Room Temperature Neutron Crystallography of Drug Resistant HIV-1 Protease Uncovers Limitations of X-ray Structural Analysis at 100 K

Abstract

HIV-1 protease inhibitors are crucial for treatment of HIV-1/AIDS, but their effectiveness is thwarted by rapid emergence of drug resistance. To better understand binding of clinical inhibitors to resistant HIV-1 protease, we used room-temperature joint X-ray/Neutron (XN) crystallography to obtain an atomic-resolution structure of the protease triple mutant (V32I/I47V/V82I) in complex with amprenavir. The XN structure reveals a D + ion located midway between the inner Oδ1 oxygen atoms of the catalytic aspartic acid residues. Comparison of the current XN structure with our previous XN structure of the wild-type HIV-1 protease-amprenavir complex suggests that the three mutations do not significantly alter the drug-enzyme interactions. This is in contrast to the observations in previous 100K X-ray structures of these complexes that indicated loss of interactions by the drug with the triple mutant protease. These findings, thus, uncover limitations of structural analysis of drug binding using X-ray structures obtained at 100K. Graphical abstractCorresponding Author Information: To whom correspondence should be addressed: kovalevskyay@ornl.gov, 505-310-4184 (AK). PDB ID Codes: the joint XN structure of PR TM -APV complex has been deposited to the Protein Data Bank with the code 5T8H. Authors will release the atomic coordinates and experimental data upon article publication.

Drug Resistance Mutation L76V Alters Nonpolar Interactions at the Flap–Core Interface of HIV-1 Protease

“… 29 Similar effects were observed for the neutron structure of amprenavir complexed with PR mutant V32I/I47V/V82I. 30 No neutron crystal structures have been reported for the inhibitor complexes in this study; hence, hydrogen bonds for L76V complexes are described by the same criteria as for previously published wild-type complexes ( Figure 3 ).…”

“… 29 Subsequent neutron studies of 1 and amprenavir complexes with a mutant PR demonstrated that the location of the two protons varies, depending on the pH, inhibitor, and mutated residues. 30 , 31 In our X-ray structures of inhibitor-bound PR L76V , we cannot distinguish which hydrogen bond interactions occur with the inhibitor hydroxyl group. In the absence of neutron structures corresponding to the PR complexes with the inhibitors described here, we have indicated distances for four possible interactions between the hydroxyl oxygen of the inhibitor and carboxylate oxygens of Asp25 and 25′ in Figure 3 .…”

Neutron scattering in the biological sciences: progress and prospects

“…One very useful consequence of this is that the oxidation states of metal centers or redox cofactors (such as in flavoenzymes) are not altered during neutron crystallographic data collection, so that the protonation, geometric and electronic coordination environments can be correctly assigned to the known redox state of the center (Bodenheimer et al, 2017;O'Dell et al, 2017;Golden et al, 2017;Casadei et al, 2014). The lack of radiation damage also means that neutron crystallography can be used to determine structures at physiological temperatures, avoiding the artifacts that might be induced by the rapid flash-cooling to 100 K usually used for X-ray work (and the eponymously named cryo-EM; Gerlits, Keen et al, 2017;Deacon et al, 1997).…”

“…Example (2015)(2016)(2017)(2018) Protonation states to elucidate enzymatic mechanism Xylose isomerase (Meilleur, Snell et al, 2006), chlorite dismutase (Schaffner et al, 2017), T4 lysozyme (Hiromoto et al, 2017), phosphoactive yellow protein (Yonezawa et al, 2017), RAS (Knihtila et al, 2015), Cel45A (Nakamura et al, 2015), phycocyanobilin:ferredoxin oxidoreductase (Unno et al, 2015) Protein-ligand interaction/protein-drug complex Galectin 3C (Manzoni et al, 2018), xylose isomerase (Munshi et al, 2014), PKG (Gerlits et al, 2018), -lactamase (Langan et al, 2018), trypsin (Schiebel et al, 2017), GCN5-related N-acetyltransferase (Kumar et al, 2018), pyridoxal 5 0 -phosphate enzyme (Dajnowicz et al, 2017), concanavalin A (Gerlits, Coates et al, 2017), MTAN (Banco et al, 2016), farnesyl pyrophosphate synthase (Yokoyama et al, 2015) Titration (pH studies) HIV (Gerlits et al, 2016), RAS (Knihtila, 2016), xylanase (Wan et al, 2015) Hydration and water binding at the active site of enzymes Carbonic anhydrase (Kovalevsky et al, 2018), RAS (Knihtila et al, 2015), H-FABP (Howard et al, 2016), carbohydrate-binding module (Fisher et al, 2015), hydronium ion identification (Kovalevsky et al, 2011) Metalloprotein/redox chemistry Lytic polysaccharide monooxygenase (Bacik et al, 2017;O'Dell et al, 2017), cholesterol oxidase (Golden et al, 2017), ascorbate peroxidase (Kwon et al, 2016), cytochrome c peroxidase (Kwon et al, 2016) Room ('physiological') temperature structures † HIV (Gerlits, Keen et al, 2017) † Most neutron crystallographic data are collected at room temperature. Here, we list proteins for which neutron diffraction at room temperature revealed st...…”

Membrane-protein crystals for neutron diffraction

“…Understanding the protonation state of aminoacid residues and the transfer of protons between them, or to cofactors and substrates, is key to understanding catalytic processes in biology (Oksanen et al, 2017). Determining H-atom positions in enzyme active sites or at drug-binding sites may enhance our prediction power in a structure-based drug-design process (Weber et al, 2013;Gerlits et al, 2017).…”