Side-chain to main-chain hydrogen bonding controls the intrinsic backbone dynamics of the amyloid precursor protein transmembrane helix.

Many transmembrane helices contain serine and/or threonine residues whose side chains form intrahelical H-bonds with upstream carbonyl oxygens. Here, we investigated the impact of threonine side-chain/main-chain backbonding on the backbone dynamics of the amyloid precursor protein transmembrane helix. This helix consists of a N-terminal dimerization region and a C-terminal cleavage region, which is processed by γ-secretase to a series of products. Threonine mutations within this transmembrane helix are known to alter the cleavage pattern, which can lead to early-onset Alzheimer's disease. Circular dichroism spectroscopy and amide exchange experiments of synthetic transmembrane domain peptides reveal that mutating threonine enhances the flexibility of this helix. Molecular dynamics simulations show that the mutations reduce intrahelical amide H-bonding and H-bond lifetimes. In addition, the removal of side-chain/main-chain backbonding distorts the helix, which alters bending and rotation at a diglycine hinge connecting the dimerization and cleavage regions. We propose that the backbone dynamics of the substrate profoundly affects the way by which the substrate is presented to the catalytic site within the enzyme. Changing this conformational flexibility may thus change the pattern of proteolytic processing.

The backbone dynamics of the amyloid precursor protein transmembrane helix provides a rationale for the sequential cleavage mechanism of γ-secretase.

The etiology of Alzheimer's disease depends on the relative abundance of different amyloid-ß (Aß) peptide species. These peptides are produced by sequential proteolytic cleavage within the transmembrane helix of the 99 residue C-terminal fragment of the amyloid precursor protein (C99) by the intramembrane protease γ-secretase. Intramembrane proteolysis is thought to require local unfolding of the substrate helix, which has been proposed to be cleaved as a homodimer. Here, we investigated the backbone dynamics of the substrate helix. Amide exchange experiments of monomeric recombinant C99 and of synthetic transmembrane domain peptides reveal that the N-terminal Gly-rich homodimerization domain exchanges much faster than the C-terminal cleavage region. MD simulations corroborate the differential backbone dynamics, indicate a bending motion at a diglycine motif connecting dimerization and cleavage regions, and detect significantly different H-bond stabilities at the initial cleavage sites. Our results are consistent with the following hypotheses about cleavage of the substrate: First, the GlyGly hinge may precisely position the substrate within γ-secretase such that its catalytic center must start proteolysis at the known initial cleavage sites. Second, the ratio of cleavage products formed by subsequent sequential proteolysis could be influenced by differential extents of solvation and by the stabilities of H-bonds at alternate initial sites. Third, the flexibility of the Gly-rich domain may facilitate substrate movement within the enzyme during sequential proteolysis. Fourth, dimerization may affect substrate processing by decreasing the dynamics of the dimerization region and by increasing that of the C-terminal part of the cleavage region.

Structural diversity in the atomic resolution 3D fingerprint of the titin M-band segment.

In striated muscles, molecular filaments are largely composed of long protein chains with extensive arrays of identically folded domains, referred to as "beads-on-a-string". It remains a largely unresolved question how these domains have developed a unique molecular profile such that each carries out a distinct function without false-positive readout. This study focuses on the M-band segment of the sarcomeric protein titin, which comprises ten identically folded immunoglobulin domains. Comparative analysis of high-resolution structures of six of these domains ‒ M1, M3, M4, M5, M7, and M10 ‒ reveals considerable structural diversity within three distinct loops and a non-conserved pattern of exposed cysteines. Our data allow to structurally interpreting distinct pathological readouts that result from titinopathy-associated variants. Our findings support general principles that could be used to identify individual structural/functional profiles of hundreds of identically folded protein domains within the sarcomere and other densely crowded cellular environments.

Novel Flp pilus biogenesis-dependent natural transformation.

Natural transformation has been described in bacterial species spread through nearly all major taxonomic groups. However, the current understanding of the structural components and the regulation of competence development is derived from only a few model organisms. Although natural transformation was discovered in members of the Actinobacteria (high GC Gram-positive bacteria) more than four decades ago, the structural components or the regulation of the competence system have not been studied in any representative of the entire phylum. In this report we identify a new role for a distinct type of pilus biogenesis genes (tad genes, for tight adherence), which so far have been connected only with biofilm formation, adherence and virulence traits. The tad-like genes found in the genome of Micrococcus luteus were shown to be required for genetic transformation in this actinobacterial species. We generated and analyzed individual knockout mutants for every open reading frame of the two predicted tad gene clusters as well as for a potential prepilin processing peptidase and identified the major component of the putative pili. By expressing a tagged variant of the major prepilin subunit and immunofluorescence microscopy we visualized filamentous structures extending from the cell surface. Our data indicate that the two tad gene islands complementarily contribute to the formation of a functional competence pilus in this organism. It seems likely that the involvement of tad genes in natural transformation is not unique only for M. luteus but may also prove to be the case in other representatives of the Actinobacteria, which contains important medically and biotechnologically relevant species.