Than Laboratory

Protein crystallography

Structure function relationship of proteins central to neurodegenerative diseases, aging and proteolytic (pro)protein processing

Projects

Alzheimer's Disease (AD) is the most frequent dementia worldwide, occurring predominantly in the elderly population. The disease-typical senile plaques contain as the main component the neurotoxic amyloid β-peptide (Aβ), which is proteolytically derived from the large type I transmembrane protein β-amyloid precursor prorein (APP). One key step is the cleavage of an intermediate inside the membrane by the large proteinase complex γ-secretase, finally liberating Aβ. While the overall processes leading to the formation of Aβ have been elucidated in many labs over the last years at the cell-biological and biochemical level, rather little is known about the detailed atomic structures of many involved molecules, their interactions and physiologic functions, setting the starting point for many research projects in our group.

 



» Crystal structure of the entire E1-domain of APP. For more details see Dahms et al (2010) Proc Natl Acad Sci, 107, 5381-5386.

Our crystal structure of the entire E1-domain of APP shows that the two constituting subdomains called growth factor like domain (GFLD) and copper binding domain (CuBD) form one rigid structural and hence functional entity. At slightly acidic (vesicular) pH the two subdomains are tightly connected. Interestingly, this interaction shows some plasticity at neutral to slightly basic (extracellular) pH, suggesting that this interaction might depend on the cellular localization of APP. As shown biochemically as well as seen in the crystal, two such E1-units form a tight dimer. This E1-based dimerization of APP is induced and stabilized by the interaction with heparin, probably mimicking the physiologically more abundant heparan sufate proteglycanes (HSPGs).

 



» Crystal structure of the metal-bound E2-domain of APP. For more details see Dahms et al (2010) J Mol Biol. in press.

Our biochemical and crystallographic data on the E2-domain of APP identified a metal-dependent molecular switch located within this part of APP. Four evolutionary highly conserved histidine residues bind specifically and with high affinity to copper and zinc at physiological concentrations. Metal specific coordination spheres induce large conformational changes and enforce distinct structural states, most likely regulating the physiological function of APP and its processing in AD.


 

 



» Crystal structure of the ligand binding region of death receptor six (DR6). For more details see Kuester et al (2011) J Mol Biol, 409, 189-201.
We recently solved the crystal structure of the extracellular domain of the orphan death receptor six (DR6), as part of our investigations on the molecular interaction of APP with signalling partners and to understand how this interaction might lead to the development of AD. DR6 belongs to the tumor necrosis factor (TNF) receptor superfamily whose family members are intimately involved in the signal transduction pathways during apoptosis, stress response and cellular survival. It was shown to act as receptor for soluble, N-terminal APP-species resulting in axonal pruning and neuronal apoptosis. The cysteine-rich domain of DR6, the usual ligand binding region of TNFR-family members, shows the typical fold of this domain and exposes a DR6-specific surface responsible for the selective recognition of its ligands. Comparison of APP to known, typical ligands of TNFR-family members, however, shows an unprecedented symmetry and structure suggesting that its interaction with DR6 is different from prototype TNFR ligands.

 

 



» The movie shows the 3D structure of our pro-furin model, where the active site cleft of furin is blocked by the tightly bound pro- domain (white). For more details see Henrich et al (2005) J Mol Biol, 345, 211-227.
In eucaryotes, many secreted proteins and peptides are proteolytically excised from larger precursors by a specific class of serine proteases, the Proprotein/Prohormone Convertases (PCs). This cleavage is essential for the activation of the respective substrates, ranging from peptide hormones (such as insulin), extracellular proteases, growth and differentiation factors (implicated in neurodegenerative diseases, tumor growth and metastasis) to bacterial toxins and viral coat proteins, making the PCs a very interesting pharmacological target. Based on our crystal structure of furin and other studies of various groups, we are currently beginning to understand how furin and more generally this class of unusually specific endoproteinases recognize and cleave their substrates. In the future, we want to extend our structural understanding also to other family members, some of them showing quite different substrate specificities, and to push the rational structure-based development of inhibitors.

 

 

Our protein target oriented research often results in the necessity to establish, extend or to adapt new protein-crystallographic methods such as the development of an element-specific electron density map, calculated from anomalous differences collected at the K-absorption edge of calcium. This map unequivocally defined the exact number and spatial localization of Ca²⁺ ions bound to furin. Recently we employed this method to investigate specific metal binding sites in APP. In addition, we have used the transformation of protein crystals by tightly controlled humidity changes to improve the internal order of protein crystals, hereby enabling us to solve the respective structure.

The Research Group

Recent selected publications

• Dahms SO, Könnig I, Roeser D, Gührs KH, Mayer MC, Kaden D, Multhaup G, Than ME (2011) Metal binding dictates conformation and function of the Amyloid Precursor Protein (APP) E2 domain. J Mol Biol. [epub ahead of print]
• Kuester M, Kemmerzehl S, Dahms SO, Roeser D, Than ME (2011) The Crystal Structure of Death Receptor 6 (DR6): A potential receptor of the amyloid precursor protein (APP). J Mol Biol, 409, 189-201. [PubMed]
• Sielaff F, Than ME, Bevec D, Lindberg I, Steinmetzer T (2011) New furin inhibitors based on weakly basic amidinohydrazones. Bioorg Med Chem Lett, 21, 836-840. [PubMed]
• Dahms SO, Hoefgen S, Roeser D, Schlott B, Gührs K-H, Than ME (2010) Structure and Biochemical Analysis of the Heparin-induced E1-Dimer of the Amyloid Precursor Protein (APP). Proc Natl Acad Sci U S A, 107, 5381-5386. [PubMed]
• Becker GL, Sielaff F, Than ME, Lindberg I, Routhier S, Day R, Lu Y, Garten W, Steinmetzer T (2010) Potent inhibitors of furin and furin-like proprotein convertases containing decarboxylated P1 arginine mimetics. J Med Chem, 53, 1067-1075 [PubMed]
• Henrich S, Lindberg I, Bode W, Than ME (2005) Proprotein convertase models based on the crystal structures of furin and kexin: explanation of their specificity. J Mol Biol, 345, 211-227. [PubMed]
• Than ME, Henrich S, Bourenkov GP, Bartunik HD, Huber R, Bode W (2005) The endoproteinase furin contains two essential Ca²⁺ ions stabilizing its N-terminus and the unique S1 specificity pocket. Acta Crystallogr D Biol Crystallogr, 61, 505-512. [PubMed]
• Henrich S, Cameron A, Bourenkov GP, Kiefersauer R, Huber R, Lindberg I, Bode W, Than ME (2003) The crystal structure of the proprotein processing proteinase furin explains its stringent specificity. Nat Struct Biol, 10, 520-526. [PubMed]