Biology subjects

Hitzenberger, M.

Publications and source records attributed to Hitzenberger, M..

4 recordsLinked to original sources

bioRxiv2023

Unravelling ATP processing by the AAA+ protein p97 at the atomic level

The human enzyme p97 regulates various cellular pathways by unfolding hundreds of protein substrates in an ATP-dependent manner, making it an essential component of homeostasis and impactful pharmacological target. The hexameric complex undergoes substantial conformational changes in the course of its catalytic cycle. Here, we elucidate the molecular motions that occur at the active site in the temporal window immediately before and after ATP hydrolysis by merging cryo-EM, NMR spectroscopy and MD simulations. p97 populates a metastable reaction intermediate, the ADP.Pi state, which is poised between hydrolysis and product release. Detailed snapshots reveal that the active site is finely tuned to trap and eventually discharge the cleaved phosphate. Signalling pathways originating at the active site coordinate the action of the hexamer subunits and couple hydrolysis with allosteric conformational changes. Our multidisciplinary approach enables a glimpse into the sophisticated spatial and temporal orchestration of ATP handling by a prototype AAA+ protein.

biophysics↗

bioRxiv2022

Intra-membrane client recognition potentiates the chaperone functions of Calnexin

One third of the human proteome are membrane proteins. They are particularly vulnerable to misfolding, often requiring assistance by molecular chaperones. Calnexin (CNX), one of the most abundant ER chaperones, plays an important role in membrane protein biogenesis and engages clients via its sugar-binding lectin domain. Using mass spectrometric analyses, we show that Calnexin (CNX) interacts with a large number of non-glycosylated membrane proteins, suggesting additional binding modes. We find that misfolded membrane proteins are preferentially bound by CNX and that CNX uses its single transmembrane domain (TMD) for client recognition. Combining experimental and computational approaches, we systematically dissect signatures for intramembrane client recognition by CNX and identify sequence motifs within the CNX TMD region that mediate client binding. Building on this, we show that intramembrane client binding potentiates the chaperone functions of CNX. Together, this study reveals a widespread role of CNX client recognition in the lipid bilayer, which synergizes with its established lectin-based substrate binding. Molecular chaperones thus can combine different interaction modes to support the biogenesis of the diverse eukaryotic membrane proteome.

cell biology↗

bioRxiv2019

Uncovering the Binding Mode of γ-Secretase Inhibitors

Knowledge of how transition state inhibitors bind to{gamma} -secretase is of major importance for the design of new Alzheimers disease therapies. Based on the known structure of{gamma} -secretase in complex with a fragment of the amyloid precursor protein we have generated a structural model of{gamma} -secretase in complex with the effective L-685,458 transition state inhibitor. The predicted binding mode is in excellent agreement with experimental data, mimicking all enzyme-substrate interactions at the active site and forming the relevant transition state geometry with the active site aspartate residues. In addition, we found that the stability of the complex is very likely also sensitive to the pH value. Comparative simulations on the binding of L-685,458 and the epimer L682,679 allowed us to explain the strongly reduced affinity of the epimer for{gamma} -secretase. The structural model could form a valuable basis for the design of new or modified{gamma} -secretase inhibitors.

biophysics↗

bioRxiv2018

Structural Modeling of Gamma-Secretase Aβn ComplexFormation and Substrate Processing

The intra-membrane aspartyl protease{gamma} -secretase (GSEC) cleaves single-span transmembrane helices including the C-terminal fragment of the amyloid precursor protein (APP). This substrate is initially cleaved at the {varepsilon}-site followed by successive processing (trimming) events mostly in steps of three amino acids. GSEC is responsible for the formation of N-terminal APP amyloid-{beta} (A {beta}) peptides of different length (e.g. A{beta}42) that can form aggregates involved in Alzheimers disease pathogenesis. The molecular mechanism of GSEC-APP substrate recognition is key for understanding how different peptide products are formed and could help in designing APP-selective modulators. Based on the known structure of apo GSEC and the APP-C99 fragment we have generated putative structural models of the initial binding in three different possible modes using extensive Molecular Dynamics (MD) simulations. The binding mode with the substrate helix located in a cleft between the transmembrane helices 2 and 3 of the presenilin subunit was identified as a most likely binding mode. Based on this arrangement the processing steps were investigated using restraint MD simulations to pull the scissile bond (for each processing step) into a transition like (cleavable) state. This allowed us to analyze in detail the motions and energetic contributions of participating residues. The structural model agrees qualitatively well with the influence of many mutations in GSEC and C99. It also explains the effects of inhibitors, cross-linking as well as spectroscopic data on GSEC substrate binding and can serve as working model for the future planning of structural and biochemical studies.

biophysics↗