Carsten Baldauf

Shear-induced unfolding activates von Willebrand factor A2 domain for proteolysis

Summary.  Background: To avoid pathological platelet aggregation by von Willebrand factor (VWF), VWF multimers are regulated in size and reactivity for adhesion by ADAMTS13‐mediated proteolysis in a shear flow dependent manner. Objective and methods: We examined whether tensile stress in VWF under shear flow activates the VWF A2 domain for cleavage by ADAMTS13 using molecular dynamics simulations. We generated a full length mutant VWF featuring a homologous disulfide bond in A2 (N1493C and C1670S), in an attempt to lock A2 against unfolding. Results: We indeed observed stepwise unfolding of A2 and exposure of its deeply buried ADAMTS13 cleavage site. Interestingly, disulfide bonds in the adjacent and highly homologous VWF A1 and A3 domains obstruct their mechanical unfolding. We find this mutant A2 (N1493C and C1670S) to feature ADAMTS13‐resistant behavior in vitro. Conclusions: Our results yield molecular‐detail evidence for the force‐sensing function of VWF A2, by revealing how tension in VWF due to shear flow selectively exposes the A2 proteolysis site to ADAMTS13 for cleavage while keeping the folded remainder of A2 intact and functional. We find the unconventional ‘knotted’ Rossmann fold of A2 to be the key to this mechanical response, tailored for regulating VWF size and activity. Based on our model we discuss the pathomechanism of some natural mutations in the VWF A2 domain that significantly increase the cleavage by ADAMTS13 without shearing or chemical denaturation, and provide with the cleavage‐activated A2 conformation a structural basis for the design of inhibitors for VWF type 2 diseases.

Photodissociation of Conformer-Selected Ubiquitin Ions Reveals Site-Specific Cis/Trans Isomerization of Proline Peptide Bonds

Ultraviolet photodissociation (UVPD) of gas-phase proteins has attracted increased attention in recent years. This growing interest is largely based on the fact that, in contrast to slow heating techniques such as collision induced dissociation (CID), the cleavage propensity after absorption of UV light is distributed over the entire protein sequence, which can lead to a very high sequence coverage as required in typical top-down proteomics applications. However, in the gas phase, proteins can adopt a multitude of distinct and sometimes coexisting conformations, and it is not clear how this three-dimensional structure affects the UVPD fragmentation behavior. Using ion mobility-UVPD-mass spectrometry in conjunction with molecular dynamics simulations, we provide the first experimental evidence that UVPD is sensitive to the higher order structure of gas-phase proteins. Distinct UVPD spectra were obtained for different extended conformations of 11(+) ubiquitin ions. Assignment of the fragments showed that the majority of differences arise from cis/trans isomerization of one particular proline peptide bond. Seen from a broader perspective, these data highlight the potential of UVPD to be used for the structural analysis of proteins in the gas phase.

Helices in peptoids of α- and β-peptides

Peptoids of α- and β-peptides (α- and β-peptoids) can be obtained by shifting the amino acid side chains from the backbone carbon atoms of the monomer constituents to the peptide nitrogen atoms. They are, therefore, N-substituted poly-glycines and poly-β-alanines, respectively. Due to the substituted nitrogen atoms, the ability for hydrogen bond formation between peptide bonds gets lost. It may be very interesting to see whether such non-natural oligomers could be regarded as foldamers, which fold into definite backbone conformers. In this paper, we provide a complete overview on helix formation in α- and β-peptoids on the basis of systematic theoretical conformational analyses employing the methods of ab initio molecular orbital (MO) theory. It can be shown that the α- and β-peptoid structures form helical structures with both trans and cis peptide bonds despite the missing hydrogen bonds. Obviously, the conformational properties of the backbone are more important for folding than the possibility of hydrogen bonding. There are close relationships between the helices of α-peptoids and poly-glycine and poly-proline helices of α-peptides, whereas the helices of β-peptoids correspond to the well-known helical structures of β-peptides as, for instance, the 31-helix of β-peptides with 14-membered hydrogen-bonded rings. Thus, α- and β-peptoids enrich the field of foldamers and may be used as useful tools in peptide and protein design.

von Willebrand factor directly interacts with DNA from neutrophil extracellular traps.

Objective—Inflammatory conditions provoke essential processes in the human vascular system. It leads to the formation of ultralarge von Willebrand factor (VWF) fibers, which are immobilized on the endothelial cell surface and transform to highly adhesive strings under shear conditions. Furthermore, leukocytes release a meshwork of DNA (neutrophil extracellular traps) during the process of the recently discovered cell death program NETosis. In the present study, we characterized the interaction between VWF and DNA and possible binding sites to underline the role of VWF in thrombosis and inflammation besides its function in platelet adhesion. Approach and Results—Both functionalized surfaces and intact cell layers of human umbilical vein endothelial cells were perfused with isolated, protein-free DNA or leukocytes from whole blood at distinct shear rates. DNA–VWF interaction was monitored using fluorescence microscopy, ELISA-based assays, molecular dynamics simulations, and electrostatic potential calculations. Isolated DNA, as well as DNA released by stimulated leukocytes, was able to bind to shear-activated, but not inactivated, VWF. However, DNA–VWF binding does not alter VWF degradation by a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13. Moreover, DNA–VWF interaction can be blocked using unfractionated and low-molecular-weight heparin, and DNA–VWF complexes attenuate platelet binding to VWF. These findings were supported using molecular dynamics simulations and electrostatic calculations of the A1- and A2-domains. Conclusions—Our findings suggest that VWF directly binds and immobilizes extracellular DNA released from leukocytes. Therefore, we hypothesize that VWF might act as a linker for leukocyte adhesion to endothelial cells, supporting leukocyte extravasation and inflammation.

ParaDockS: a framework for molecular docking with population-based metaheuristics.

Molecular docking is a simulation technique that aims to predict the binding pose between a ligand and a receptor. The resulting multidimensional continuous optimization problem is practically unsolvable in an exact way. One possible approach is the combination of an optimization algorithm and an objective function that describes the interaction. The software ParaDockS is designed to hold different optimization algorithms and objective functions. At the current stage, an adapted particle-swarm optimizer (PSO) is implemented. Available objective functions are (i) the empirical objective function p-Score and (ii) an adapted version of the knowledge-based potential PMF04. We tested the docking accuracy in terms of reproducing known crystal structures from the PDBbind core set. For 73% of the test instances the native binding mode was found with an rmsd below 2 A. The virtual screening efficiency was tested with a subset of 13 targets and the respective ligands and decoys from the directory of useful decoys (DUD). ParaDockS with PMF04 shows a superior early enrichment. The here presented approach can be employed for molecular docking experiments and virtual screenings of large compound libraries in academia as well as in industrial research and development. The performance in terms of accuracy and enrichment is close to the results of commercial software solutions.

A novel calcium-binding site of von Willebrand factor A2 domain regulates its cleavage by ADAMTS13

The proteolysis of VWF by ADAMTS13 is an essential step in the regulation of its hemostatic and thrombogenic potential. The cleavage occurs at strand β4 in the structural core of the A2 domain of VWF, so unfolding of the A2 domain is a prerequisite for cleavage. In the present study, we present the crystal structure of an engineered A2 domain that exhibits a significant difference in the α3-β4 loop compared with the previously reported structure of wild-type A2. Intriguingly, a metal ion was detected at a site formed mainly by the C-terminal region of the α3-β4 loop that was later identified as Ca(²+) after various biophysical and biochemical studies. Force-probe molecular dynamic simulations of a modeled structure of the wild-type A2 featuring the discovered Ca(²+)-binding site revealed that an increase in force was needed to unfold strand β4 when Ca(²+) was bound. Cleavage assays consistently demonstrated that Ca(²+) binding stabilized the A2 domain and impeded its unfolding, and consequently protected it from cleavage by ADAMTS13. We have revealed a novel Ca(²+)-binding site at the A2 domain of VWF and demonstrated a relationship between Ca(²+) and force in the regulation of VWF and primary hemostasis.

Synthesis and Structure of α/δ-Hybrid Peptides—Access to Novel Helix Patterns in Foldamers

Stimulated by an overview on all periodic folding patterns of alpha/delta-hybrid peptides with 1:1 alternating backbone provided by ab initio molecular orbital theory, the first representatives of this foldamer class were synthesized connecting novel C-linked carbo-delta-amino acid constituents and L-Ala. In agreement with theoretical predictions, extensive NMR spectroscopic analyses confirm the formation of new motifs of 13/11-mixed helical patterns in these peptides supported by the rigidity of the D-xylose side chain in the selected delta-amino acid constituents. Relationships between possible helix types in alpha/delta-hybrid peptides and their counterparts in other 1:1 hybrid peptide classes and native alpha-peptides are discussed; these indicate the high potential of these foldamers to mimic native peptide secondary structures. The design of alpha/delta-hybrid peptides provides an opportunity to expand the domain of foldamers and allows the introduction of desired functionalities through the alpha-amino acid constituents.

A β/γ Motif to Mimic α-Helical Turns in Proteins

The attempt to construct nature’s architecture from nonnatural building blocks has challenged scientists for many decades. One goal of this field of study is to overcome the intrinsic protease susceptibility of natural peptides as it limits their clinical use. Peptides composed of homologous amino acids, those that have additional backbone methylene units compared to the natural a-amino acids, are at present among the most widely studied biomimetic oligomers that adopt well-defined conformations (foldamers). The wide variety of specific secondary structures that can be adopted by band g-peptides becomes especially valuable for the design of higher levels of organization such as tertiary or quaternary structures. Previous efforts towards this goal employing b-amino acid building blocks led to the discovery of both homomeric and heteromeric helix bundles and helical inhibitors of protein–protein interactions; however, there are significant differences between the packing observed in these artificial quaternary assemblies and that in the corresponding natural assemblies. This phenomenon has thus far impeded the combination of both classes into compact protein-like chimeric structures. The aim of the current study was to identify extended sequences of band g-amino acids that can be incorporated into an a-helical coiled coil to produce artificial chimeric folding motifs. Such artificial motifs with their orthogonal structural elements are great candidates for incorporation into natural helical proteins. Because protein–protein interactions involving helical domains determine specificity for important biological processes such as transcriptional control, cellular differentiation, and replication, selective disruption should be an excellent strategy for drug discovery. We were inspired by previous reports in which the principle of “equal backbone atoms” was suggested. Those designs were based on either unsubstituted or conformationally constrained amino acids. In particular b/g-hybrid peptides appear to be well-suited to mimic an a-helical conformation, thus we focused on preserving the natural side chains for the purpose of accurately imitating the natural packing in order to lend stability to the assembly. The a-helical coiled coil is a well-conserved and versatile folding motif that can serve as a model for tertiary and quaternary protein structures. This motif features a canonical heptad repeat, (abcdefg)n, in which hydrophobic residues occupy the a and d positions; these side chains make up the hydrophobic core of the interhelical interface. Charged residues at e and g generally form the second molecular recognition motif by interhelical ionic interactions. One such characteristic heptad, comprising three 13-atom hydrogen-bonded turns of the helix, can be substituted by a pentad repeat of alternating band g-amino acids with retention of the helix dipole and the formation of two 13-membered helix turns. The peptide model system described here comprises a basic a-peptide “Base-pp” which has a high propensity for heterooligomerization to an a-helical coiled coil in the presence of the acidic peptide “Acid-pp” (Figure 1 A). Heterooligomerization is driven by the burial of hydrophobic surface area, primarily contributed by Leu, and is directed by electrostatic interactions between Lys and Glu residues that flank the hydrophobic core. To evaluate b/g-hybrid peptides as a-helix mimics, the two central turns of Base-pp (positions 15–21) were replaced by a pentad of alternating band g-amino acid residues in the chimera B3b2g (Figure 1 B and C). CD spectroscopy (Figure 2 A) indicates random coil and mostly unfolded conformations for B3b2g and Acid-pp, respectively, as was expected based on the design of positions e and g. In contrast, an equimolar mixture of B3b2g and Acid-pp shows significant a-helical structure formation with two well defined minima at 208 and 222 nm. Analysis of the ellipticity at 222 nm as a function of the mole fraction of B3b2g reveals a global minimum at 0.5 (inset in Figure 2 A), which corresponds to the presence of a heteromeric assembly between Acid-pp and B3b2g with 1:1 stoichiometry. Size exclusion chromatography (SEC) was performed to characterize the oligomerization states of the peptides described above. Comparison of retention times with the peptides GCN4-p1, GCN4-pII, and GCN4-pLI as investigated by Harbury et al. suggests the presence of monomeric species (64 min) for the individual peptides Acid-pp, Base-pp and B3b2g, but the formation of four-helix-bundles (57 min) in the equimolar mixtures Acid-pp/Base-pp and Acid-pp/B3b2g (Figure 2 B). Also, [a] R. Rezaei Araghi, M. Salwiczek, S. C. Wagner, S. Wieczorek, Prof. Dr. B. Koksch Institute of Chemistry and Biochemistry, Freie Universit t Berlin Takustraße 3, 14195 Berlin (Germany) Fax: (+ 49) 30-83855644 E-mail : koksch@chemie.fu-berlin.de [b] Dr. C. J ckel Laboratory of Organic Chemistry, Eidgençssische Technische Hochschule Wolfgang-Paulistrasse 10, 8093 Z rich (Switzerland) [c] Dr. H. Cçlfen, A. Vçlkel Department of Colloid Chemistry, Max-Planck-Institute of Colloids and Interfaces 14424 Potsdam (Germany) [d] Dr. C. Baldauf BioQuant, Ruprecht-Karls-Universit t Heidelberg Im Neuenheimer Feld 267, 69120 Heidelberg (Germany) [e] Dr. C. Baldauf MPG-CAS Partner Institute for Computational Biology 320 Yue Yang Road, 200031 Shanghai (P. R. China) Fax: (+ 86) 21-54920451 E-mail : carsten@picb.ac.cn Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.200900700.

Side-chain control of folding of the homologous α-, β-, and γ-peptides into “mixed” helices (β-helices)

A systematic analysis of the substituent influence on the formation of the unique secondary structure type of “mixed” helices in the homologous α‐, β‐, and γ‐peptides was performed on the basis of ab initio molecular orbital theory. Contrary to the common periodic peptide helices, mixed helices have an alternating periodicity and their hydrogen‐bonding pattern is similar to those of β‐sheets. They belong, therefore, to the family of β‐helices. It is shown that folding of peptide sequences into mixed helices is energetically preferred over folding into their periodic counterparts in numerous cases. The influence of entropy and solvents on the formation of the various competitive mixed and periodic helix types is discussed. Among the oligomers of the various homologous amino acids, β‐peptides show the highest tendency to form β‐helices. The rules of substituent influence derived from the analysis of a wide variety of backbone substitution patterns might be helpful for a rational design of mixed helix structures, which could be important for mimicking membrane channels.

Conformations of prolyl-peptide bonds in the bradykinin 1-5 fragment in solution and in the gas phase

The dynamic nature of intrinsically disordered peptides makes them a challenge to characterize by solution-phase techniques. In order to gain insight into the relation between the disordered state and the environment, we explore the conformational space of the N-terminal 1-5 fragment of bradykinin (BK[1-5](2+)) in the gas phase by combining drift tube ion mobility, cold-ion spectroscopy, and first-principles simulations. The ion-mobility distribution of BK[1-5](2+) consists of two well-separated peaks. We demonstrate that the conformations within the peak with larger cross-section are kinetically trapped, while the more compact peak contains low-energy structures. This is a result of cis-trans isomerization of the two prolyl-peptide bonds in BK[1-5](2+). Density-functional theory calculations reveal that the compact structures have two very different geometries with cis-trans and trans-cis backbone conformations. Using the experimental CCSs to guide the conformational search, we find that the kinetically trapped species have a trans-trans configuration. This is consistent with NMR measurements performed in a solution, which show that 82% of the molecules adopt a trans-trans configuration and behave as a random coil.