Katrine Kirkeby Skeby

The influence of cholesterol on membrane protein structure, function, and dynamics studied by molecular dynamics simulations.

The plasma membrane, which encapsulates human cells, is composed of a complex mixture of lipids and embedded proteins. Emerging knowledge points towards the lipids as having a regulating role in protein function. Furthermore, insight from protein crystallography has revealed several different types of lipids intimately bound to membrane proteins and peptides, hereby possibly pointing to a site of action for the observed regulation. Cholesterol is among the lipid membrane constituents most often observed to be co-crystallized with membrane proteins, and the cholesterol levels in cell membranes have been found to play an essential role in health and disease. Remarkably little is known about the mechanism of lipid regulation of membrane protein function in health as well as in disease. Herein, we review molecular dynamics simulation studies aimed at investigating the effect of cholesterol on membrane protein and peptide properties. This article is part of a Special Issue entitled: Lipid-protein interactions.

Identification of a common binding mode for imaging agents to amyloid fibrils from molecular dynamics simulations.

Amyloid diseases are characterized by the misfolding and deposition of proteins in the body in the form of insoluble amyloid fibrils. Alzheimers disease and type 2 diabetes mellitus are two examples of amyloid diseases which are closely related both with respect to the atomic structures of the amyloid fibrils and the disease pathology. Alzheimers disease is very difficult to diagnose, and much research is being performed to develop noninvasive diagnostic methods, such as imaging with small-molecule agents. The interactions between amyloid fibrils and imaging agents are challenging to examine experimentally due to the insoluble nature of amyloid fibrils. This study uses molecular dynamics simulations to investigate the interactions between 13 aromatic amyloid imaging agents, entailing 4 different organic scaffolds, and a model of an amyloid fibril. Clustering analysis combined with free energy calculations are used to categorize and rank the resulting complexes. Several binding modes are identified across the different ligand scaffolds, however a common favorable binding mode can be identified in which the agent is placed in surface grooves along the amyloid fibril axis. The existence of multiple binding modes for imaging agents is proposed to originate from subtle differences in amino acid composition of the surface grooves on an amyloid fibril, resulting in fine tuning of the binding affinities for a specific amyloid fibril.

The importance of being capped: Terminal capping of an amyloidogenic peptide affects fibrillation propensity and fibril morphology.

The formation of aggregated fibrillar β-sheet structures has been proposed to be a generic feature of proteins. Aggregation propensity is highly sequence dependent, and often only part of the protein is incorporated into the fibril core. Therefore, shorter peptide fragments corresponding to the fibril core are attractive fibrillation models. The use of peptide models introduces new termini into the fibrils, yet little attention has been paid to the role these termini may play in fibrillation. Here, we report that terminal modifications of a 10-residue peptide fragment of human islet amyloid polypeptide strongly affect fibrillation kinetics and the resulting fibril morphology. Capping of the N-terminus abolishes fibrillation, while C-terminal capping results in fibrils with a twisted morphology. Peptides with either both termini free or both termini capped form flat fibrils. Molecular dynamics simulations reveal that the N-terminal acetyl cap folds up and interacts with the peptides hydrophobic side chains, while the uncapped N-terminus in the C-terminally capped version results in twisting of the fibrils due to charge repulsion from the free N-termini. Our results highlight the role of terminal interactions in fibrillation of small peptides and provide molecular insight into the consequences of C-terminal modifications frequently found in peptide hormones in vivo.

Conformational Dynamics of the Human Islet Amyloid Polypeptide in a Membrane Environment: Toward the Aggregation Prone Form

Human islet amyloid polypeptide (hIAPP) is a 37-residue peptide hormone, which upon misfolding changes from the physiologically active monomer into pathological amyloid fibril aggregates in the pancreas of type 2 diabetes mellitus patients. During this process, the insulin-producing pancreatic β-cells are damaged; however, the underlying mechanism of this mode of cytotoxicity remains elusive. It is known that anionic lipids accelerate amyloid fibril formation, implicating the importance of the cellular membrane in the process, and that a pH close to the level in the β-cell secretory granules (pH 5.5) inhibits amyloid fibril formation. Using all-atom molecular dynamics simulations, we have investigated the membrane-associated monomer state of α-helical hIAPP, analyzed specific interactions of hIAPP with a mixed anionic-zwitterionic lipid membrane and examined the influence of pH on the structure and dynamics of hIAPP and its interaction with the membrane. We find that hIAPP primarily interacts with the membrane by forming favorable interactions between anionic lipids and the positively charged residues in the N-terminal part of the peptide. Rationalizing experimental findings, the simulations show that the N-terminal part of the peptide interacts with the membrane in the lipid headgroup region. At neutral pH, the C-terminal part of the peptide, which contains the residues that initiate fibril formation, displays a highly dynamic, unfolded state, which interacts with the membrane significantly less than the N-terminal part. Such an unfolded form can be proposed to contribute to the acceleration of fibril formation. At low pH, protonation of His18 mediates a stronger interaction of the C-terminal part with the membrane, resulting in the immobilization of the C-terminal part on the membrane surface that might constitute a mechanism by which low pH inhibits fibril formation.

Identification of Key Interactions in the Initial Self-Assembly of Amylin in a Membrane Environment

Islet amyloid polypeptide, also known as amylin, forms aggregates that reduce the amount of insulin-producing cells in patients with type II diabetes mellitus. Much remains unknown about the process of aggregation and cytotoxicity, but it is known that certain cell membrane components can alter the rate of aggregation. Using atomistic molecular dynamics simulations combined with the highly mobile membrane mimetic model incorporating enhanced sampling of lipid diffusion, we investigate interaction of amylin peptides with the membrane components as well as the self-assembly of amylin. Consistent with experimental evidence, we find that an initial membrane-bound α-helical state folds into stable β-sheet structures upon self-assembly. Our results suggest the following mechanism for the initial phase of amylin self-assembly. The peptides move around on the membrane with the positively charged N-terminus interacting with the negatively charged lipid headgroups. When the peptides start to interact, they partly unfold and break some of the contacts with the membrane. The initial interactions between the peptides are dominated by aromatic and hydrophobic interactions. Oligomers are formed showing both intra- and interpeptide β-sheets, initially with interactions mainly in the C-terminal domain of the peptides. Decreasing the pH to 5.5 is known to inhibit amyloid formation. At low pH, His18 is protonated, adding a fourth positive charge at the peptide. With His18 protonated, no oligomerization is observed in the simulations. The additional charge gives a strong midpoint anchoring of the peptides to negatively charged membrane components, and the peptides experience additional interpeptide repulsion, thereby preventing interactions.

Structural Basis for Simvastatin Competitive Antagonism of Complement Receptor 3

The complement system is an important part of the innate immune response to infection but may also cause severe complications during inflammation. Small molecule antagonists to complement receptor 3 (CR3) have been widely sought, but a structural basis for their mode of action is not available. We report here on the structure of the human CR3 ligand-binding I domain in complex with simvastatin. Simvastatin targets the metal ion-dependent adhesion site of the open, ligand-binding conformation of the CR3 I domain by direct contact with the chelated Mg2+ ion. Simvastatin antagonizes I domain binding to the complement fragments iC3b and C3d but not to intercellular adhesion molecule-1. By virtue of the I domains wide distribution in binding kinetics to ligands, it was possible to identify ligand binding kinetics as discriminator for simvastatin antagonism. In static cellular experiments, 15–25 μm simvastatin reduced adhesion by K562 cells expressing recombinant CR3 and by primary human monocytes, with an endogenous expression of this receptor. Application of force to adhering monocytes potentiated the effects of simvastatin where only a 50–100 nm concentration of the drug reduced the adhesion by 20–40% compared with untreated cells. The ability of simvastatin to target CR3 in its ligand binding-activated conformation is a novel mechanism to explain the known anti-inflammatory effects of this compound, in particular because this CR3 conformation is found in pro-inflammatory environments. Our report points to new designs of CR3 antagonists and opens new perspectives and identifies druggable receptors from characterization of the ligand binding kinetics in the presence of antagonists.

Amyloid and Amyloid Fibrils

When proteins do not fold correctly, it can lead to very serious diseases. One such group of diseases is the amyloid diseases, of which Alzheimer’s disease (AD), Parkinson’s disease, and type 2 diabetes mellitus (T2DM) are members. The amyloid diseases are characterized by the aggregation of a specific protein into amyloid fibrils. During this process, a cytotoxic event occurs which can be a serious actor in the evolvement of the disease. This thesis is concerned with elucidating the biological processes concerning amyloid proteins, more specifically, the peptide hormone human islet amyloid polypeptide (hIAPP), which is involved in glucose homeostasis and deposits in the pancreas of T2DM patients.

Coarse Grained Study of Amyloid Protofibril Aggregation

This study is described in the manuscript entitled “Protofibrillar Assembly Toward the Formation of Amyloid Fibrils”. The study was mainly conducted by Jesper Sorensen.

Effect of Terminal Capping on Aggregation of Peptide Fragments

This section summarizes and discusses the results from the submitted manuscript entitled “The importance of being capped: Terminal capping of an amyloidogenic peptide affects fibril formation propensity and fibril morphology”.

Determining the Aggregation Prone Structure of hIAPP

This section presents the results described in the manuscript entitled “Conformational Dynamics of the Human Islet Amyloid Polypeptide in a Membrane Environment: Toward the Aggregation Prone Form”. The study was initiated during my stay at the University of Illinois, Urbana-Champaign, and was continued after my return to Aarhus.