by CF3 Group: Structural Diversity of Fluorinated β-Proline Dimers

β-Proline-functionalized dimers consisting of homochiral monomeric units were synthesized by a non-peptidic coupling method for the first time. The applied synthetic methodology is based on 1,3-dipolar cycloaddition chemistry of azomethine ylides and provides absolute control over the β-proline backbone stereogenic centers. An o-(trifluoromethyl)phenyl substituent contributes to appropriate stabilization of the definite acrylamide chiral cis conformation and to achieve the dipole reactivity that is not observed for aryl groups lacking strong electronegative character.

Theoretical and NMR Conformational Studies of β-Proline Oligopeptides With Alternating Chirality of Pyrrolidine Units

Synthetic β-peptides are potential functional mimetics of native α-proteins. A recently developed, novel, synthetic approach provides an effective route to the broad group of β-proline oligomers with alternating patterns of stereogenic centers. Conformation of the pyrrolidine ring, Z/E isomerism of β-peptide bonds, and hindered rotation of the neighboring monomers determine the spatial structure of this group of β-proline oligopeptides. Preferences in their structural organization and corresponding thermodynamic properties are determined by NMR spectroscopy, restrained molecular dynamics and quantum mechanics. The studied β-proline oligopeptides exist in dimethyl sulfoxide solution in a limited number of conformers, with compatible energy of formation and different spatial organization. In the β-proline tetrapeptide with alternating chirality of composing pyrrolidine units, one of three peptide bonds may exist in an E configuration. For the alternating β-proline pentapeptide, the presence of an E configuration for at least of one β-peptide bond is mandatory. In this case, three peptide bonds synchronously change their configurations. Larger polypeptides may only exist in the presence of several E configurations of β-peptide bonds forming a wave-like extended structure.