Publications by authors named "Martin Zacharias"

223 Publications

Molecular mechanism of amyloidogenic mutations in hypervariable regions of antibody light chains.

J Biol Chem 2021 Jan 25:100334. Epub 2021 Jan 25.

Center for Integrated Protein Science Munich at the Department Chemie, Technische Universität München, Lichtenbergstr. 4, 85748 Garching, Germany. Electronic address:

Systemic light chain (AL) amyloidosis is a fatal protein misfolding disease in which excessive secretion, misfolding, and subsequent aggregation of free antibody light chains eventually leads to deposition of amyloid plaques in various organs. Patient-specific mutations in the antibody V domain are closely linked to the disease, but the molecular mechanisms by which certain mutations induce misfolding and amyloid aggregation of antibody domains are still poorly understood. Here, we compare a patient V domain with its non-amyloidogenic germline counterpart and show that, out of the five mutations present, two of them strongly destabilize the protein and induce amyloid fibril formation. Surprisingly, the decisive, disease-causing mutations are located in the highly variable complementarity determining regions (CDRs) but exhibit a strong impact on the dynamics of conserved core regions of the patient V domain. This effect seems to be based on a deviation from the canonical CDR structures of CDR2 and CDR3 induced by the substitutions. The amyloid-driving mutations are not necessarily involved in propagating fibril formation by providing specific side chain interactions within the fibril structure. Rather, they destabilize the V domain in a specific way, increasing the dynamics of framework regions, which can then change their conformation to form the fibril core. These findings reveal unexpected influences of CDR-framework interactions on antibody architecture, stability, and amyloid propensity.
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http://dx.doi.org/10.1016/j.jbc.2021.100334DOI Listing
January 2021

Proteolysis of Rab32 by GtgE induces an inactive GTPase conformation.

iScience 2021 Jan 15;24(1):101940. Epub 2020 Dec 15.

Department of Biochemistry and Signaltransduction, University Medical Centre Hamburg-Eppendorf (UKE), Martinistrasse 52, 20246 Hamburg, Germany.

Rab GTPases are central regulators of intracellular vesicular trafficking. They are frequently targeted by bacterial pathogens through post-translational modifications. secretes the cysteine protease GtgE during infection, leading to a regioselective proteolytic cleavage of the regulatory switch I loop in the small GTPases of the Rab32 subfamily. Here, using a combination of biochemical methods, molecular dynamics simulations, NMR spectroscopy, and single-pair Förster resonance energy transfer, we demonstrate that the cleavage of Rab32 causes a local increase of conformational flexibility in both switch regions. Cleaved Rab32 maintains its ability to interact with the GDP dissociation inhibitor (GDI). Interestingly, the Rab32 cleavage enables GDI binding also with an active GTP-bound Rab32 . Furthermore, the Rab32 proteolysis provokes disturbance in the interaction with its downstream effector VARP. Thus, the proteolysis of Rab32 is not a globally degradative mechanism but affects various biochemical and structural properties of the GTPase in a diverse manner.
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http://dx.doi.org/10.1016/j.isci.2020.101940DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7779776PMC
January 2021

Targeting Telomeres: Molecular Dynamics and Free Energy Simulation of Gold-Carbene Binding to DNA.

Biophys J 2021 Jan 5;120(1):101-108. Epub 2020 Dec 5.

Physics Department T38, Technical University of Munich, Garching, Germany. Electronic address:

DNA sequences in regulatory regions and in telomers at the ends of chromosomes frequently contain tandem repeats of guanine nucleotides that can form stacked structures stabilized by Hoogsten pairing and centrally bound monovalent cations. The replication and elongation of telomeres requires the disruption of these G-quadruplex structures. Hence, drug molecules such as gold (Au)-carbene that stabilize G-quadruplexes may also interfere with the elongation of telomeres and, in turn, could be used to control cell replication and growth. To better understand the molecular mechanism of Au-carbene binding to G-quadruplexes, we employed molecular dynamics simulations and free energy simulations. Whereas very restricted mobility of two Au-carbene ligands was found upon binding as a doublet to one side of the G-quadruplex, much larger translational and orientational mobility was observed for a single Au-carbene binding at the second G-quadruplex surface. Comparative simulations on duplex DNA in the presence of Au-carbene ligands indicates a preference for the minor groove and weaker unspecific and more salt-dependent binding than to the G-quadruplex surface. Analysis of energetic contributions reveals a dominance of nonpolar and van der Waals interactions to drive binding. The simulations can also be helpful for proposing possible modifications that could improve Au-carbene affinity and specificity for G-quadruplex binding.
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http://dx.doi.org/10.1016/j.bpj.2020.11.2263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820797PMC
January 2021

Altered Hinge Conformations in APP Transmembrane Helix Mutants May Affect Enzyme-Substrate Interactions of γ-Secretase.

ACS Chem Neurosci 2020 12 24;11(24):4426-4433. Epub 2020 Nov 24.

Institute for Biological Interfaces 4, Karlsruhe Institute of Technology, P.O. Box 3640, 76021 Karlsruhe, Germany.

Cleavage of substrates by γ-secretase is an inherently slow process where substrate-enzyme affinities cannot be broken down into specific sequence requirements in contrast to soluble proteases. Nevertheless, despite its apparent sequence tolerance single point mutations in amyloid precursor protein can severely affect cleavage efficiencies and change product line preferences. We have determined by NMR spectroscopy the structures of the transmembrane domain of amyloid precursor protein in TFE/water and compared it to that of four mutants: two FAD mutants, V44M and I45T, and the two diglycine hinge mutants, G38L and G38P. In accordance with previous publications, the transmembrane domain is composed of two helical segments connected by the diglycine hinge. Mutations alter kink angles and structural flexibility. Furthermore, to our surprise, we observe different, but specific mutual orientations of N- and C-terminal helical segments in the four mutants compared to the wildtype. We speculate that the observed orientations for G38L, G38P, V44M, and I45T lead to unfavorable interactions with γ-secretase exosites during substrate movement to the enzyme's active site in presenilin and/or for the accommodation into the substrate-binding cavity of presenilin.
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http://dx.doi.org/10.1021/acschemneuro.0c00640DOI Listing
December 2020

Base-Pairing and Base-Stacking Contributions to Double-Stranded DNA Formation.

Authors:
Martin Zacharias

J Phys Chem B 2020 11 6;124(46):10345-10352. Epub 2020 Nov 6.

Physics Department T38, Technical University of Munich, 85748 Garching, Germany.

Double-stranded (ds)DNA formation and dissociation are of fundamental biological importance. The negative DNA charge influences the dsDNA stability. However, the base pairing and the stacking between neighboring bases are responsible for the sequence-dependent stability of dsDNA. The stability of a dsDNA molecule can be estimated from empirical nearest-neighbor models based on contributions assigned to base-pair steps along the DNA and additional parameters because of DNA termini. In efforts to separate contributions, it has been concluded that base stacking dominates dsDNA stability, whereas base pairing contributes negligibly. Using a different model for dsDNA formation, we reanalyze dsDNA stability contributions and conclude that base stacking contributes already at the level of separate ssDNAs but that pairing contributions drive the dsDNA formation. The theoretical model also predicts that stability contributions of base-pair steps that contain only guanine/cytosine, mixed steps, and steps with only adenine/thymine follow the order 6:5:4, respectively, as expected based on the formed hydrogen bonds. The model is fully consistent with the available stacking data and the nearest-neighbor dsDNA parameters. It allows assigning a narrowly distributed value for the effective free energy contribution per formed hydrogen bond during dsDNA formation of -0.72 kcal·mol based entirely on the experimental data.
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http://dx.doi.org/10.1021/acs.jpcb.0c07670DOI Listing
November 2020

Rapid Design of Potential Cyclic Peptide Binders Targeting Protein-Protein Interfaces.

Front Chem 2020 8;8:573259. Epub 2020 Oct 8.

Physics Department T38, Technical University of Munich, Garching, Germany.

Rational design of specific inhibitors of protein-protein interactions is desirable for drug design to control cellular signal transduction but also for studying protein-protein interaction networks. We have developed a rapid computational approach to rationally design cyclic peptides that potentially bind at desired regions of the interface of protein-protein complexes. The methodology is based on comparing the protein backbone structure of short peptide segments (epitopes) at the protein-protein interface with a collection of cyclic peptide backbone structures. A cyclic peptide that matches the backbone structure of the segment is used as a template for a binder by adapting the amino acid side chains to the side chains found in the target complex. For a small library of cyclic peptides with known high resolution structures we found for the majority (~82%) of 154 protein-protein complexes at least one very well fitting match for a cyclic peptide template to a protein-protein interface segment. The majority of the constructed protein-cyclic peptide complexes was very stable during Molecular Dynamics simulations and showed an interaction energy score that was typically more favorable compared to interaction scores of typical peptide-protein complexes. Our cPEPmatch approach could be a promising approach for rapid suggestion of cyclic peptide binders that could be tested experimentally and further improved by chemical modification.
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http://dx.doi.org/10.3389/fchem.2020.573259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7578414PMC
October 2020

Efficient Refinement and Free Energy Scoring of Predicted Protein-Protein Complexes Using Replica Exchange with Repulsive Scaling.

J Chem Inf Model 2020 Nov 19;60(11):5552-5562. Epub 2020 Oct 19.

Physik-Department T38, Technische Universität München, James-Franck-Str. 1, 85748 Garching, Germany.

Accurate prediction and evaluation of protein-protein complex structures are of major importance to understand the cellular interactome. Typically, putative complexes are predicted based on docking methods, and simple force field or knowledge-based scoring functions are applied to evaluate single complex structures. We have extended a replica-exchange-based scheme employing different levels of a repulsive biasing between partners in each replica simulation (RS-REMD) to simultaneously refine and score protein-protein complexes. The bias acts specifically on the intermolecular interactions based on an increase in effective pairwise van der Waals radii (repulsive scaling (RS)-REMD) without affecting interactions within each protein or with the solvent. The method provides a free energy score that correlates quite well with experimental binding free energies on a set of 36 complexes with correlation coefficients of 0.77 and 0.55 in explicit and implicit solvent simulations, respectively. For a large set of docked decoy complexes, significant improvement of docked complexes was found in many cases with the starting structure in the vicinity (within 20 Å) of the native complex. In the majority of cases (14 out of 20 in explicit solvent), near native docking solutions were identified as the best scoring complexes. The approach is computational demanding but may offer a route for refinement and realistic ranking of predicted protein-protein docking geometries.
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http://dx.doi.org/10.1021/acs.jcim.0c00853DOI Listing
November 2020

Ligand binding and global adaptation of the GlnPQ substrate binding domain 2 revealed by molecular dynamics simulations.

Protein Sci 2020 12 3;29(12):2482-2494. Epub 2020 Nov 3.

Physik-Department T38, Technische Universität München, Garching, Germany.

Substrate-binding domains (SBD) are important structural elements of substrate transporters mediating the transport of essential molecules across the cell membrane. The SBD2 domain of the glutamine (GLN) transporter from bacteria consists of two domains D1 and D2 that bind GLN in the space between the domains in a closed conformation. In the absence of ligand, SBD2 adopts an open conformation with increased domain distance. In molecular dynamics (MD) simulations in the absence of ligands, no closing of the open conformation was observed on the MD time scale. Addition of GLN resulted in several reversible binding and unbinding events of GLN at the binding site on the D1 domain but did not induce domain closing indicating that binding and global domain closing do not occur simultaneously. The SBD2 structure remained in a closed state when starting from the GLN-bound closed crystal structure and opened quickly to reach the open state upon removal of the GLN ligand. Free energy simulations to induce opening to closing indicated a barrier for closing in the absence and presence of ligand and a significant penalty for closing without GLN in the binding pocket. Simulations of a Leu480Ala mutation also indicate that an interaction of a C-terminal D1-tail with a D2-helix (not contacting the substrate-binding region) plays a decisive role for controlling the barrier of conformational switching in the SBD2 protein. The results allow us to derive a model of the molecular mechanism of substrate binding to SBD2 and associated conformational changes.
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http://dx.doi.org/10.1002/pro.3981DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7679957PMC
December 2020

Occurrence of SARS-CoV-2 in the intraocular milieu.

Exp Eye Res 2020 12 28;201:108273. Epub 2020 Sep 28.

Department of Ophthalmology, Medical University of Graz Auenbruggerplatz 4, 8036, Graz, Austria.

The purpose of this research is to study the intraocular occurrence of SARS-CoV-2. In postmortem examinations, aqueous humor and the vitreous samples were collected. All individuals were previously positive in nasopharyngeal swabbing and cause of death was respiratory failure due to SARS-CoV-2 infection. Testing was done using quantitative RT-PCR. We included 16 aqueous humor and 16 vitreous samples for PCR testing. None of the results was positive for SARS-CoV-2. Human GAPDH genes to verify the presence of RNA was present in all aqueous humor samples (16/16, 100%) and 15/16 (93.8%) vitreous samples. In conclusion, this case series found no evidence of SARS-CoV-2 in the intraocular milieu.
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http://dx.doi.org/10.1016/j.exer.2020.108273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7521885PMC
December 2020

How Mutations Perturb γ-Secretase Active Site Studied by Free Energy Simulations.

ACS Chem Neurosci 2020 10 6;11(20):3321-3332. Epub 2020 Oct 6.

Physik-Department T38,Techniche Universität München, James-Franck-Strasse 1, 85748 Garching, Germany.

γ-Secretase is involved in processing of the amyloid precursor protein (APP) and generation of short A peptides that may play a key role in neurodegenerative diseases such as Alzheimer's disease (AD). Several mutations in γ-secretase influence its activity, resulting in early AD onset (Familial AD or FAD mutations). The molecular details of how mutations, not located close to the active site, can affect enzyme activity is not understood. In molecular dynamics simulations of γ-secretase in the absence of substrate (apo), we identified two active site conformational states characterized by a direct contact between catalytic Asp residues (closed state) and an open water-bridged state. In the presence of substrate, only conformations compatible with the open active site geometry are accessible. Systematic free energy simulations on wild type and FAD mutations indicate a free energy difference between closed and open states that is significantly modulated by FAD mutations and correlates with the corresponding experimental activity. For mutations with reduced activity, an increased penalty for open-state transitions was found. Only for two mutations located at the active site a direct perturbation of the open-state geometry was observed that could directly explain the drop of enzyme activity. The simulations suggest that modulation of the closed/open equilibrium and perturbation of the open (active) catalytic geometry are possible mechanisms of how FAD mutations affect γ-secretase activity. The results also offer an explanation for the experimental finding that FAD mutations, although not located at the interface to the substrate, mainly destabilize the enzyme-substrate complex.
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http://dx.doi.org/10.1021/acschemneuro.0c00440DOI Listing
October 2020

CHARMM-GUI supports the Amber force fields.

J Chem Phys 2020 Jul;153(3):035103

Departments of Biological Sciences, Chemistry, and Bioengineering, Lehigh University, Bethlehem, Pennsylvania 18015, USA.

As part of our ongoing efforts to support diverse force fields and simulation programs in CHARMM-GUI, this work presents the development of FF-Converter to prepare Amber simulation inputs with various Amber force fields within the current CHARMM-GUI workflow. The currently supported Amber force fields are ff14SB/ff19SB (protein), Bsc1 (DNA), OL3 (RNA), GLYCAM06 (carbohydrate), Lipid17 (lipid), GAFF/GAFF2 (small molecule), TIP3P/TIP4P-EW/OPC (water), and 12-6-4 ions, and more will be added if necessary. The robustness and usefulness of this new CHARMM-GUI extension are demonstrated by two exemplary systems: a protein/N-glycan/ligand/membrane system and a protein/DNA/RNA system. Currently, CHARMM-GUI supports the Amber force fields only for the Amber program, but we will expand the FF-Converter functionality to support other simulation programs that support the Amber force fields.
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http://dx.doi.org/10.1063/5.0012280DOI Listing
July 2020

From monomer to fibril: Abeta-amyloid binding to Aducanumab antibody studied by molecular dynamics simulation.

Proteins 2020 12 3;88(12):1592-1606. Epub 2020 Aug 3.

Physics Department T38, Technical University of Munich, Garching, Germany.

Alzheimer's disease is one of the most common causes of dementia. It is believed that the aggregation of short Aβ-peptides to form oligomeric and protofibrillar amyloid assemblies plays a central role for disease-relevant neurotoxicity. In recent years, passive immunotherapy has been introduced as a potential treatment strategy with anti-amyloid antibodies binding to Aβ-amyloids and inducing their subsequent degradation by the immune system. Although so far mostly unsuccessful in clinical studies, the high-dosed application of the monoclonal antibody Aducanumab has shown therapeutic potential that might be attributed to its much greater affinity to Aβ-aggregates vs monomeric Aβ-peptides. In order to better understand how Aducanumab interacts with aggregated Aβ-forms compared to monomers, we have generated structural model complexes based on the known structure of Aducanumab in complex with an Aβ -eptitope. Structural models of Aducanumab bound to full-sequence Aβ -monomers, oligomers, protofilaments and mature fibrils were generated and investigated using extensive molecular dynamics simulations to characterize the flexibility and possible additional interactions. Indeed, an aggregate-specific N-terminal binding motif was found in case of Aducanumab binding to oligomers, protofilaments and fibrils that is located next to but not overlapping with the epitope binding site found in the crystal structure with Aβ . Analysis of binding energetics indicates that this motif binds weaker than the epitope but likely contributes to Aducanumab's preference for aggregated Aβ-species. The predicted aggregate-specific binding motif could potentially serve as a basis to reengineer Aducanumab for further enhanced preference to bind Aβ-aggregates vs monomers.
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http://dx.doi.org/10.1002/prot.25978DOI Listing
December 2020

Folding and Unfolding of the Short Light-Triggered β-Hairpin Peptide AzoChignolin Occurs within 100 ns.

J Phys Chem B 2020 06 16;124(25):5113-5121. Epub 2020 Jun 16.

Department of Organic Chemistry and Center for Integrated Protein Science, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, München 81377, Germany.

To map the underlying molecular mechanisms of folding dynamics in proteins, light-operated peptides have emerged as promising tools. In this study, we reveal the complete sequence of light-induced structural changes of AzoChignolin, a short β-hairpin peptide containing an azobenzene photoswitch in its loop region. Light-triggered structural changes were monitored by time-resolved IR spectroscopy. Formation and destruction of the hairpin structure is very fast and occurs within 100 ns for AzoChignolin in methanol. Atomistic molecular dynamics simulations using two explicit solvents, methanol and water, revealed the underlying molecular processes and allowed us to gain further insight into the reaction mechanism. Despite its rapid reaction time, hairpin formation in these solvents is not force-driven by the molecular switch but proceeded via formation of interstrand hydrogen bonds and contacts between aromatic residues. Moreover, the combined experimental and theoretical study demonstrates that the solvent (methanol vs water) does not dictate the velocity of β-hairpin formation in the AzoChignolin peptide comprising only a few hydrophobic residues in the strands.
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http://dx.doi.org/10.1021/acs.jpcb.0c02021DOI Listing
June 2020

The dynamics of γ-secretase and its substrates.

Semin Cell Dev Biol 2020 09 16;105:86-101. Epub 2020 May 16.

Physics of Synthetic Biological Systems (E14), Technical University of Munich, D-85748 Garching, Germany. Electronic address:

γ-Secretase is an intramembrane aspartyl-protease catalyzing the final step in the regulated intramembrane proteolysis of a large number of single-span type-1 transmembrane proteins. The most extensively studied substrates are the amyloid-β precursor protein (APP) and the NOTCH receptors. An important technique for the characterization of interactions and conformational changes enabling γ-secretase to perform hydrolysis within the confines of the membrane are molecular dynamics simulations on different time and length scales. Here, we review structural and dynamical features of γ-secretase and its substrates including flexibility descriptions from simulations and experiments. We address (1) conformational sampling of apo-enzyme and unbound substrates (exemplified for APP, NOTCH1 and the apparent non-substrate integrin β1), (2) substrate recruitment pathways, (3) conformational changes associated with the formation of the recognition complex, (4) cleavage-site unfolding upon interaction with the enzyme's active site, (5) substrate processing after endoproteolysis, and (6) inhibition and modulation of γ-secretase. We conclude with still open questions and suggest further investigations in order to advance our understanding on how γ-secretase selects and processes substrates. This knowledge will improve the ability to better target substrates selectively for therapeutic applications.
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http://dx.doi.org/10.1016/j.semcdb.2020.04.008DOI Listing
September 2020

How global DNA unwinding causes non-uniform stress distribution and melting of DNA.

PLoS One 2020 15;15(5):e0232976. Epub 2020 May 15.

Physics Department T38, Technical University of Munich, Garching, Germany.

DNA unwinding is an important process that controls binding of proteins, gene expression and melting of double-stranded DNA. In a series of all-atom MD simulations on two DNA molecules containing a transcription start TATA-box sequence we demonstrate that application of a global restraint on the DNA twisting dramatically changes the coupling between helical parameters and the distribution of deformation energy along the sequence. Whereas only short range nearest-neighbor coupling is observed in the relaxed case, long-range coupling is induced in the globally restrained case. With increased overall unwinding the elastic deformation energy is strongly non-uniformly distributed resulting ultimately in a local melting transition of only the TATA box segment during the simulations. The deformation energy tends to be stored more in cytidine/guanine rich regions associated with a change in conformational substate distribution. Upon TATA box melting the deformation energy is largely absorbed by the melting bubble with the rest of the sequences relaxing back to near B-form. The simulations allow us to characterize the structural changes and the propagation of the elastic energy but also to calculate the associated free energy change upon DNA unwinding up to DNA melting. Finally, we design an Ising model for predicting the local melting transition based on empirical parameters. The direct comparison with the atomistic MD simulations indicates a remarkably good agreement for the predicted necessary torsional stress to induce a melting transition, for the position and length of the melted region and for the calculated associated free energy change between both approaches.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0232976PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7228070PMC
August 2020

Compensatory Mechanisms in Temperature Dependence of DNA Double Helical Structure: Bending and Elongation.

J Chem Theory Comput 2020 Apr 31;16(4):2857-2863. Epub 2020 Mar 31.

Department of Informatics and Chemistry, University of Chemistry and Technology Prague, Technická 5, 166 28 Prague, Czech Republic.

Changes in the structure of double-stranded (ds) DNA with temperature affect processes in thermophilic organisms and are important for nanotechnological applications. Here we investigate temperature-dependent conformational changes of dsDNA at the scale of several helical turns and at the base pair step level, inferred from extensive all-atom molecular dynamics simulations of DNA at temperatures from 7 to 47 °C. Our results suggest that, contrary to twist, the overall bending of dsDNA without A-tracts depends only very weakly on temperature, due to the mutual compensation of directional local bends. Investigating DNA length as a function of temperature, we find that the sum of distances between base pair centers (the wire length) exhibits a large expansion coefficient of ∼2 × 10 °C, similar to values reported for thermoplastic materials. However, the wire length increase with temperature is absorbed by expanding helix radius, so the length measured along the helical axis (the spring length) seems to suggest a very small negative thermal expansion coefficient. These compensatory mechanisms contribute to thermal stability of DNA structure on the biologically relevant scale of tens of base pairs and longer.
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http://dx.doi.org/10.1021/acs.jctc.0c00037DOI Listing
April 2020

Structures of peptide-free and partially loaded MHC class I molecules reveal mechanisms of peptide selection.

Nat Commun 2020 03 11;11(1):1314. Epub 2020 Mar 11.

European Molecular Biology Laboratory, Hamburg Outstation, Hamburg, Germany.

Major Histocompatibility Complex (MHC) class I molecules selectively bind peptides for presentation to cytotoxic T cells. The peptide-free state of these molecules is not well understood. Here, we characterize a disulfide-stabilized version of the human class I molecule HLA-A*02:01 that is stable in the absence of peptide and can readily exchange cognate peptides. We present X-ray crystal structures of the peptide-free state of HLA-A*02:01, together with structures that have dipeptides bound in the A and F pockets. These structural snapshots reveal that the amino acid side chains lining the binding pockets switch in a coordinated fashion between a peptide-free unlocked state and a peptide-bound locked state. Molecular dynamics simulations suggest that the opening and closing of the F pocket affects peptide ligand conformations in adjacent binding pockets. We propose that peptide binding is co-determined by synergy between the binding pockets of the MHC molecule.
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http://dx.doi.org/10.1038/s41467-020-14862-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7066147PMC
March 2020

Fatal amyloid formation in a patient's antibody light chain is caused by a single point mutation.

Elife 2020 03 10;9. Epub 2020 Mar 10.

Center for Integrated Protein Science Munich at the Department Chemie, Technische Universität München, Garching, Germany.

In systemic light chain amyloidosis, an overexpressed antibody light chain (LC) forms fibrils which deposit in organs and cause their failure. While it is well-established that mutations in the LC's V domain are important prerequisites, the mechanisms which render a patient LC amyloidogenic are ill-defined. In this study, we performed an in-depth analysis of the factors and mutations responsible for the pathogenic transformation of a patient-derived λ LC, by recombinantly expressing variants in . We show that proteolytic cleavage of the patient LC resulting in an isolated V domain is essential for fibril formation. Out of 11 mutations in the patient V, only one, a leucine to valine mutation, is responsible for fibril formation. It disrupts a hydrophobic network rendering the C-terminal segment of V more dynamic and decreasing domain stability. Thus, the combination of proteolytic cleavage and the destabilizing mutation trigger conformational changes that turn the LC pathogenic.
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http://dx.doi.org/10.7554/eLife.52300DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7064341PMC
March 2020

Prediction of protein-protein complexes using replica exchange with repulsive scaling.

J Comput Chem 2020 06 9;41(15):1436-1447. Epub 2020 Mar 9.

Physik-Department T38, Technische Universität München, Garching, Germany.

The realistic prediction of protein-protein complex structures is import to ultimately model the interaction of all proteins in a cell and for the design of new protein-protein interactions. In principle, molecular dynamics (MD) simulations allow one to follow the association process under realistic conditions including full partner flexibility and surrounding solvent. However, due to the many local binding energy minima at the surface of protein partners, MD simulations are frequently trapped for long times in transient association states. We have designed a replica-exchange based scheme employing different levels of a repulsive biasing between partners in each replica simulation. The bias acts only on intermolecular interactions based on an increase in effective pairwise van der Waals radii (repulsive scaling (RS)-REMD) without affecting interactions within each protein or with the solvent. For a set of five protein test cases (out of six) the RS-REMD technique allowed the sampling of near-native complex structures even when starting from the opposide site with respect to the native binding site for one partner. Using the same start structures and same computational demand regular MD simulations sampled near native complex structures only for one case. The method showed also improved results for the refinement of docked structures in the vicinity of the native binding geometry compared to regular MD refinement.
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http://dx.doi.org/10.1002/jcc.26187DOI Listing
June 2020

Molecular mechanism of Be-ion binding to HLA-DP2: tetrahedral coordination, conformational changes and multi-ion binding.

Phys Chem Chem Phys 2020 Jan;22(2):799-810

Department of Applied Chemistry, Cochin University of Science and Technology, Thrikakkara, Kochi 682 022, India.

The chemistry of beryllium is rather unusual, however, less explored as compared to other main group elements. This is mainly attributed to the high toxicity of beryllium, leading to chronic granulomatous pneumonitis, called chronic beryllium disease (CBD). It has been reported that Be2+-ion binding to the human leukocyte antigen protein (HLA-DP2) and peptide (M2) results in favorable interaction with the T-cell receptor protein (TCR), which initiates immune-mediated toxicity. We have carried out molecular dynamics (MD) simulations combined with quantum mechanical/molecular mechanical (QM/MM) studies to explore the binding nature of Be2+ with a HLA-DP2 protein and M2 peptide. The interaction between the negatively charged M2 peptide and the negatively charged binding cleft of HLA-DP2 is unfavorable. However, this interaction is stabilized by one Be2+ and two Na+-ions bridged by negatively charged carboxyl groups of glutamate residues (β26E and β69E) of the β-chain of HLA-DP2 and one glutamate (p7E) and one aspartate residue (p4D) of the M2 peptide. This multi-ion cavity consists of tetrahedrally coordinated static Be2+ and Na+-ions, as well as one dynamically exchangeable Na+-ion. The smaller size and higher charge of the Be2+-ion as compared to the Na+-ion reduce the distance between the M2 peptide and the β-chain of HLA-DP2, which results in conformational change suitable for TCR binding. However, the replacement of the Be2+ by the Na+-ion could not generate a suitable binding site for TCR.
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http://dx.doi.org/10.1039/c9cp05695eDOI Listing
January 2020

The structure and oxidation of the eye lens chaperone αA-crystallin.

Nat Struct Mol Biol 2019 12 2;26(12):1141-1150. Epub 2019 Dec 2.

Center for Integrated Protein Science Munich at the Department Chemie, Technische Universität München, Garching, Germany.

The small heat shock protein αA-crystallin is a molecular chaperone important for the optical properties of the vertebrate eye lens. It forms heterogeneous oligomeric ensembles. We determined the structures of human αA-crystallin oligomers by combining cryo-electron microscopy, cross-linking/mass spectrometry, NMR spectroscopy and molecular modeling. The different oligomers can be interconverted by the addition or subtraction of tetramers, leading to mainly 12-, 16- and 20-meric assemblies in which interactions between N-terminal regions are important. Cross-dimer domain-swapping of the C-terminal region is a determinant of αA-crystallin heterogeneity. Human αA-crystallin contains two cysteines, which can form an intramolecular disulfide in vivo. Oxidation in vitro requires conformational changes and oligomer dissociation. The oxidized oligomers, which are larger than reduced αA-crystallin and destabilized against unfolding, are active chaperones and can transfer the disulfide to destabilized substrate proteins. The insight into the structure and function of αA-crystallin provides a basis for understanding its role in the eye lens.
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http://dx.doi.org/10.1038/s41594-019-0332-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7115824PMC
December 2019

Coarse-grained and atomic resolution biomolecular docking with the ATTRACT approach.

Proteins 2020 08 13;88(8):1018-1028. Epub 2019 Dec 13.

Physik-Department T38, Technische Universität München, Garching, Germany.

The ATTRACT protein-protein docking program has been employed to predict protein-protein complex structures in CAPRI rounds 38-45. For 11 out of 16 targets acceptable or better quality solutions have been submitted (~70%). It includes also several cases of peptide-protein docking and the successful prediction of the geometry of carbohydrate-protein interactions. The option of combining rigid body minimization and simultaneous optimization in collective degrees of freedom based on elastic network modes was employed and systematically evaluated. Application to a large benchmark set indicates a modest improvement in docking performance compared to rigid docking. Possible further improvements of the docking approach in particular at the scoring and the flexible refinement steps are discussed.
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http://dx.doi.org/10.1002/prot.25860DOI Listing
August 2020

Global Dynamics of Yeast Hsp90 Middle and C-Terminal Dimer Studied by Advanced Sampling Simulations.

Front Mol Biosci 2019 27;6:93. Epub 2019 Sep 27.

Physics Department T38, Technical University of Munich, Garching, Germany.

The Hsp90 protein complex is one of the most abundant molecular chaperone proteins that assists in folding of a variety of client proteins. During its functional cycle it undergoes large domain rearrangements coupled to the hydrolysis of ATP and association or dissociation of domain interfaces. In order to better understand the domain dynamics comparative Molecular Dynamics (MD) simulations of a sub-structure of Hsp90, the dimer formed by the middle (M) and C-terminal domain (C), were performed. Since this MC dimer lacks the ATP-binding N-domain it allows studying global motions decoupled from ATP binding and hydrolysis. Conventional (c)MD simulations starting from several different closed and open conformations resulted in only limited sampling of global motions. However, the application of a Hamiltonian Replica exchange (H-REMD) method based on the addition of a biasing potential extracted from a coarse-grained elastic network description of the system allowed much broader sampling of domain motions than the cMD simulations. With this multiscale approach it was possible to extract the main directions of global motions and to obtain insight into the molecular mechanism of the global structural transitions of the MC dimer.
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http://dx.doi.org/10.3389/fmolb.2019.00093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6798034PMC
September 2019

Cryo-EM structure of a transthyretin-derived amyloid fibril from a patient with hereditary ATTR amyloidosis.

Nat Commun 2019 11 1;10(1):5008. Epub 2019 Nov 1.

Institute of Protein Biochemistry, Ulm University, 89081, Ulm, Germany.

ATTR amyloidosis is one of the worldwide most abundant forms of systemic amyloidosis. The disease is caused by the misfolding of transthyretin protein and the formation of amyloid deposits at different sites within the body. Here, we present a 2.97 Å cryo electron microscopy structure of a fibril purified from the tissue of a patient with hereditary Val30Met ATTR amyloidosis. The fibril consists of a single protofilament that is formed from an N-terminal and a C-terminal fragment of transthyretin. Our structure provides insights into the mechanism of misfolding and implies the formation of an early fibril state from unfolded transthyretin molecules, which upon proteolysis converts into mature ATTR amyloid fibrils.
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http://dx.doi.org/10.1038/s41467-019-13038-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6825171PMC
November 2019

The Architecture of Talin1 Reveals an Autoinhibition Mechanism.

Cell 2019 Sep;179(1):120-131.e13

Department of Structural Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany. Electronic address:

Focal adhesions (FAs) are protein machineries essential for cell adhesion, migration, and differentiation. Talin is an integrin-activating and tension-sensing FA component directly connecting integrins in the plasma membrane with the actomyosin cytoskeleton. To understand how talin function is regulated, we determined a cryoelectron microscopy (cryo-EM) structure of full-length talin1 revealing a two-way mode of autoinhibition. The actin-binding rod domains fold into a 15-nm globular arrangement that is interlocked by the integrin-binding FERM head. In turn, the rod domains R9 and R12 shield access of the FERM domain to integrin and the phospholipid PIP at the membrane. This mechanism likely ensures synchronous inhibition of integrin, membrane, and cytoskeleton binding. We also demonstrate that compacted talin1 reversibly unfolds to an ∼60-nm string-like conformation, revealing interaction sites for vinculin and actin. Our data explain how fast switching between active and inactive conformations of talin could regulate FA turnover, a process critical for cell adhesion and signaling.
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http://dx.doi.org/10.1016/j.cell.2019.08.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6856716PMC
September 2019

Phosphorylation of Ser111 in Rab8a Modulates Rabin8-Dependent Activation by Perturbation of Side Chain Interaction Networks.

Biochemistry 2019 08 8;58(33):3546-3554. Epub 2019 Aug 8.

Physics Department T38 , Technical University of Munich , James-Franck-Strasse 1 , 85748 Garching , Germany.

GTPases are key players during cellular signaling. Phosphorylation of Rab proteins, which belong to the Ras superfamily of small GTPases regulating intracellular transport, has been implicated in the pathogenesis of Parkinson's disease. For Rab8a, it was shown that serine 111 phosphorylation (pS111) is dependent on the protein kinase PINK1 and that mimicking the phosphorylation at S111 by a serine/glutamate substitution (S111E) impaired Rab8a activation by its cognate nucleotide exchange factor (GEF) Rabin8. However, Ser111 is not part of the interface of the Rab8a:Rabin8 complex. Here, we performed comparative molecular dynamics and free energy simulations on Rab8a and Rab8a:Rabin8 complexes to elucidate the molecular details of how pS111 and S111E may influence the interaction with Rabin8. The simulations indicate that S111E and pS111 establish an intramolecular interaction with arginine 79 (R79). The interaction persists in the complex and perturbs a favorable intermolecular salt-bridge contact between R79 in Rab8a and aspartate 187 in Rabin8. Binding free energy analysis reveals that S111E and pS111, as well as the R79A mutation, drastically decrease the binding affinity for Rabin8. Combining the R79A mutation with S111E or pS111 nearly diminishes Rab8a-Rabin8 binding. experiments confirm our computational results showing a >80% decrease in the nucleotide exchange rate of the respective Rab8a mutants in the presence of Rabin8 compared to that of the wild type. In addition to insights into how S111 phosphorylation of Rab8a influences GEF-mediated activation, the simulations demonstrate how side chain modifications in general can allosterically influence the surface side chain interaction network between binding partners.
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http://dx.doi.org/10.1021/acs.biochem.9b00516DOI Listing
August 2019

Empty peptide-receptive MHC class I molecules for efficient detection of antigen-specific T cells.

Sci Immunol 2019 07;4(37)

Department of Health Technology, Technical University of Denmark (DTU), Denmark.

The peptide-dependent stability of MHC class I molecules poses a substantial challenge for their use in peptide-MHC multimer-based approaches to comprehensively analyze T cell immunity. To overcome this challenge, we demonstrate the use of functionally empty MHC class I molecules stabilized by a disulfide bond to link the α and α helices close to the F pocket. Peptide-loaded disulfide-stabilized HLA-A*02:01 shows complete structural overlap with wild-type HLA-A*02:01. Peptide-MHC multimers prepared using disulfide-stabilized HLA-A*02:01, HLA-A*24:02, and H-2K can be used to identify antigen-specific T cells, and they provide a better staining index for antigen-specific T cell detection compared with multimers prepared with wild-type MHC class I molecules. Disulfide-stabilized MHC class I molecules can be loaded with peptide in the multimerized form without affecting their capacity to stain T cells. We demonstrate the value of empty-loadable tetramers that are converted to antigen-specific tetramers by a single-step peptide addition through their use to identify T cells specific for mutation-derived neoantigens and other cancer-associated antigens in human melanoma.
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http://dx.doi.org/10.1126/sciimmunol.aau9039DOI Listing
July 2019

Uncovering the Binding Mode of γ -Secretase Inhibitors.

ACS Chem Neurosci 2019 08 25;10(8):3398-3403. Epub 2019 Jun 25.

Physics Department T38 , Technical University of Munich , James-Frank-Strasse 1 , 85748 Garching , Germany.

Knowledge of how transition state inhibitors bind to γ-secretase is of major importance for the design of new Alzheimer's disease therapies. On the basis of the known structure of γ-secretase in complex with a fragment of the amyloid precursor protein, we generated a structural model of γ-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. The model also indicates the possible location and nature of the amino acid residues forming the proposed binding pockets S1', S2', and S3' near the active site that are occupied by chemical groups of the inhibitor. In addition, we found that the stability of the complex is very likely 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 γ-secretase. The structural model could form a valuable basis for the design of new or modified γ-secretase inhibitors.
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http://dx.doi.org/10.1021/acschemneuro.9b00272DOI Listing
August 2019

Prognostic value of cyclin A2 and B1 expression in lung carcinoids.

Pathology 2019 Aug 20;51(5):481-486. Epub 2019 Jun 20.

Medical University of Graz, Diagnostic and Research Institute of Pathology, Graz, Austria.

Carcinoid classification in the lung is still based on morphological criteria. Although there are many studies investigating the role of Ki-67 proliferation index in the classification of lung neuroendocrine tumours, it is still not used in routine diagnostics. Interestingly, cyclins, which have a crucial role in controlling the cell cycle, have not been thoroughly studied in lung neuroendocrine tumours. The aim of our study was to investigate the correlation of cyclin A2 and B1 expression with prognosis, Ki-67 proliferation index, and carcinoid morphology. A cohort of 134 resected typical and atypical carcinoids was stained with antibodies against Ki-67, cyclin A2 and B1. The positive nuclear reaction was assessed in hot spot areas and expressed as the percentage of tumour cells. Univariate analyses found the highest relative hazard between low and high cyclin A2 expression both with respect to overall survival [hazard ratio (HR)=16; 95% confidence interval (CI) 4.8-51; p=0.0000054], and relapse (HR=8; 95% CI 3.1-21; p=0.00002). In multivariate analysis for overall survival cyclin A2 (HR=10; 95% CI 2.5->100; p=0.0082) and B1 (HR=6.5; 95% CI 1.5-35; p=0.02) remained significant when adjusted for other risk factors, whereas Ki-67 was no longer significant (HR=0.64; 95% CI 0.003-5.5; p=0.65). This suggests that Ki-67 is closer to conventional risk factors for survival than cyclin A2 and B1. Furthermore, the analysis revealed 4 mitoses per 2 mm as a more powerful prognostic cut-off than currently accepted 2 mitoses. We have clearly demonstrated that application of cyclin A2 and cyclin B1 might bring additional value regarding the overall and progression-free survival of patients with carcinoids of the lung.
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http://dx.doi.org/10.1016/j.pathol.2019.03.011DOI Listing
August 2019

Extracellular interface between APP and Nicastrin regulates Aβ length and response to γ-secretase modulators.

EMBO J 2019 06 20;38(12). Epub 2019 May 20.

VIB-KU Leuven Center for Brain & Disease Research, Leuven, Belgium

γ-Secretase complexes (GSECs) are multimeric membrane proteases involved in a variety of physiological processes and linked to Alzheimer's disease (AD). Presenilin (PSEN, catalytic subunit), Nicastrin (NCT), Presenilin Enhancer 2 (PEN-2), and Anterior Pharynx Defective 1 (APH1) are the essential subunits of GSECs. Mutations in PSEN and the Amyloid Precursor Protein (APP) cause early-onset AD GSECs successively cut APP to generate amyloid-β (Aβ) peptides of various lengths. AD-causing mutations destabilize GSEC-APP/Aβ interactions and thus enhance the production of longer Aβs, which elicit neurotoxic events underlying pathogenesis. Here, we investigated the molecular strategies that anchor GSEC and APP/Aβ during the sequential proteolysis. Our studies reveal that a direct interaction between NCT ectodomain and APP influences the stability of GSEC-Aβn assemblies and thereby modulates Aβ length. The data suggest a potential link between single-nucleotide variants in and AD risk. Furthermore, our work indicates that an extracellular interface between the protease (NCT, PSEN) and the substrate (APP) represents the target for compounds (GSMs) modulating Aβ length. Our findings may guide future rationale-based drug discovery efforts.
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http://dx.doi.org/10.15252/embj.2019101494DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6576158PMC
June 2019