@article{milikisiyants_wang_munro_donohue_ward_bolton_brown_smirnova_ladizhansky_smirnov_2017, title={Oligomeric Structure of Anabaena Sensory Rhodopsin in a Lipid Bilayer Environment by Combining Solid-State NMR and Long-range DEER Constraints}, volume={429}, ISSN={["1089-8638"]}, url={https://doi.org/10.1016/j.jmb.2017.05.005}, DOI={10.1016/j.jmb.2017.05.005}, abstractNote={Oligomerization of membrane proteins is common in nature. Here, we combine spin-labeling double electron-electron resonance (DEER) and solid-state NMR (ssNMR) spectroscopy to refine the structure of an oligomeric integral membrane protein, Anabaena sensory rhodopsin (ASR), reconstituted in a lipid environment. An essential feature of such a combined approach is that it provides structural distance restraints spanning a range of ca 3-60Å while using the same sample preparation (i.e., mutations, paramagnetic labeling, and reconstitution in lipid bilayers) for both ssNMR and DEER. Direct modeling of the multispin effects on DEER signal allowed for the determination of the oligomeric order and for obtaining long-range DEER distance restraints between the ASR trimer subunits that were used to refine the ssNMR structure of ASR. The improved structure of the ASR trimer revealed a more compact packing of helices and side chains at the intermonomer interface, compared to the structure determined using the ssNMR data alone. The extent of the refinement is significant when compared with typical helix movements observed for the active states of homologous proteins. Our combined approach of using complementary DEER and NMR measurements for the determination of oligomeric structures would be widely applicable to membrane proteins where paramagnetic tags can be introduced. Such a method could be used to study the effects of the lipid membrane composition on protein oligomerization and to observe structural changes in protein oligomers upon drug, substrate, and co-factor binding.}, number={12}, journal={JOURNAL OF MOLECULAR BIOLOGY}, publisher={Elsevier BV}, author={Milikisiyants, Sergey and Wang, Shenlin and Munro, Rachel A. and Donohue, Matthew and Ward, Meaghan E. and Bolton, David and Brown, Leonid S. and Smirnova, Tatyana I. and Ladizhansky, Vladimir and Smirnov, Alex I.}, year={2017}, month={Jun}, pages={1903–1920} }
 @article{young_donohue_smirnova_smirnov_zhou_2013, title={The UDP-diacylglucosamine Pyrophosphohydrolase LpxH in Lipid A Biosynthesis Utilizes Mn2+ Cluster for Catalysis}, volume={288}, ISSN={["1083-351X"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84884550173&partnerID=MN8TOARS}, DOI={10.1074/jbc.m113.497636}, abstractNote={Background: LpxH is a novel pyrophosphate hydrolase in lipid A biosynthesis. Results: Enzymatic and EPR studies reveal a catalytically important Mn2+ cluster within LpxH. Conclusion: LpxH is a Mn2+-dependent lipid A enzyme with an active site similar to calcineurin-like phosphatases, not Nudix family hydrolases. Significance: Unmasking the true nature of LpxH catalysis represents an important step toward structural characterization and development of antibiotics. In Escherichia coli and the majority of β- and γ-proteobacteria, the fourth step of lipid A biosynthesis, i.e. cleavage of the pyrophosphate group of UDP-2,3-diacyl-GlcN, is carried out by LpxH. LpxH has been previously suggested to contain signature motifs found in the calcineurin-like phosphoesterase (CLP) family of metalloenzymes; however, it cleaves a pyrophosphate bond instead of a phosphoester bond, and its substrate contains nucleoside diphosphate moieties more common to the Nudix family rather than to the CLP family. Furthermore, the extent of biochemical data fails to demonstrate a significant level of metal activation in enzymatic assays, which is inconsistent with the behavior of a metalloenzyme. Here, we report cloning, purification, and detailed enzymatic characterization of Haemophilus influenzae LpxH (HiLpxH). HiLpxH shows over 600-fold stimulation of hydrolase activity in the presence of Mn2+. EPR studies reveal the presence of a Mn2+ cluster in LpxH. Finally, point mutants of residues in the conserved metal-binding motifs of the CLP family greatly inhibit HiLpxH activity, highlighting their importance in enzyme function. Contrary to previous analyses of LpxH, we find HiLpxH does not obey surface dilution kinetics. Overall, our work unambiguously establishes LpxH as a calcineurin-like phosphoesterase containing a Mn2+ cluster coordinated by conserved residues. These results set the scene for further structural investigation of the enzyme and for design of novel antibiotics targeting lipid A biosynthesis.}, number={38}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Young, Hayley E. and Donohue, Matthew P. and Smirnova, Tatyana I. and Smirnov, Alex I. and Zhou, Pei}, year={2013}, month={Sep}, pages={26987–27001} }