Crystal Structure and NMR of an α,δ-Peptide Foldamer Helix Shows Side-Chains are Well Placed for Bifunctional Catalysis: Application as a Minimalist Aldolase Mimic.

We report the first NMR and X-ray diffraction (XRD) structures of an unusual 13/11-helix (alternating i, i+1 {NH-O=C} and i, i+3 {C=O-H-N} H-bonds) formed by a heteromeric 1 : 1 sequence of α- and δ-amino acids, and demonstrate the application of this framework towards catalysis. Whilst intramolecular hydrogen bonds (IMHBs) are the clear driver of helix formation in this system, we also observe an apolar interaction between the ethyl residue of one δ-amino acid and the cyclohexyl group of the next δ-residue in the sequence that seems to stabilize one type of helix over another. To the best of our knowledge this type of additional stabilization leading to a specific helical preference has not been observed before. Critically, the helix type realized places the α-residue functionalities in positions proximal enough to engage in bifunctional catalysis as demonstrated in the application of our system as a minimalist aldolase mimic.

Benchmarking polarizable and non-polarizable force fields for Ca2+-peptides against a comprehensive QM dataset.

Explicit description of atomic polarizability is critical for the accurate treatment of inter-molecular interactions by force fields (FFs) in molecular dynamics (MD) simulations aiming to investigate complex electrostatic environments such as metal-binding sites of metalloproteins. Several models exist to describe key monovalent and divalent cations interacting with proteins. Many of these models have been developed from ion-amino-acid interactions and/or aqueous-phase data on cation solvation. The transferability of these models to cation-protein interactions remains uncertain. Herein, we assess the accuracy of existing FFs by their abilities to reproduce hierarchies of thousands of Ca2+-dipeptide interaction energies based on density-functional theory calculations. We find that the Drude polarizable FF, prior to any parameterization, better approximates the QM interaction energies than any of the non-polarizable FFs. Nevertheless, it required improvement in order to address polarization catastrophes where, at short Ca2+-carboxylate distances, the Drude particle of oxygen overlaps with the divalent cation. To ameliorate this, we identified those conformational properties that produced the poorest prediction of interaction energies to reduce the parameter space for optimization. We then optimized the selected cation-peptide parameters using Boltzmann-weighted fitting and evaluated the resulting parameters in MD simulations of the N-lobe of calmodulin. We also parameterized and evaluated the CTPOL FF, which incorporates charge-transfer and polarization effects in additive FFs. This work shows how QM-driven parameter development, followed by testing in condensed-phase simulations, may yield FFs that can accurately capture the structure and dynamics of ion-protein interactions.

Structure and Reactivity of Al-O(H)-Al Moieties in Siloxide Frameworks: Solution and Gas-Phase Model Studies.

Even though aluminas and aluminosilicates have found widespread application, a consistent molecular understanding of their surface heterogeneity and the behavior of defects resulting from hydroxylation/dehydroxylation remains unclear. Here, we study the well-defined molecular model compound, [Al3 (µ2 -OH)3 (THF)3 (PhSi(OSiPh2 O)3 )2 ], 1, to gain insight into the acid-base reactivity of cyclic trinuclear Al3 (µ2 -OH)3 moieties at the atomic level. We find that, like zeolites, they are sufficiently acidic to catalyze the isomerization of olefins. DFT and gas phase vibrational spectroscopy on solvent-free and deprotonated 1 show that the six-membered ring structure of its Al3 (µ2 -OH)3 core is unstable with respect to deprotonation of one of its hydroxy groups and rearranges into two edge-sharing four-membered rings. This renders AlIV -O(H)-AlIV units strong acid sites, and all results together suggest that their acidity is similar to that of zeolitic SiIV -O(H)-AlIV groups.

The Structure of the Protonated Serine Octamer.

The amino acid serine has long been known to form a protonated "magic-number" cluster containing eight monomer units that shows an unusually high abundance in mass spectra and has a remarkable homochiral preference. Despite many experimental and theoretical studies, there is no consensus on a Ser8H+ structure that is in agreement with all experimental observations. Here, we present the structure of Ser8H+ determined by a combination of infrared spectroscopy and ab initio molecular dynamics simulations. The three-dimensional structure that we determine is ∼25 kcal mol-1 more stable than the previous most stable published structure and explains both the homochiral preference and the experimentally observed facile replacement of two serine units.

von Willebrand factor is dimerized by protein disulfide isomerase.

Multimeric von Willebrand factor (VWF) is essential for primary hemostasis. The biosynthesis of VWF high-molecular-weight multimers requires spatial separation of each step because of varying pH value requirements. VWF is dimerized in the endoplasmic reticulum by formation of disulfide bonds between the C-terminal cysteine knot (CK) domains of 2 monomers. Here, we investigated the basic question of which protein catalyzes the dimerization. We examined the putative interaction of VWF and the protein disulfide isomerase PDIA1, which has previously been used to visualize endoplasmic reticulum localization of VWF. Excitingly, we were able to visualize the PDI-VWF dimer complex by high-resolution stochastic optical reconstruction microscopy and atomic force microscopy. We proved and quantified direct binding of PDIA1 to VWF, using microscale thermophoresis and fluorescence correlation spectroscopy (dissociation constants KD = 236 ± 66 nM and KD = 282 ± 123 nM by microscale thermophoresis and fluorescence correlation spectroscopy, respectively). The similar KD (258 ± 104 nM) measured for PDI interaction with the isolated CK domain and the atomic force microscopy images strongly indicate that PDIA1 binds exclusively to the CK domain, suggesting a key role of PDIA1 in VWF dimerization. On the basis of protein-protein docking and molecular dynamics simulations, combined with fluorescence microscopy studies of VWF CK-domain mutants, we suggest the following mechanism of VWF dimerization: PDI initiates VWF dimerization by forming the first 2 disulfide bonds Cys2771-2773' and Cys2771'-2773. Subsequently, the third bond, Cys2811-2811', is formed, presumably to protect the first 2 bonds from reduction, thereby rendering dimerization irreversible. This study deepens our understanding of the mechanism of VWF dimerization and the pathophysiological consequences of its inhibition.

Probing chirality recognition of protonated glutamic acid dimers by gas-phase vibrational spectroscopy and first-principles simulations.

The homochirality of the amino acid metabolism still puzzles biochemists. Vibrational spectroscopy of mass-selected gas-phase amino acids and their clusters can precisely reveal their conformation and might ultimately help to decode the interactions responsible for chirality recognition. Infrared photodissociation (IRPD) and conformer-selective IR-IR hole burning spectra of protonated glutamic acid dimers (LL-/LD-Glu2H+) recorded in the fingerprint and XH stretch ranges (1100-1900 and 2600-3600 cm-1) provide direct insight into their stereospecific interactions. Glu2H+ dimers are generated by electrospray ionization and stored in a cryogenic quadrupole ion trap held at 10 K. The assignment of the IRPD spectra is supported by vibrational analysis using many-body dispersion-corrected hybrid density-functional theory. Sampling of the conformational space is accomplished by basin hopping and replica-exchange molecular dynamics simulations. The most stable LD-Glu2H+ dimer (LD1) is predicted to be more stable than the most stable LL-Glu2H+ dimer (LL1) by ΔE0 = 4.0 kJ mol-1, which relies on stronger secondary interactions in LD1 as demonstrated by the noncovalent interaction method. IR-IR hole burning spectroscopy reveals the coexistence of at least four LD-Glu2H+ and three LL-Glu2H+ conformers. Their IR-dip spectra are assigned to the most stable conformers at room and cryogenic temperature, revealing incomplete thermalization of the ions by kinetic trapping in the cold trap. We observe different population ratios of LL and LD conformers of Glu2H+, as revealed by specific νNH2 and νCO intensities (fingerprints of chirality recognition).

Mutation G1629E Increases von Willebrand Factor Cleavage via a Cooperative Destabilization Mechanism.

The large multimeric glycoprotein von Willebrand Factor (VWF) plays a pivotal adhesive role during primary hemostasis. VWF is cleaved by the protease ADAMTS13 as a down-regulatory mechanism to prevent excessive VWF-mediated platelet aggregation. For each VWF monomer, the ADAMTS13 cleavage site is located deeply buried inside the VWF A2 domain. External forces in vivo or denaturants in vitro trigger the unfolding of this domain, thereby leaving the cleavage site solvent-exposed and ready for cleavage. Mutations in the VWF A2 domain, facilitating the cleavage process, cause a distinct form of von Willebrand disease (VWD), VWD type 2A. In particular, the VWD type 2A Gly1629Glu mutation drastically accelerates the proteolytic cleavage activity, even in the absence of forces or denaturants. However, the effect of this mutation has not yet been quantified, in terms of kinetics or thermodynamics, nor has the underlying molecular mechanism been revealed. In this study, we addressed these questions by using fluorescence correlation spectroscopy, molecular dynamics simulations, and free energy calculations. The measured enzyme kinetics revealed a 20-fold increase in the cleavage rate for the Gly1629Glu mutant compared with the wild-type VWF. Cleavage was found cooperative with a cooperativity coefficient n = 2.3, suggesting that the mutant VWF gives access to multiple cleavage sites of the VWF multimer at the same time. According to our simulations and free energy calculations, the Gly1629Glu mutation causes structural perturbation in the A2 domain and thereby destabilizes the domain by ∼10 kJ/mol, promoting its unfolding. Taken together, the enhanced proteolytic activity of Gly1629Glu can be readily explained by an increased availability of the ADAMTS13 cleavage site through A2-domain-fold thermodynamic destabilization. Our study puts forward the Gly1629Glu mutant as a very efficient enzyme substrate for ADAMTS13 activity assays.

Mutual A domain interactions in the force sensing protein von Willebrand factor.

The von Willebrand factor (VWF) is a glycoprotein in the blood that plays a central role in hemostasis. Among other functions, VWF is responsible for platelet adhesion at sites of injury via its A1 domain. Its adjacent VWF domain A2 exposes a cleavage site under shear to degrade long VWF fibers in order to prevent thrombosis. Recently, it has been shown that VWF A1/A2 interactions inhibit the binding of platelets to VWF domain A1 in a force-dependent manner prior to A2 cleavage. However, whether and how this interaction also takes place in longer VWF fragments as well as the strength of this interaction in the light of typical elongation forces imposed by the shear flow of blood remained elusive. Here, we addressed these questions by using single molecule force spectroscopy (SMFS), Brownian dynamics (BD), and molecular dynamics (MD) simulations. Our SMFS measurements demonstrate that the A2 domain has the ability to bind not only to single A1 domains but also to VWF A1A2 fragments. SMFS experiments of a mutant [A2] domain, containing a disulfide bond which stabilizes the domain against unfolding, enhanced A1 binding. This observation suggests that the mutant adopts a more stable conformation for binding to A1. We found intermolecular A1/A2 interactions to be preferred over intramolecular A1/A2 interactions. Our data are also consistent with the existence of two cooperatively acting binding sites for A2 in the A1 domain. Our SMFS measurements revealed a slip-bond behavior for the A1/A2 interaction and their lifetimes were estimated for forces acting on VWF multimers at physiological shear rates using BD simulations. Complementary fitting of AFM rupture forces in the MD simulation range adequately reproduced the force response of the A1/A2 complex spanning a wide range of loading rates. In conclusion, we here characterized the auto-inhibitory mechanism of the intramolecular A1/A2 bond as a shear dependent safeguard of VWF, which prevents the interaction of VWF with platelets.

Kinetically Trapped Liquid-State Conformers of a Sodiated Model Peptide Observed in the Gas Phase.

We investigate the peptide AcPheAla5LysH+, a model system for studying helix formation in the gas phase, in order to fully understand the forces that stabilize the helical structure. In particular, we address the question of whether the local fixation of the positive charge at the peptide's C-terminus is a prerequisite for forming helices by replacing the protonated C-terminal Lys residue by Ala and a sodium cation. The combination of gas-phase vibrational spectroscopy of cryogenically cooled ions with molecular simulations based on density-functional theory (DFT) allows for detailed structure elucidation. For sodiated AcPheAla6, we find globular rather than helical structures, as the mobile positive charge strongly interacts with the peptide backbone and disrupts secondary structure formation. Interestingly, the global minimum structure from simulation is not present in the experiment. We interpret that this is due to high barriers involved in rearranging the peptide-cation interaction that ultimately result in kinetically trapped structures being observed in the experiment.

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.

Assessing the stability of alanine-based helices by conformer-selective IR spectroscopy.

Polyalanine based peptides that carry a lysine at the C-terminus ([Ac-AlanLys + H](+)) are known to form α-helices in the gas phase. Three factors contribute to the stability of these helices: (i) the interaction between the helix macro dipole and the charge, (ii) the capping of dangling C[double bond, length as m-dash]O groups by lysine and (iii) the cooperative hydrogen bond network. In previous studies, the influence of the interaction between the helix dipole and the charge as well as the impact of the capping was studied intensively. Here, we complement these findings by systematically assessing the third parameter, the H-bond network. In order to selectively remove one H-bond along the backbone, we use amide-to-ester substitutions. The resulting depsi peptides were analyzed by ion-mobility and m/z-selective infrared spectroscopy as well as theoretical calculations. Our results indicate that peptides which contain only one ester bond still maintain the helical conformation. We conclude that the interaction between the charge and the helix macro-dipole is most crucial for the formation of the α-helical conformation and a single backbone H-bond has only little influence on the overall stability.

Force-sensitive autoinhibition of the von Willebrand factor is mediated by interdomain interactions.

Von Willebrand factor (VWF) plays a central role in hemostasis. Triggered by shear-stress, it adheres to platelets at sites of vascular injury. Inactivation of VWF has been associated to the shielding of its adhesion sites and proteolytic cleavage. However, the molecular nature of this shielding and its coupling to cleavage under shear-forces in flowing blood remain unknown. In this study, we describe, to our knowledge, a new force-sensory mechanism for VWF-platelet binding, which addresses these questions, based on a combination of molecular dynamics (MD) simulations, atomic force microscopy (AFM), and microfluidic experiments. Our MD simulations demonstrate that the VWF A2 domain targets a specific region at the VWF A1 domain, corresponding to the binding site of the platelet glycoprotein Ibα (GPIbα) receptor, thereby causing its blockage. This implies autoinhibition of the VWF for the binding of platelets mediated by the A1-A2 protein-protein interaction. During force-probe MD simulations, a stretching force dissociated the A1A2 complex, thereby unblocking the GPIbα binding site. Dissociation was found to be coupled to the unfolding of the A2 domain, with dissociation predominantly occurring before exposure of the cleavage site in A2, an observation that is supported by our AFM experiments. This suggests that the A2 domain prevents platelet binding in a force-dependent manner, ensuring that VWF initiates hemostasis before inactivation by proteolytic cleavage. Microfluidic experiments with an A2-deletion VWF mutant resulted in increased platelet binding, corroborating the key autoinhibitory role of the A2 domain within VWF multimers. Overall, autoinhibition of VWF mediated by force-dependent interdomain interactions offers the molecular basis for the shear-sensitive growth of VWF-platelet aggregates, and might be similarly involved in shear-induced VWF self-aggregation and other force-sensing functions in hemostasis.

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.

First-Principles Molecular Structure Search with a Genetic Algorithm.

The identification of low-energy conformers for a given molecule is a fundamental problem in computational chemistry and cheminformatics. We assess here a conformer search that employs a genetic algorithm for sampling the low-energy segment of the conformation space of molecules. The algorithm is designed to work with first-principles methods, facilitated by the incorporation of local optimization and blacklisting conformers to prevent repeated evaluations of very similar solutions. The aim of the search is not only to find the global minimum but to predict all conformers within an energy window above the global minimum. The performance of the search strategy is (i) evaluated for a reference data set extracted from a database with amino acid dipeptide conformers obtained by an extensive combined force field and first-principles search and (ii) compared to the performance of a systematic search and a random conformer generator for the example of a drug-like ligand with 43 atoms, 8 rotatable bonds, and 1 cis/trans bond.

Coiled-coils in phage display screening: insight into exceptional selectivity provided by molecular dynamics.

Involved in numerous key biological functions, protein helix-helix interactions follow a well-defined intermolecular recognition pattern. The characteristic structure of the α-helical coiled-coil allows for the specific randomization of clearly defined interaction partners within heteromeric systems. In this work, a rationally designed heterodimeric coiled-coil was used to investigate potential factors influencing the sequence selectivity in interhelical interactions.

Native like helices in a specially designed ß peptide in the gas phase.

In the natural peptides, helices are stabilized by hydrogen bonds that point backward along the sequence direction. Until now, there is only little evidence for the existence of analogous structures in oligomers of conformationally unrestricted ß amino acids. We specifically designed the ß peptide Ac-(ß(2)hAla)6-LysH(+) to form native like helical structures in the gas phase. The design follows the known properties of the peptide Ac-Ala6-LysH(+) that forms a α helix in isolation. We perform ion-mobility mass-spectrometry and vibrational spectroscopy in the gas phase, combined with state-of-the-art density-functional theory simulations of these molecular systems in order to characterize their structure. We can show that the straightforward exchange of alanine residues for the homologous ß amino acids generates a system that is generally capable of adopting native like helices with backward oriented H-bonds. By pushing the limits of theory and experiments, we show that one cannot assign a single preferred structure type due to the densely populated energy landscape and present an interpretation of the data that suggests an equilibrium of three helical structures.

Exploring the conformational preferences of 20-residue peptides in isolation: Ac-Ala19-Lys + H(+)vs. Ac-Lys-Ala19 + H(+) and the current reach of DFT.

Reliable, quantitative predictions of the structure of peptides based on their amino-acid sequence information are an ongoing challenge. We here explore the energy landscapes of two unsolvated 20-residue peptides that result from a shift of the position of one amino acid in otherwise the same sequence. Our main goal is to assess the performance of current state-of-the-art density-functional theory for predicting the structure of such large and complex systems, where weak interactions such as dispersion or hydrogen bonds play a crucial role. For validation of the theoretical results, we employ experimental gas-phase ion mobility-mass spectrometry and IR spectroscopy. While unsolvated Ac-Ala19-Lys + H(+) will be shown to be a clear helix seeker, the structure space of Ac-Lys-Ala19 + H(+) is more complicated. Our first-principles structure-screening strategy using the dispersion-corrected PBE functional (PBE + vdW(TS)) identifies six distinctly different structure types competing in the low-energy regime (≈16 kJ mol(-1)). For these structure types, we analyze the influence of the PBE and the hybrid PBE0 functional coupled with either a pairwise dispersion correction (PBE + vdW(TS), PBE0 + vdW(TS)) or a many-body dispersion correction (PBE + MBD*, PBE0 + MBD*). We also take harmonic vibrational and rotational free energy into account. Including this, the PBE0 + MBD* functional predicts only one unique conformer to be present at 300 K. We show that this scenario is consistent with both experiments.

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.

System-Specific Parameter Optimization for Nonpolarizable and Polarizable Force Fields.

The accuracy of classical force fields (FFs) has been shown to be limited for the simulation of cation-protein systems despite their importance in understanding the processes of life. Improvements can result from optimizing the parameters of classical FFs or by extending the FF formulation by terms describing charge transfer (CT) and polarization (POL) effects. In this work, we introduce our implementation of the CTPOL model in OpenMM, which extends the classical additive FF formula by adding CT and POL. Furthermore, we present an open-source parametrization tool, called FFAFFURR, that enables the (system-specific) parametrization of OPLS-AA and CTPOL models. The performance of our workflow was evaluated by its ability to reproduce quantum chemistry energies and by molecular dynamics simulations of a zinc-finger protein.

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 ß4 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-ß4 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-ß4 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 ß4 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.