The N-Terminal Amphipathic Helix of the Topological Specificity Factor MinE Is Associated with Shaping Membrane Curvature

Pole-to-pole oscillations of the Min proteins in Escherichia coli are required for the proper placement of the division septum. Direct interaction of MinE with the cell membrane is critical for the dynamic behavior of the Min system. In vitro, this MinE-membrane interaction led to membrane deformation; however, the underlying mechanism remained unclear. Here we report that MinE-induced membrane deformation involves the formation of an amphipathic helix of MinE2–9, which, together with the adjacent basic residues, function as membrane anchors. Biochemical evidence suggested that the membrane association induces formation of the helix, with the helical face, consisting of A2, L3, and F6, inserted into the membrane. Insertion of this helix into the cell membrane can influence local membrane curvature and lead to drastic changes in membrane topology. Accordingly, MinE showed characteristic features of protein-induced membrane tubulation and lipid clustering in in vitro reconstituted systems. In conclusion, MinE shares common protein signatures with a group of membrane trafficking proteins in eukaryotic cells. These MinE signatures appear to affect membrane curvature

Atomic Force Microscopy Characterization of Protein Fibrils Formed by the Amyloidogenic Region of the Bacterial Protein MinE on Mica and a Supported Lipid Bilayer

<div><p>Amyloid fibrils play a crucial role in many human diseases and are found to function in a range of physiological processes from bacteria to human. They have also been gaining importance in nanotechnology applications. Understanding the mechanisms behind amyloid formation can help develop strategies towards the prevention of fibrillation processes or create new technological applications. It is thus essential to observe the structures of amyloids and their self-assembly processes at the nanometer-scale resolution under physiological conditions. In this work, we used highly force-sensitive frequency-modulation atomic force microscopy (FM-AFM) to characterize the fibril structures formed by the N-terminal domain of a bacterial division protein MinE in solution. The approach enables us to investigate the fibril morphology and protofibril organization over time progression and in response to changes in ionic strength, molecular crowding, and upon association with different substrate surfaces. In addition to comparison of the fibril structure and behavior of MinE^1-31 under varying conditions, the study also broadens our understanding of the versatile behavior of amyloid-substrate surface interactions.</p></div

Organization of protein domains in MinE and model of the cross-β structure formed by the amyloidogenic region of MinE.

<p><b>(A)</b> The MinE protein can be divided into three functional domains, a membrane-binding domain that contains a membrane-induced amphipathic helix and basic residues, a bifunctional domain that interacts with MinD in an α-helical conformation and self-assembles in a β-stranded conformation, and a dimerization domain at the C-terminus. The dimerization domain is also known as the topological specificity domain. <b>(B)</b> Illustration of the cross-β structure formed by the amyloidogenic region of MinE (19–28); the alternating β strands are colored green and yellow for clarity. <b>(C)</b> RMSD plots of α-carbon and main-chain atoms from a 5-ns simulation to demonstrate conformational equilibrium. <b>(D)</b> Frontal view of the cross-β structure of the amyloidogenic region of MinE^1-31; only the backbone of the molecule and the side chains facing the hydrophobic interface are shown. <b>(E)</b> Top view of the model showing anti-parallel arrangements of the residues in the amyloidogenic region; residues containing side chains facing the hydrophobic interface of two β sheets are shown in red.</p

Self-assembly of MinE^1-31 on mica under different buffer conditions.

<p>AFM images of MinE^1-31 self-assembled into straight <b>(A</b> and <b>D)</b>, bent <b>(B</b> and <b>E)</b> and highly curved fibrils <b>(C</b> and <b>F)</b> in imaging buffers A, B and C, respectively. Graphs <b>(G–I)</b> show the height profiles corresponding to the green lines in images (D), (E) and (F). The four line profiles (I1–4) associated with (F) show alternating variations in the height (h_c = 3.5 ± 0.3 nm, <i>n</i> = 34; h_c' = 4.7 ± 0.3 nm, <i>n</i> = 32; green double arrows) along the curved fibrillar structure. White arrows in (D) and (E) indicate where protofibrils are visible. (E) High resolution image of the outlined square region in (B). Purple arrow indicates newly growing fibril. It should be noted that the feature of double fibrils in (E) is not a result of the double-tip artifact, because triple fibrils are also observed, as indicated by yellow arrows. Red double arrows in (G, H, I1 and 3) indicate the separation distance between protofibrils (D) and fibrils (E and F). h_c and h_c' denote the height of fibrils in imaging buffer C. The peptide concentration used in imaging buffers A, B and C, was 24, 41, and 18 μM, respectively.</p

Self-assembly of MinE^1-31 on SLBs.

<p><b>(A–C)</b> Assembly of MinE^1-31 on a high-coverage area of the SLBs. The peptide concentration was 24 μM. <b>(E–G)</b> Assembly of MinE^1-31 on broken SLBs. The time for each set of data is indicated at the upper-right hand corner of the images. Notice that (G) was taken before (E) and (F). Newly assembled fibrils can be observed between (A) and (B) and between (E) and (F). The peptide concentration used was 12 μM. Graphs (B) and (C) and (F) and (G) are images of (A) and (B) at higher resolution, respectively. Graphs (D) and (H) show the height profiles corresponding to the green lines in images (C) and (G). Experiments were performed in imaging buffer A. Red double arrows indicate the separation distance between protofibrils. Green double arrow indicates the separation distance between fibrils. Purple arrows indicate the newly formed fibrils comparing to the previous images. Blue line indicates the thickness of supported lipid bilayer.</p

Growth of MinE^1-31 fibrils on mica.

<p><b>(A–D)</b> Elongation of straight fibrils in imaging buffer A. The peptide concentration was 12 μM. <b>(E–H)</b> Elongation of highly curved fibrils in imaging buffer C. The peptide concentration was 18 μM. The time interval in an image sequence depended on the scanning speed, which was determined by the instrument configuration and the size of the region of interest. Only selected images in each time sequence are shown. Red arrows indicate the direction of fibril elongation; yellow arrows indicate annealing between fibrils; white arrow indicates protofibrils growing along the existing fibrils; green arrow indicates a node-like region for branch formation. Occasionally we can see growth from single fibrils to paired fibrils. One is indicated by a purple arrow in (F) and (G).</p

Trans-ethnic fine mapping identifies a novel independent locus at the 3' end of CDKAL1 and novel variants of several susceptibility loci for type 2 diabetes in a Han Chinese population.

Aims/hypothesisCandidate gene and genome-wide association studies have identified ∼60 susceptibility loci for type 2 diabetes. A majority of these loci have been discovered and tested only in European populations. The aim of this study was to assess the presence and extent of trans-ethnic effects of these loci in an East Asian population.MethodsA total of 9,335 unrelated Chinese Han individuals, including 4,535 with type 2 diabetes and 4,800 non-diabetic ethnically matched controls, were genotyped using the Illumina 200K Metabochip. We tested 50 established loci for type 2 diabetes and related traits (fasting glucose, fasting insulin, 2 h glucose). Disease association with the additive model of inheritance was analysed with logistic regression.ResultsWe found that 14 loci significantly transferred to the Chinese population, with two loci (p = 5.7 × 10(-12) for KCNQ1; p = 5.0 × 10(-8) for CDKN2A/B-CDKN2BAS) reaching independent genome-wide statistical significance. Five of these 14 loci had similar lead single-nucleotide polymorphisms (SNPs) as were found in the European studies while the other nine were different. Further stepwise conditional analysis identified a total of seven secondary signals and an independent novel locus at the 3' end of CDKAL1.Conclusions/interpretationThese results suggest that many loci associated with type 2 diabetes are commonly shared between European and Chinese populations. Identification of population-specific SNPs may increase our understanding of the genetic architecture underlying type 2 diabetes in different ethnic populations

Many-Body Effect of Antimicrobial Peptides: On the Correlation Between Lipid's Spontaneous Curvature and Pore Formation

Recently we have shown that the free energy for pore formation induced by antimicrobial peptides contains a term representing peptide-peptide interactions mediated by membrane thinning. This many-body effect gives rise to the cooperative concentration dependence of peptide activities. Here we performed oriented circular dichroism and x-ray diffraction experiments to study the lipid dependence of this many-body effect. In particular we studied the correlation between lipid's spontaneous curvature and peptide's threshold concentration for pore formation by adding phosphatidylethanolamine and lysophosphocholine to phosphocholine bilayers. Previously it was argued that this correlation exhibited by magainin and melittin supported the toroidal model for the pores. Here we found similar correlations exhibited by melittin and alamethicin. We found that the main effect of varying the spontaneous curvature of lipid is to change the degree of membrane thinning, which in turn influences the threshold concentration for pore formation. We discuss how to interpret the lipid dependence of membrane thinning