We present an approach to assess antibody CDR-H3 loops according to their dynamic properties using molecular dynamics simulations. We selected six antibodies in three pairs differing substantially in their individual promiscuity respectively specificity. For two pairs of antibodies crystal structures are available in different states of maturation and used as starting structures for the analyses. For a third pair we chose two antibody CDR sequences obtained from a synthetic library and predicted the respective structures. For all three pairs of antibodies we performed metadynamics simulations to overcome the limitations in conformational sampling imposed by high energy barriers. Additionally, we used classic molecular dynamics simulations to describe nano- to microsecond flexibility and to estimate up to millisecond kinetics of captured conformational transitions. The methodology represents the antibodies as conformational ensembles and allows comprehensive analysis of structural diversity, thermodynamics of conformations and kinetics of structural transitions. Referring to the concept of conformational selection we investigated the link between promiscuity and flexibility of the antibodies' binding interfaces. The obtained detailed characterization of the binding interface clearly indicates a link between structural flexibility and binding promiscuity for this set of antibodies.

Thermosensitive polymers such as poly(Nisopropylacrylamide) (PNIPAM) undergo a phase transition in aqueous solution from a random-coil structural ensemble to a globule structural ensemble at the lower critical solution temperature (LCST). Above this temperature, PNIPAM agglomerates and becomes insoluble, whereas it is soluble below the temperature. Thus, thermosensitive polymers represent essential targets for several applications, e.g., in drug delivery. Although their ability to change structure in response to a temperature alteration is highly relevant for industrial processes, their thermodynamic properties are mostly qualitatively understood, and the quantitative thermodynamic picture is still elusive. In this study, we used a combined atomistic molecular dynamics and well-tempered metadynamics simulation approach to estimate coil−globule transition thermodynamics. An isotactic 30-mer of PNIPAM was investigated over a broad temperature range between 200 and 360 K. The transition from the globule to the random-coil structure was observed with well-tempered metadynamics. For the first time, the free energy surface of PNIPAM was estimated and it is shown that the simulation results are in line with the experimentally observed thermosensitive behavior. Below the LCST, the random-coil ensemble represents the global energy minimum and is thermodynamically favored by 21 ± 9 kJ/mol compared to the globule ensemble; both are separated by a barrier of 49 ± 14 kJ/ mol. In contrast, above the LCST, the globule ensemble is thermodynamically favored by 21 ± 8 kJ/mol over the random-coil ensemble. The barrier from random-coil to globule is 17 ± 10 kJ/mol.

Whereas enzymes in the fumarylacetoacetate hydrolase (FAH) superfamily catalyze several distinct chemical reactions, the structural basis for their multi-functionality remains elusive. As a well-studied example, human FAH domain-containing protein 1 (FAHD1) is a mitochondrial protein displaying both acylpyruvate hydrolase (ApH) and oxaloacetate decarboxylase (ODx) activity. As mitochondrial ODx, FAHD1 acts antagonistically to pyruvate carboxylase, a key metabolic enzyme. Despite its importance for mitochondrial function, very little is known about the catalytic mechanisms underlying FAHD1 enzymatic activities, and the architecture of its ligated active site is currently ill defined. We present crystallographic data of human FAHD1 that provide new insights into the structure of the catalytic center at high resolution, featuring a flexible ‘lid’-like helical region which folds into a helical structure upon binding of the ODx inhibitor oxalate. The oxalate-driven structural transition results in the generation of a potential catalytic triad consisting of E33, H30 and an associated water molecule. In silico docking studies indicate that the substrate is further stabilized by a complex hydrogen-bond network, involving amino acids Q109 and K123, identified herein as potential key residues for FAHD1 catalytic activity. Mutation of amino acids H30, E33 and K123 each had discernible influence on the ApH and/or ODx activity of FAHD1, suggesting distinct catalytic mechanisms for both activities. The structural analysis presented here provides a defined structural map of the active site of FAHD1 and contributes to a better understanding of the FAH superfamily of enzymes.

To characterize the thermosensitive coil–globule transition in atomistic detail, the conformational dynamics of linear polymer chains of acrylamide-based polymers have been investigated at multiple temperatures. Therefore, molecular dynamic simulations of 30mers of polyacrylamide (AAm), poly- N -methylacrylamide (NMAAm), poly- N -ethylacrylamide (NEAAm), and poly- N -isopropylacrylamide (NIPAAm) have been performed at temperatures ranging from 250 to 360 K for 2 μs. While two of the polymers are known to exhibit thermosensitivity (NEAAm, NIPAAm), no thermosensitivity is observed for AAm and NMAAm in aqueous solution. Our computer simulations consistently reproduce these properties. To understand the thermosensitivity of the respective polymers, the conformational ensembles at different temperatures have been separated according to the coil–globule transition. The coil and globule conformational ensembles were exhaustively analyzed in terms of hydrogen bonding with the solvent, the change of the solvent accessible surface, and enthalpic contributions. Surprisingly, independent of different thermosensitive properties of the four polymers, the surface affinity to water of coil conformations is higher than for globule conformations. Therefore, polymer–solvent interactions stabilize coil conformations at all temperatures. Nevertheless, the enthalpic contributions alone cannot explain the differences in thermosensitivity. This clearly implies that entropy is the distinctive factor for thermosensitivity. With increasing side chain length, the lifetime of the hydrogen bonds between the polymer surface and water is extended. Thus, we surmise that a longer side chain induces a larger entropic penalty due to immobilization of water molecules.

Prokaryotic and eukaryotic fumarylacetoacetate hydrolase (FAH) superfamily members, sharing conserved regions that form the so-called FAH-domain, catalyze a remarkable variety of reactions. These enzymes are essential in the metabolic pathways to degrade aromatic compounds in prokaryotes and eukaryotes. It appears that prokaryotic FAH superfamily members evolved mainly to allow microbes to generate energy and useful metabolites from complex carbon sources. We review recent findings, indicating that both prokaryotic and eukaryotic members of the FAH superfamily also display oxaloacetate decarboxylase (ODx) activity. The identification of human FAH domain-containing protein 1 as mitochondrial ODx regulating mitochondrial function supports the new concept that, during evolution, eukaryotic FAH superfamily members have acquired important regulatory functions beyond catabolism of complex carbon sources. Molecular studies on the evolution and function of FAH superfamily members are expected to provide new mechanistic insights in their physiological roles.