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Instruments / Technical Area	Publication Details	Published	Actions
I24-Microfocus Macromolecular Crystallography	X‐ray crystallography and free energy calculations reveal the binding mechanism of A2A adenosine receptor antagonists Willem Jespers , Gregory Verdon , Jhonny Azuaje , Maria Majellaro , Henrik Keränen , Xerardo García‐mera , Miles Congreve , Francesca Deflorian , Chris De Graaf , Andrei Zhukov , Andrew S. Dore , Jonathan S. Mason , Johan Åqvist , Robert M. Cooke , Eddy Sotelo , Hugo Gutiérrez‐de‐terán Angewandte Chemie International Edition DOI: 10.1002/anie.202003788 Diamond Proposal Number(s): [17185] Open Access Show Abstract ▼ Abstract: We present a robust protocol based on iterations of free energy perturbation (FEP) calculations, chemical synthesis, biophysical mapping and X‐ray crystallography to reveal the binding mode of an antagonist series to the A2A adenosine receptor (AR). Eight A2AAR binding site mutations from biophysical mapping experiments were initially analyzed with sidechain FEP simulations, performed on alternate binding modes. The results distinctively supported one binding mode, which was subsequently used to design new chromone derivatives. Their affinities for the A2AAR were experimentally determined and investigated through a cycle of ligand‐FEP calculations, validating the binding orientation of the different chemical substituents proposed. Subsequent X‐ray crystallography of the A2AAR with a low and a high affinity chromone derivative confirmed the predicted binding orientation. The new molecules and structures here reported were driven by free energy calculations, and provide new insights on antagonist binding to the A2AAR, an emerging target in immuno‐oncology.	Jul 2020
I03-Macromolecular Crystallography	Structural mechanism of AadA, a dual specificity aminoglycoside adenyl transferase from Salmonella enterica Ana Laura Stern , Sander Egbert Van Der Verren , Sandesh Kanchugal Puttaswamy , Joakim Näsvall , Hugo Gutierrez De Teran , Maria Selmer Journal Of Biological Chemistry DOI: 10.1074/jbc.RA118.003989 Diamond Proposal Number(s): [11171] Open Access Show Abstract ▼ Abstract: Streptomycin and spectinomycin are antibiotics that bind to the bacterial ribosome and perturb protein synthesis. The clinically most prevalent bacterial resistance mechanism is their chemical modification by aminoglycoside-modifying enzymes such as aminoglycoside nucleotidyltransferases (ANTs). AadA from Salmonella enterica is an aminoglycoside (3’’)(9) adenylyl transferase that O-adenylates position 3” of streptomycin and position 9 of spectinomycin. We previously reported the apo AadA structure with a closed active site. To clarify how AadA binds ATP and its two chemically distinct drug substrates, we here report crystal structures of wildtype AadA complexed with ATP, magnesium, and streptomycin and of an active-site mutant, E87Q, complexed with ATP and streptomycin or the closely related dihydrostreptomycin. These structures revealed that ATP binding induces a conformational change that positions the two domains for drug binding at the interdomain cleft and disclosed the interactions between both domains and the three rings of streptomycin. Spectinomycin docking followed by molecular dynamics simulations suggested that despite the limited structural similarities with streptomycin, spectinomycin makes similar interactions around the modification site, and, in agreement with mutational data, critically interacts with fewer residues. Using structure-guided sequence analyses of ANT(3”)(9) enzymes acting on both substrates and ANT(9) enzymes active only on spectinomycin, we identified sequence determinants for activity on each substrate. We experimentally confirmed that Trp-173 and Asp-178 are essential only for streptomycin resistance. Activity assays indicated that Glu-87 is the catalytic base in AadA and that the non-adenylating E87Q mutant can hydrolyze ATP in the presence of streptomycin.	Jun 2018

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