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Proceedings of the National Academy of Sciences of the United States of America 117(37), 22841 - 22848 (2020). doi:10.1073/pnas.2010317117, Quantum mechanics/molecular mechanics (QM/MM) maturation of an immunoglobulin (Ig) powered by supercomputation delivers novel functionality to this catalytic template and facilitates artificial evolution of biocatalysts. We here employ density functional theory-based (DFT-b) tight binding and funnel metadynamics to advance our earlier QM/MM maturation of A17 Ig-paraoxonase (WTIgP) as a reactibody for organophosphorus toxins. It enables regulation of biocatalytic activity for tyrosine nucleophilic attack on phosphorus. The single amino acid substitution l-Leu47Lys results in 340-fold enhanced reactivity for paraoxon. The computed ground-state complex shows substrate-induced ionization of the nucleophilic l-Tyr37, now H-bonded to l-Lys47, resulting from repositioning of l-Lys47. Multiple antibody structural homologs, selected by phenylphosphonate covalent capture, show contrasting enantioselectivities for a P-chiral phenylphosphonate toxin. That is defined by crystallographic analysis of phenylphosphonylated reaction products for antibodies A5 and WTIgP. DFT-b analysis using QM regions based on these structures identifies transition states for the favored and disfavored reactions with surprising results. This stereoselection analysis is extended by funnel metadynamics to a range of WTIgP variants whose predicted stereoselectivity is endorsed by experimental analysis. The algorithms used here offer prospects for tailored design of highly evolved, genetically encoded organophosphorus scavengers and for broader functionalities of members of the Ig superfamily, including cell surface-exposed receptors., Published by National Acad. of Sciences, Washington, DC

It is proposed to perform quantum mechanical/molecular dynamics calculations of chemical reactions that are planned to be catalyzed by antibodies and then conduct a virtual screening of the library of potential antibody mutants to select an optimal biocatalyst. We tested the effectiveness of this approach by the example of hydrolysis of organophosphorus toxicant paraoxon using kinetic approaches and X-ray analysis of the antibody biocatalyst designed de novo.

In striated muscles, molecular filaments are largely composed of long protein chains with extensive arrays of identically folded domains, referred to as “beads-on-a-string”. It remains a largely unresolved question how these domains have developed a unique molecular profile such that each carries out a distinct function without false-positive readout. This study focuses on the M-band segment of the sarcomeric protein titin, which comprises ten identically folded immunoglobulin domains. Comparative analysis of high-resolution structures of six of these domains ‒ M1, M3, M4, M5, M7, and M10 ‒ reveals considerable structural diversity within three distinct loops and a non-conserved pattern of exposed cysteines. Our data allow to structurally interpreting distinct pathological readouts that result from titinopathy-associated variants. Our findings support general principles that could be used to identify individual structural/functional profiles of hundreds of identically folded protein domains within the sarcomere and other densely crowded cellular environments.

Expression of insulin-like growth factor 1 (IGF-1) mRNAs splice forms was recently shown to be stimulated by myofibrillar proteins released from the damaged muscle. In this study, we report that individual subfragments of titin and myomesin composed of Fn type III and Ig-like domains can activate expression of two IGF-1 splice forms in cultured myoblasts, both at protein and mRNA levels. Competition studies showed that each of the domain-types interacts with its own receptor. Induction of IGF-1 expression caused by domains of different types showed dissimilar sensitivity to inhibitors of regulatory cascades. The effect of Fn type III domains was more sensitive to inhibition of Ca(2+)/calmodulin dependent protein kinase, whereas the effect of Ig-like domains showed greater sensitivity to the inhibition of the adenylyl cyclase-cAMP-PKA pathway.

The enantioselectivity of β-cyclodextrin (β-CD) towards L- and D-N-acetyltryptophan (NAcTrp) has been studied in aqueous solution and the crystalline state. NMR studies in solution show that β-CD forms complexes of very similar but not identical geometry with both L- and D-NAcTrp and exhibits stronger binding with L-NAcTrp. In the crystalline state, only β-CD–L-NAcTrp crystallizes readily from aqueous solutions as a dimeric complex (two hosts enclosing two guest molecules). In contrast, crystals of the complex β-CD–D-NAcTrp were never obtained, although numerous conditions were tried. In aqueous solution, the orientation of the guest in both complexes is different than in the β-CD–L-NAcTrp complex in the crystal. Overall, the study shows that subtle differences observed between the β-CD–L,D-NAcTrp complexes in aqueous solution are magnified at the onset of crystallization, as a consequence of accumulation of many soft host–guest interactions and of the imposed crystallographic order, thus resulting in very dissimilar propensity of each enantiomer to produce crystals with β-CD.

The enantioselectivity of β-cyclodextrin (β-CD) towards L- and D-N-acetyltryptophan (NAcTrp) has been studied in aqueous solution and the crystalline state. NMR studies in solution show that β-CD forms complexes of very similar but not identical geometry with both L- and D-NAcTrp and exhibits stronger binding with L-NAcTrp. In the crystalline state, only β-CD–L-NAcTrp crystallizes readily from aqueous solutions as a dimeric complex (two hosts enclosing two guest molecules). In contrast, crystals of the complex β-CD–D-NAcTrp were never obtained, although numerous conditions were tried. In aqueous solution, the orientation of the guest in both complexes is different than in the β-CD–L-NAcTrp complex in the crystal. Overall, the study shows that subtle differences observed between the β-CD–L,D-NAcTrp complexes in aqueous solution are magnified at the onset of crystallization, as a consequence of accumulation of many soft host–guest interactions and of the imposed crystallographic order, thus resulting in very dissimilar propensity of each enantiomer to produce crystals with β-CD.

We report continuous-wave electron-paramagnetic-resonance (EPR) spectra of the high-spin FeII complex Fe[(SPPh2)2N]2 at 275.7 GHz, 94.1 GHz and 9.5 GHz. Combined analysis of these EPR spectra shows that the complex occurs in multiple conformations. For two main conformations the spin-Hamiltonian parameters, which reflect the electronic structure of the complex, are accurately determined: (1) D = 9.17 cm - 1 (275 GHz), E / D = 0.021 and (2) D = 8.87 cm - 1 (266 GHz), E / D = 0.052 . The EPR spectra obtained at 275.7 GHz on single crystals of the complex are essential for the analysis and in addition they reveal that the two main conformations occur at two magnetically distinguishable sites.

Science advances 2(10), e1501695 - e1501695(2016). doi:10.1126/sciadv.1501695, In vitro selection of antibodies from large repertoires of immunoglobulin (Ig) combining sites using combinatorial libraries is a powerful tool, with great potential for generating in vivo scavengers for toxins. However, addition of a maturation function is necessary to enable these selected antibodies to more closely mimic the full mammalian immune response. We approached this goal using quantum mechanics/molecular mechanics (QM/MM) calculations to achieve maturation in silico. We preselected A17, an Ig template, from a naïve library for its ability to disarm a toxic pesticide related to organophosphorus nerve agents. Virtual screening of 167,538 robotically generated mutants identified an optimum single point mutation, which experimentally boosted wild-type Ig scavenger performance by 170-fold. We validated the QM/MM predictions via kinetic analysis and crystal structures of mutant apo-A17 and covalently modified Ig, thereby identifying the displacement of one water molecule by an arginine as delivering this catalysis., Published by Assoc., Washington, DC [u.a.]

High throughput next-generation sequencing techniques have made whole genome sequencing accessible in clinical practice; however, the abundance of variation in the human genomes makes the identification of a disease-causing mutation on a background of benign rare variants challenging.Here we combine whole genome sequencing with linkage analysis in a 3-generation family affected by cardiomyopathy with features of autosomal dominant left ventricular noncompaction cardiomyopathy. A missense mutation in the giant protein titin is the only plausible disease-causing variant that segregates with disease among the 7 surviving affected individuals, with interrogation of the entire genome excluding other potential causes. This A178D missense mutation, affecting a conserved residue in the second immunoglobulin-like domain of titin, was introduced in a bacterially expressed recombinant protein fragment and biophysically characterized in comparison to its wild-type counterpart. Multiple experiments, including size exclusion chromatography, small-angle x ray scattering, and circular dichroism spectroscopy suggest partial unfolding and domain destabilization in the presence of the mutation. Moreover, binding experiments in mammalian cells show that the mutation markedly impairs binding to the titin ligand telethonin.Here we present genetic and functional evidence implicating the novel A178D missense mutation in titin as the cause of a highly penetrant familial cardiomyopathy with features of left ventricular noncompaction. This expands the spectrum of titin's roles in cardiomyopathies. It furthermore highlights that rare titin missense variants, currently often ignored or left uninterpreted, should be considered to be relevant for cardiomyopathies and can be identified by the approach presented here.

The Zn[(OPPh2)(SePPh2)N]2 complex was prepared by a metathetical reaction between a Zn(II) salt and the deprotonated form of the dichalcogenoimidodiphosphinato ligand [(OPPh2)(SePPh2)N]−. X-ray crystallography revealed a pseudo-tetrahedral MO2Se2 coordination sphere, owed to the asymmetric (O,Se) nature of the chelating ligand. A comparison between the structural features of the M[(OPPh2)(SePPh2)N]2 complexes, M = Co, Ni, Zn, shows that the three complexes are isomorphous. The IR and 31P NMR properties of Zn[(OPPh2)(SePPh2)N]2 are analyzed in the framework of its crystallographic structure, and are compared with data on similar systems. The above three complexes, along with the (OPPh2)(SePPh2)NH ligand and SeO2, are investigated as inhibitors of the Platelet Activating Factor (PAF) and thrombin activities. The paramagnetic M[(OPPh2)(SePPh2)N]2 complexes, M = Co, Ni, exhibit an approximately 65-fold increased activity, compared to diamagnetic Zn[(OPPh2)(SePPh2)N]2, in both the PAF- and the thrombin-induced aggregation of washed rabbit platelets (WRP). The above three complexes show a comparable significant inhibitory activity in the PAF-induced aggregation of rabbit platelet rich plasma (RPR). Remarkably, SeO2, a form of Se present in blood, exhibits a very strong and selective inhibitory activity towards the PAF-induced aggregation of WPR. Our studies extend the dataset of metal complexes investigated as inhibitors of PAF and thrombin.

The inclusion complex of a salicylaldehyde Schiff base (anil) in β-cyclodextrin (βCD) is reported for the first time. UV–vis spectroscopic and fluorescence studies show that N -(1-adamantyl)salicylaldimine (ASA), which is thermochromic as a free crystalline compound, becomes photochromic upon encapsulation into βCD. Characterisation of the complex has been performed by NMR spectroscopy in aqueous solution and by UV and fluorescence spectroscopy and powder X-ray diffraction in the crystalline state. The NMR studies indicate host:guest ratio of 1:1 and prove that only the adamantyl moiety of the guest is enclosed into the host cavity, whereas the hydroxyphenyl part is outside. Trials to grow single crystals of the complex produced a novel βCD lattice, which can be rationalized by applying the inclusion mode suggested by NMR spectroscopy. βCD in the crystal lattice seems to act as a medium allowing the cis to trans isomerisation of ASA, a volume-demanding process necessary for the expression of photochromism in this class of molecules. The study demonstrates that βCD nanocavities can lead to supramolecular control of the photochemical behaviour of appropriately designed anils, a property with many potential applications.

Sulfur-containing mono- or bidentate types of ligands, usually form square planar Ni((II))S(4) complexes. However, it has already been established that the bidentate L(-) dithioimidodiphosphinato ligands, [R(2)P(S)NP(S)R'(2)](-), R, and R' = aryl or alkyl, can afford both tetrahedral and square planar, NiS(4)-containing, homoleptic Ni(R,R')L(2) complexes, owing to an apparent structural flexibility, which has not, so far, been probed. In this work, the literature tetrahedral Ni[R(2)P(S)NP(S)R(2)](2) complexes, R = Ph (Ni(Ph,Ph)L(2), 1(Td)) and R = (i)Pr (Ni(iPr,iPr)L(2), 2) as well as the newly synthesized Ni[(i)Pr(2)P(S)NP(S)Ph(2)](2) complex (Ni(iPr,Ph)L(2), 3), have been studied by UV-vis, IR, and (31)P NMR spectroscopy. Complex 3 was shown by X-ray crystallography to be square planar, and magnetic studies confirmed that it is diamagnetic in the solid state. However, it becomes paramagnetic in solution, as it shows a similar UV-vis spectrum to one of the tetrahedral 1(Td) and 2 complexes. The crystal structure of the potassium salt of the asymmetric ligand, [(i)Pr(2)P(S)NP(S)Ph(2)]K, has also been determined and compared to those of the protonated (i)Pr(2)P(S)NHP(S)Ph(2) ligand and complex 3. All three, 1(Td), 2, and 3, Ni(R,R')L(2) complexes show strong paramagnetic effects in their solution (31)P NMR spectra. The magnetic properties of paramagnetic complexes 1 and 2 in the solid state were investigated on oriented crystals, and their analysis afforded remarkably small values of the spin-orbit coupling constant (lambda) and orbital reduction factor (k) parameters, implying significant delocalization of unpaired electronic density toward the ligands. The above experimental findings are combined with data from standard density functional theory and correlated multiconfiguration ab initio theoretical methods, in an effort to investigate the interplay between the square planar and tetrahedral geometries of the NiS(4) core, the mechanistic pathway for the spin-state interconversion, the degree of covalency of the Ni-S bonds, and the distribution of the spin density in this type of system. The analysis provides justification for the structural flexibility of such ligands, affording Ni(R,R')L(2) complexes with variable metallacycle conformation and NiS(4) core geometries. Of particular importance are the large zero-field splitting values estimated by both experimental and theoretical means, which have not, as yet, been verified by direct methods, such as electron paramagnetic resonance spectroscopy. The findings of our work confirm earlier observations on the feasibility of synthesizing either tetrahedral or square planar NiS(4) complexes containing the same type of ligands. They can also form the basis of investigating structure-properties relationships in other NiS(4)-containing systems.

The reaction between Co(II) and the deprotonated tetraphenylimidodiphosphinic acid, [(OPPh2)2N]−, afforded a binuclear [Co2{(OPPh2)2N}4] complex, as revealed by X-ray crystallography. The structure contains two monometallic biconnective [(OPPh2)2N]− ligands, with an averaged Co−O bond length of 2.008 A. The other two [(OPPh2)2N]− act as bimetallic triconnective ligands, forming two oxygen atom bridges, with the four Co−O(bridging) distances averaged at 2.155 A, and the two Co−O(non-bridging) distances at 1.973 A. The two approximately trigonal bipyramidal Co(II) ions are at a Co⋯Co distance of 3.301 A. The analysis of the magnetic properties of [Co2{(OPPh2)2N}4] is consistent with an unusual ferromagnetic interaction, in a binuclear Co(II) system, between the two S = 3/2 Co(II) ions.

Catalytic antibody variants with κ and λ light-chain constant domains show differences in their crystal structures which lead to subtle changes in catalytic efficiency and thermodynamic parameters as well as in their affinity for peptide substrates., The engineering of catalytic function in antibodies requires precise information on their structure. Here, results are presented that show how the antibody domain structure affects its functionality. The previously designed organophos­phate-metabolizing reactibody A17 has been re-engineered by replacing its constant κ light chain by the λ chain (A17λ), and the X-ray structure of A17λ has been determined at 1.95 Å resolution. It was found that compared with A17κ the active centre of A17λ is displaced, stabilized and made more rigid owing to interdomain interactions involving the CDR loops from the VL and VH domains. These VL/VH domains also have lower mobility, as deduced from the atomic displacement parameters of the crystal structure. The antibody elbow angle is decreased to 126° compared with 138° in A17κ. These structural differences account for the subtle changes in catalytic efficiency and thermodynamic parameters determined with two organophosphate ligands, as well as in the affinity for peptide substrates selected from a combinatorial cyclic peptide library, between the A17κ and A17λ variants. The data presented will be of interest and relevance to researchers dealing with the design of antibodies with tailor-made functions.

The complete resolution of 2,2[prime or minute]-dihydroxy-1,1[prime or minute]-binaphthyl into its enantiomers by inclusion complexation with a commercially available derivative of choline, is reported. The two enantiomers are recovered in99% ee from the inclusion complexes by simple dissolution in a diethyl ether-water medium and the resolving agent can be recycled.

Twelve Schiff bases of methoxy-substituted salicylaldehyde have been examined by crystallographic and spectroscopic methods, as well as by DFT theoretical calculations in order to investigate the effect of the substituent's position on the keto-enol equilibrium in the crystalline state. Four out of the 10 structurally characterized compounds with methoxy substitution on the para and/or ortho positions with respect to the aldimine bridge and deriving from aliphatic amines or alkylarylamines are found as cis-keto tautomers and form dimers. In contrast, the five pure enol tautomers derive either from aliphatic or alkylarylamines and are meta substituted or from aniline or benzylamine and are para and/or ortho methoxy substituted. The DFT calculations support the crystallographic results and, moreover, they have shown that keto and enol tautomers are affected differently by the relative arrangement of the monomers. Overall, the DFT calculations point to a plausible hypothesis for the stabilization of the keto form in the crystalline state: In cases with a sufficiently low enol-keto energy difference of the isolated monomers, as when the methoxy group is at ortho and/or para positions with respect to the aldimino group, extra stabilization of the keto form is derived from molecular association, thus leading to its crystallization.

by hybrid structural biology Matthias Wilmanns, Petr Konarev, Dmitri Svergun, Gabriel Zoldak, Matthias Rief, Fabienne Beuron, Ed Morris, & Spyros D. Chatziefthimiou, European Molecular Biology Laboratory Hamburg Unit, Hamburg, Germany, Institute for Biophysics and Munich Center for Integrated Protein Science, Technical University of Munich, Munich, Germany, Section of Structural Biology, The Institute of Cancer Research, Chester Beatty Laboratories, London, United Kingdom. E-mail: wilmanns@embl-hamburg.de

The inclusion of the long conjugated 4-pyridinealdazine molecule in β-cyclodextrin results in a novel trimer association of β-cyclodextrin macrocycles observed for the first time and an unusual face-to-face assembly of hydrated guest dimers inside the cavity.