Fig. 1: CROSSTALK AT THE BORDER. Four different functionally described systems with a protein:membrane interplay and acknowledged links to disease have been selected. (Left) SN is an IDP, but can adapt to a structured state (amphipathic helix) in contact with membranes. Recent (unpublished) research reveals how membrane vicinity modulates the folding landscape of SN and changes the aggregation propensity. SN is believed to sense membrane curvature and engages in exocytosis. (Middle left) Activation of FVII into FVIIa is mediated by TF, but also dependent on the presence of phosphatidyl serine in the lipid membrane. The exact mechanism including the required microscopic stoichiometric conditions in terms of FVII/FVIIa, TF and PS are not understood. (Middle right) GHR forms a homo dimer with contacts in the single-pass membrane helices and with a recently described membrane interaction domain in the ICD (Haxholm et al., Biochem J, 2015). The cooperation between these interactions and signaling regulation is unknown. (Right) NHE1 is a 12 TM protein with a long C-terminal ICD displaying extensive intrinsic disorder. NHE1 interacts with multiple anionic membrane lipids, resides partially in lipid rafts/caveolae, and is thought to be regulated by membrane cholesterol. In red are shown phosphatidyl serines (PS) and in orange cholesterol.

Research topics in SYNERGY

The majority of drugs approved by the FDA target membrane proteins. These are easily accessible and are thus highly efficient drug targets. Unfortunately, discoveries of novel drug targets have virtually stalled. Here, we suggest a conceptual expansion of the definition of a drug target, recognizing that the lipid membrane and its protein constituents comprise an integrated, highly dynamic and complex environment, where membrane proteins and lipids cannot be considered independent entities. We argue that they engage in synergistic reciprocal CO-STRUCTURES via conventional or FUZZY interactions. This emerging concept in molecular recognition  - denoted FUZZINESS - evolves from the discovery that many complexes contain highly dynamic functional hotspots. A structural ambiguity is thus necessary for functionality and involves conformational equilibria and/or flexibility of the binding interface. These properties render fuzzy interactions fundamentally essential to the description of protein-membrane co-structures, yet they are only rudimentarily understood.

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The ultimate goal of SYNERGY is to enable rational design of therapeutics (small- or macromolecular) targeting the interactions between a given protein and the lipid bilayer environment. 

SYNERGY

THE IDEA is to explore the synergistic protein-membrane co-structural interplay including fuzziness as a new avenue for treatment of disease. We propose a unique experimental platform with a highly interdisciplinary focus on structure, function and dynamics, in vitro and in a cellular context. We will investigate this virtually unexplored area of macro-molecular relationships using 4 disease-relevant membrane proteins chosen to represent a spectrum of known lipid bilayer interactions.

HYPOTHESES

1) Protein:membrane synergy is deterministic for function
2) Fuzziness is functionally relevant in protein:membrane interactions
3) Protein:membrane interactions can be therapeutically targeted 

SYSTEMS

Due to the heterogeneous environment of the membrane and the diversity of possible protein:membrane interactions, functionally well-described, disease-relevant systems representing an array of different and variable protein:membrane contacts have been selected for study (Fig 1) as described in the following.

Membrane attachment and penetration: Parkinson's Disease (PD) is characterized by dopaminergic neurodegeneration and the presence of Lewy bodies whose major constituent is fibrillated α-synuclein (αSN). αSN is involved in synaptic exocytosis via SNARE protein complexes and is a suggested membrane curvature sensor but neither the interplay between the membrane and the many αSN structures, nor the influence of the lipid composition is currently described. In vivo cytotoxicity of pre-fibrillar αSN species acts via membrane-destabilizing protein:membrane interactions, to influence exocytosis in a poorly understood manner and a potential toxic form of αSN has been suggested to cause membrane rupture, pore formation, or membrane structural modulations. Our data reveal that a pre-fibrillar form of αSN interacts with model membranes forming protein:lipid co-aggregates (Fig 1). We will test the hypothesis that different co-structures of lipids and αSN play different roles in disease and under normal conditions. In this study we aim to understand the fuzzy αSN:membrane co-structural alterations leading to cytotoxic events and neuronal infections, essential for future rational drug design strategies.

Higher order membrane anchored complexes: Recombinant forms of activated factor VIIa (FVIIa) are central to blood coagulation, and approved as treatment for haemophilia. The tissue Factor (TF)/FVIIa complex formation relies on interplay with Ca2+ and POPS lipids for recruitment and interactions. Despite a wealth of structural information on soluble forms of TF and FVII, physiologically relevant structural information about the two complexes at the membrane remains scarce. We hypothesize that different lipids control the formation of higher order structures involving TF and hence influence the blood-clotting function. To investigate this hypothesis the nanodisc-platform with its control of the lipid environment will be employed. TF/FVIIa and TF/FVIIa/FXa/TFPI are investigated with scientists from Novo Nordisk A/S who have extensive system expertise. The ultimate goal is to understand the structure of TF/FVIIa- and TF/FVIIa/FXa/TFPI complexes and their interaction with the plasma membrane.

Single-pass protein: The growth hormone receptor (GHR)/Growth hormone (GH) system stimulates postnatal growth, is a metabolic regulator affecting hepatic metabolism and is implicated in cancer development. The receptor is a single-pass membrane protein that acts as a homo-dimer. GH binding causes a reorientation of the TMDs in the preformed dimer, implicating two different dimeric structures: the on and off states. We recently showed the ICD to be an intrinsically disordered protein, with a specific lipid interaction domain (LID) proximal to the membrane, the function of which is completely unexplored. We hypothesize that fuzzy interactions with lipids act to stabilize specific conformations of the receptor dimer

Multi-pass protein: The sodium-proton exchanger 1 (NHE1) is a 12 transmembrane (TM) helix protein with a C-terminal ICD, which we recently showed to be an intrinsically disordered protein. NHE1 interacts with anionic phospholipids (Fig 1) and with a BAR protein, and it has been assigned mechanosensor functions. NHE1 plays essential roles in regulation of cellular pH, volume, proliferation, survival, and motility, and is implicated in cancer and cardiovascular diseases. NHE1 is regulated by membrane lipid composition, incl. cholesterol, and resides in caveolae/lipid rafts and invadopodia. We will test the hypothesis that fuzzy interactions with lipids regulate NHE1 function, via at least two types of mechanisms operating on different time scales: (i) steady-state contributions to NHE1 dimerization and steady state function of the NHE1 dimer; (ii) dynamic modulation of interactions between the NHE1 ICD and the membrane.

PERSPECTIVES

Novel views of protein functional dynamics are emerging and are fundamentally changing how we envision protein function. An even broader view, as adopted in this initiative, incorporates how lipids and proteins coexist synergistically with fine-tuned co-structural dynamics, including fuzzy complexes, in the membrane. Their combined understanding demands a detailed investigation of co-structures and co-dynamics, which will enable their rational targeting. Numerous societal challenges, including amyloidosis, cancer, diabetes and epidemics, depend on protein:lipid interactions. Through a highly interdisciplinary synergistic approach, which allows us to address hitherto elusive properties of these complexes in vitro and in a cellular context, the results and insight generated by SYNERGY have strong potential to benefit human health.