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Allostery describes the functional coupling between sites in biomolecules. Recently, the role of changes in protein dynamics for allosteric communication has been highlighted. A quantitative and predictive description of allostery is fundamental for understanding biological processes. Here, we integrate an ensemble-based perturbation approach with the analysis of biomolecular rigidity and flexibility to construct a model of dynamic allostery. Our model by definition excludes the possibility of conformational changes, evaluates static, not dynamic, properties of molecular systems, and describes allosteric effects due to ligand binding in terms of a novel free energy measure. We validated our model on three distinct biomolecular systems, eglin c, protein tyrosine phosphatase 1B, and the lymphocyte function-associated antigen 1 domain. In all cases, it successfully identified key residues for signal transmission in very good agreement with experiment. It correctly and quantitatively discriminated between positively or negatively cooperative effects for one of the systems. Our model should be a promising tool for the rational discovery of novel allosteric drugs.
In J Chem Theory Comput, 2017

Pyruvate phosphate dikinase (PPDK) is an essential enzyme of C4 photosynthesis in plants, catalyzing the ATP-driven conversion of pyruvate to phosphoenolpyruvate (PEP). It is further used by some bacteria and unicellular protists in the reverse, ATP-forming direction. Many weed species use C4 photosynthesis in contrast to world’s major crops, which are C3 plants. Hence inhibitors of PPDK may be used as C4-specific herbicides. By screening a library of 80 commercially available kinase inhibitors, we identified compounds derived from bisindolylmaleimide (bisindolylmaleimide IV, IC50 = 0.76 ± 0.13 μM) and indirubin (indirubin-3’-monoxime, IC50 = 4.2 ± 0.9 μM) that showed high inhibitory potency towards PPDK and are among the most effective PPDK inhibitors described today. Physiological studies on leaf tissues of a C4 model plant confirmed in vivo inhibition of C4-driven photosynthesis by these substances. Moreover, comparative docking studies of non-inhibitory bisindolylmaleimide derivatives suggest that the selectivity towards PPDK may be increased by addition of functional groups to the core structure.
In PLOS ONE, 2017

Pyruvate phosphate dikinase (PPDK) is an essential enzyme of both the C4 photosynthetic pathway and cellular energy metabolism of some bacteria and unicellular protists. In C4 plants, it catalyzes the ATP- and Pi-dependent formation of phosphoenolpyruvate (PEP) while in bacteria and protozoa the ATP-forming direction is used. PPDK is composed out of three distinct domains and exhibits one of the largest single domain movements known today during its catalytic cycle. However, little information about potential intermediate steps of this movement was available. A recent study resolved a discrete intermediate step of PPDK’s swiveling movement, shedding light on the details of this intriguing mechanism. Here we present an additional structural intermediate that possibly represents another crucial step in the catalytic cycle of PPDK, providing means to get a more detailed understanding of PPDK’s mode of function.
In Protein Sci, 2017

eLabFTW is a free and open source electronic laboratory notebook for researchers. Once installed on a server, it allows researchers to track their experiments, but also to manage their assets in the lab (antibodies, mouse, siRNAs, proteins, etc.). Experiments can be timestamp to any RFC 3161 Time Stamping Authority, allowing solid legal proof in case of issues about a patent. It also features a scheduler to book equipment.
In JOSS, 2017

Pyruvate phosphate dikinase (PPDK) is a vital enzyme in cellular energy metabolism catalyzing the ATP- and Pi-dependent formation of phosphoenolpyruvate from pyruvate in C4 -plants, but the reverse reaction forming ATP in bacteria and protozoa. The multi-domain enzyme is considered an efficient molecular machine that performs one of the largest single domain movements in proteins. However, a comprehensive understanding of the proposed swiveling domain motion has been limited by not knowing structural intermediates or molecular dynamics of the catalytic process. Here, we present crystal structures of PPDKs from Flaveria, a model genus for studying the evolution of C4 -enzymes from phylogenetic ancestors. These structures resolve yet unknown conformational intermediates and provide the first detailed view on the large conformational transitions of the protein in the catalytic cycle. Independently performed unrestrained MD simulations and configurational free energy calculations also identified these intermediates. In all, our experimental and computational data reveal strict coupling of the CD swiveling motion to the conformational state of the NBD. Moreover, structural asymmetries and nucleotide binding states in the PPDK dimer support an alternate binding change mechanism for this intriguing bioenergetic enzyme.
In Sci Rep, 2017

The pyruvate phosphate dikinase (PPDK) is a versatile enzyme which catalyzes the reversible interconversion of pyruvate, inorganic phosphate (Pi) and adenosine triphosphate (ATP) to phosphoenolpyruvate (PEP), pyrophosphate (PPi) and adenosine monophosphate (AMP). PPDK is involved in the energy metabolism of several bacteria and protists as a glycolytic enzyme where it acts in the ATP-forming direction, boosting the energy efficiency of these organisms. The PEP-forming direction is used in C4 plants where the primary CO2 acceptor PEP is regenerated by PPDK. Hence, PPDK is one of the rate-limiting enzymes of C4 photosynthesis. Phosphoryl group transfer between the distant substrate binding domains is realized by the so-called swiveling domain mechanism which employs a swiveling movement of the central domain (CD). However, intermediate conformations of the CD have been unresolved so far. Similarly, the binding mode of nucleotides within the nucleotide binding domain (NBD) and the opening and closing motion of this domain were only postulated in analogy to other proteins with homologous nucleotide binding domains. A possible crosstalk between monomers in the functional homodimer or homotetramer has not been investigated so far. In the course of this work, a number of novel crystallographic structures of PPDK from the C4 plant Flaveria trinervia and the C3 plant Flaveria pringlei have been solved. These allow to derive a more detailed view on the domain movements of PPDK in its catalytic cycle. Two conformational intermediates of the CD have been trapped which show that the swiveling movement of the CD does not proceed in a linear interpolation between two extreme conformations, but instead proceeds via distinct sub-steps. Additionally, nucleotidebound (closed) and nucleotide-free (open) conformations of the NBD were identified in these structures. This hints at a possible alternate binding change mechanism employed in the functional dimeric or tetrameric assembly. Eventually a library of known kinase inhibitors was screened for inhibitory effects on PPDK. During this study, novel high efficacy inhibitors of PPDK have been identified which belong to the class of bisindolylmaleimides. A comparative in silico docking approach revealed high potential for structural adjustment of these inhibitors with the aim of an improved selectivity towards PPDK.
HHU Düsseldorf, 2017