Dirk Roeser

Metal Binding Dictates Conformation and Function of the Amyloid Precursor Protein (APP) E2 Domain.

The amyloid precursor protein (APP) and its neurotoxic cleavage product Aβ are key players in the development of Alzheimers disease and appear essential for neuronal development and cell homeostasis in mammals. Proteolytic processing of APP is influenced by metal ions, protein ligands and its oligomerization state. However, the structural basis and functional mechanism of APP regulation are hitherto largely unknown. Here we identified a metal-dependent molecular switch located within the E2 domain of APP containing four evolutionary highly conserved histidine residues. Three X-ray structures of the metal-bound molecule were solved at 2.6-2.0 Å resolution. Using protein crystallographic and biochemical methods, we characterized this novel high-affinity binding site within the E2 domain that binds competitively 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 Alzheimers disease.

Novel Zinc-binding Site in the E2 Domain Regulates Amyloid Precursor-like Protein 1 (APLP1) Oligomerization

Background: Oligomeric complexes of APP, APLP1, and APLP2 contribute to synapse formation and structure. Results: Zinc binding to the E2 domain of APP and APLPs promotes their oligomerization in the cell, most notably with APLP1. Conclusion: Extracellular zinc is a regulator for structure and function of APP and APLPs. Significance: Novel insight into how APP and APLP function is regulated at the molecular level. The amyloid precursor protein (APP) and the APP-like proteins 1 and 2 (APLP1 and APLP2) are a family of multidomain transmembrane proteins possessing homo- and heterotypic contact sites in their ectodomains. We previously reported that divalent metal ions dictate the conformation of the extracellular APP E2 domain (Dahms, S. O., Könnig, I., Roeser, D., Gührs, K.-H., Mayer, M. C., Kaden, D., Multhaup, G., and Than, M. E. (2012) J. Mol. Biol. 416, 438–452), but unresolved is the nature and functional importance of metal ion binding to APLP1 and APLP2. We found here that zinc ions bound to APP and APLP1 E2 domains and mediated their oligomerization, whereas the APLP2 E2 domain interacted more weakly with zinc possessing a less surface-exposed zinc-binding site, and stayed monomeric. Copper ions bound to E2 domains of all three proteins. Fluorescence resonance energy transfer (FRET) analyses examined the effect of metal ion binding to APP and APLPs in the cellular context in real time. Zinc ions specifically induced APP and APLP1 oligomerization and forced APLP1 into multimeric clusters at the plasma membrane consistent with zinc concentrations in the blood and brain. The observed effects were mediated by a novel zinc-binding site within the APLP1 E2 domain as APLP1 deletion mutants revealed. Based upon its cellular localization and its dominant response to zinc ions, APLP1 is mainly affected by extracellular zinc among the APP family proteins. We conclude that zinc binding and APP/APLP oligomerization are intimately linked, and we propose that this represents a novel mechanism for regulating APP/APLP protein function at the molecular level.

Analysis of the Overall Structure of the Multi-Domain Amyloid Precursor Protein (APP)

The amyloid precursor protein (APP) and its processing by the α-, β- and γ-secretases is widely believed to play a central role during the development of Alzheimer´s disease. The three-dimensional structure of the entire protein, its physiologic function and the regulation of its proteolytic processing remain, however, largely unclear to date. To gain a deeper understanding of the structure of APP that underlies all of its functions, we first cloned and recombinantly expressed different constructs in E. coli. Using limited proteolysis followed by mass spectrometry and Edman degradation as well as analytical gel permeation chromatography coupled static light scattering, we experimentally analyzed the structural domain boundaries and determined that the large ectodomain of APP consists of exactly two rigidly folded domains – the E1-domain (Leu18-Ala190) and the E2-domain (Ser295-Asp500). Both, the acidic domain (AcD) connecting E1 and E2 as well as the juxtamembrane region (JMR) connecting E2 to the single transmembrane helix are highly flexible and extended. We identified in-between the E1-domain and the AcD an additional domain of conservation and partial flexibility that we termed extension domain (ED, Glu191-Glu227). Using Bio-layer interferometry, pull-down assays and analytical gel filtration experiments we demonstrated that the E1-domain does not tightly interact with the E2-domain, both in the presence and in the absence of heparin. APP hence forms an extended molecule that is flexibly tethered to the membrane. Its multi-domain architecture enables together with the many known functionalities the concomitant performance of several, independent functions, which might be regulated by cellular, compartment specific pH-changes.

Heparin induced dimerization of APP is primarily mediated by E1 and regulated by its acidic domain.

The amyloid precursor protein (APP) and its cellular processing are believed to be centrally involved in the etiology of Alzheimers disease (AD). In addition, many physiological functions have been described for APP, including a role in cell-cell- and cell-ECM-adhesion as well as in axonal outgrowth. We show here the molecular determinants of the oligomerization/dimerization of APP, which is central for its cellular (mis)function. Using size exclusion chromatography (SEC), dynamic light scattering and SEC-coupled static light scattering we demonstrate that the dimerization of APP is energetically induced by a heparin mediated dimerization of the E1 domain, which results in a dimeric interaction of E2. We also show that the acidic domain (AcD) interferes with the dimerization of E1 and propose a model where both, cis- and trans-dimerization occur dependent on cellular localization and function.

Interaction of the amyloid precursor protein‐like protein 1 (APLP1) E2 domain with heparan sulfate involves two distinct binding modes

Two X-ray structures of APLP1 E2 with and without a heparin dodecasaccharide are presented, revealing two distinct binding modes of the protein to heparan sulfate. The data provide a mechanistic explanation of how APP-like proteins bind to heparan sulfates and how they specifically recognize nonreducing structures of heparan sulfates.

Novel Insights into Structure and Function of Factor XIIIa-Inhibitor Tridegin

The inhibition of the final step in blood coagulation, the factor XIIIa (FXIIIa) catalyzed cross-linking of fibrin monomers, is currently still a challenge in medicinal chemistry. We report synthesis, recombinant expression, disulfide connectivity, and biological activity of tridegin, the sole existing peptide representative displaying inhibitory activity on FXIIIa. Inhibition of the enzyme by this 66-mer cysteine-rich peptide is mediated by its C-terminal sequence, while the N-terminal part comprises structural information and contributes to inhibitor binding. Either of the production strategies examined leads to the formation of different disulfide-bridged isomers indicating the requirement of the correct fold for inhibitory activity. Molecular modeling and docking studies confirm disulfide bond isomer preference with respect to binding to FXIIIa, in turn, the knowledge of the enzyme-inhibitor interactions might bring about comprehensive ideas for the design of a suitable lead structure for addressing FXIIIa.

Identification of inhibitors of the transmembrane protease FlaK of Methanococcus maripaludis

GxGD‐type intramembrane cleaving proteases (I‐CLiPs) form a family of proteolytic enzymes that feature an aspartate‐based catalytic mechanism. Yet, they structurally and functionally largely differ from the classical pepsin‐like aspartic proteases. Among them are the archaeal enzyme FlaK, processing its substrate FlaB2 during the formation of flagella and γ‐secretase, which is centrally involved in the etiology of the neurodegenerative Alzheimers disease. We developed an optimized activity assay for FlaK and based on screening of a small in‐house library and chemical synthesis, we identified compound 9 as the first inhibitor of this enzyme. Our results show that this intramembrane protease differs from classical pepsin‐like aspartic proteases and give insights into the substrate recognition of this enzyme. By providing the needed tools to further study the enzymatic cycle of FlaK, our results also enable further studies towards a functional understanding of other GxGD‐type I‐CLiPs.

Structure-function-relationship of the APP-E1-domain

Background Alzheimer’s disease is one of the most frequent forms of dementia in the elderly population affecting about 25 % of people in the age of 80 to 90 years [1]. Due to the more and more ageing society the importance of dementia is increasing. The brain of affected patients is characterized by the deposition of senile plaques containing the neurotoxic peptide Ab40-42 that is derived out of its precursor, the Amyloid Precursor Protein (APP) [2]. Beside its role in Alzheimer’s pathology many physiological functions, like stimulation of synaptogenesis and signal transduction in a receptor-like manner are discussed for APP [3]. However, until now it was not possible to correlate the known structures of subdomains with most of the proposed physiological functions of APP.

Structural and biochemical analysis of the heparin-induced E1 dimer of the amyloid precursor protein

a widespread misperception that dominant mutations in amyloid precursor protein, presenilin 1, and presenilin 2 cause most cases of EOAD even though they explain less than 10% of all EOAD cases. Here we set out to determine the genetic contribution to the remainingw90% of EOAD cases. Methods: A liability threshold model of disease was used to estimate heritability of EOAD and late-onset AD (LOAD) using concordance for AD among parent-offspring pairs. Individuals with probable AD and detailed parental history (n 1⁄4 5,370) were identified in a research registry of 32 United States Alzheimer’s Disease Centers. Results: For LOAD (n 1⁄4 4,302), we found sex-specific parent-offspring concordance that ranged fromw10-30% resulting in a heritability of 69.8% (95% CI: 64.6-75.0%) and equal heritability for both sexes regardless of parental gender. For EOAD (n 1⁄4 702), we found that the parent-offspring concordance is 1⁄4 10% and EOAD heritability is 92-100% for all likely values of EOAD prevalence. Conclusions: We confirm LOAD is a highly polygenic disease. By contrast, the data for EOAD suggest it is an almost entirely genetically based disease, and the low rate of concordance and sex-specific pattern observed lead us to reject the hypotheses that EOAD is a purely dominant, mitochondrial, X-linked, or polygenic disorder. The most likely explanation of the data is thatw90% of EOAD cases are due to autosomal recessive causes.