Higuchi Biosciences Center - University of Kansas

Fall 2008 Science Talks
December 5, 2008

D1. Crystal Structure of Human Cytochrome P450 2E1 in Complex with a Fatty Acid Analog
Porubsky, P.R.*, Scott, E.E.
Department of Medicinal Chemistry, University of Kansas, 1251 Wescoe Hall Dr., Lawrence, KS 66045, porub998@ku.edu

Human cytochrome P450 2E1 (CYP2E1) is a xenobiotic metabolizing enzyme that is highly conserved among mammals. In addition to small molecular weight exogenous drugs like the analgesic acetaminophen and volatile anesthetic halothane, CYP2E1 is also involved in endogenous fatty acid metabolism. Recently we solved structures of CYP2E1 bound with small inhibitors indazole or 4-methylpyrazole in the active site coordinating to the heme iron. These structures revealed an active site of only 190 Å3 consistent with small molecule binding and metabolism, but too small for fatty acid binding and metabolism. In an effort to investigate the structural adaptations facilitating fatty acid binding, CYP2E1 was cocrystallized with the fatty acid analog ?-imidazoyl-decanoic acid and its structure solved to 2.7 Å. Comparison of the CYP2E1 structures shows that only small side chain movements are required for the accommodation of the much larger fatty acid analog. Future work will be focused on cocrystal structures of CYP2E1 with both longer and shorter chain analogs to better understand the ability of the enzyme to metabolize a variety of fatty acids substrates.

This research was supported by the NIH GM076343.

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D2. Cu/Zn) Superoxide dismutase interacts with calcineurin in a sequence specific fashion
Authors: *A. Agbas, D. Hui, E. K. Michaelis
Higuchi Biosciences Center, University of Kansas, Lawrence, KS

Abstract: Cu-Zn superoxide dismutase (SOD1) stabilizes calcineurin (Cn), a phenomenon that we have previously shown to be unrelated to either exposure of Cn to superoxide or to the dismutation of superoxide by SOD1 [Agbas et al., Biochem.J., 2007, 405, 51-59]. We showed that native bovine, recombinant human or rat, and two chimeras of human-rat SOD1, all activated Cn, but Mn-SOD (SOD2) did not affect Cn activity. Particularly noteworthy was the observation that a chimera of N-terminal human/C-terminal rat SOD1 had little or no detectable dismutase activity yet stimulated Cn activity the same as intact human or rat SOD1. Nevertheless, the active site of SOD1 appears to be involved in Cn activation based on observations that the activation of Cn was correlated with the prevention of Fe and Zn losses from the active site of Cn, suggesting a conformation-dependent SOD1-Cn interaction. In order to define the sequence-specific interactions of SOD1 and Cn, we generated several truncated human SOD1 constructs and have begun to analyze both the dismutation activity associated with these constructs as well as their interaction with Cn. The conformation of the SOD1 constructs was stabilized by integrating the SOD1 peptide structure within the sequence of the EGFP structure. The latter was used as a stable, base structure onto which the truncated SOD1 sequences were strategically inserted. The SOD1-EGFP species generated were purified using chitin affinity chromatography and tested for SOD-type activity and Cn activation. The molecular sites of interaction between SOD1 and Cn are being defined through this approach.

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D3. Immobilization of F₁ATP synthase for nanodevice applications
Smith, Gregory;* Settle, Jenifer K.;* Berrie, Cindy L.;* Richter, Mark L.;^† Wu, Judy Z.^‡
*Department of Chemistry, University of Kansas, Lawrence, KS
^†Department of Molecular Biosciences, University of Kansas, Lawrence, KS
^‡Department of Physics and Astronomy, University of Kansas, Lawrence, KS

The F₁-ATP synthase acts as a small rotary motor upon hydrolysis of ATP making it a desirable target for nanotechnology applications. Goals for this project include immobilizing F₁-ATPase on a surface, in the proper position and correct orientation, while maintaining activity. Tapping mode AFM is utilized to observe the conformation of the protein on a variety of different surfaces. AFM and electrical methods will be used to pattern the surface at specific locations and detect activity. Electrodes will be fabricated using an electro-less plating technique on self assembled monolayers on gold or silicon. Ultimately, learning about the requirements necessary to specifically attach F₁-ATPase to surfaces and its motor abilities will enable the production of biomolecular and nanomechanical devices.

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D4. Influence of Phosphorylation on Conformational Change of Phospholamban Cytoplasmic Domain and Its Interactions with Membranes: Molecular Dynamics Studies by Potential of Mean Force Calculations
Taehoon Kim, Jinhyuk Lee and Wonpil Im
Department of Molecular Biosciences and Center for Bioinformatics, University of Kansas

Phospholamban (PLB) is an integral membrane protein of 52 amino acids that consists of amphiphatic cytoplasmic (Met1-Ser16) and transmembrane (Gln22-Leu52) domains. PLB regulates the activity of sarcoplasmic reticulum calcium ATPase (SERCA), a calcium pump, to modulate the intercellular calcium levels at the sarcoplasmic reticulum of cardiac, slow twitch and smooth muscles. Binding of PLB to SERCA inhibits the SERCA activity and thus decreases the calcium influx into SR, which results in reduction of the cardiac relaxation rate. A conformational change of PLB upon phophorylation of its Ser16 by protein kinase A causes PLB to dissociate from SERCA. Recent experiments have illustrated the dynamic nature of PLB cytoplasmic domain upon phosphorylation; the population of unphosphorylated PLB cytoplasmic domain that interacts with membranes is about 84%, but its population reduces by 20% when phosphorylated. However, it is not clearly understood why such different conformational preferences occur depending on phosphorylation. The aim of this proposal is to characterize the underlying driving forces that govern the dynamics and conformational change of PLB cytoplasmic domain upon phosphorylation. We plan to calculate the potentials of mean force (PMFs) as a function of tilt angle of PLB cytoplasmic domain in a POPC membrane with and without phosphorylation. It is expected that this proposal will provide detailed information on energetics of conformational change of PLB cytoplasmic domain as well as its interactions with membranes.

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D5. Mechanisms of a prokaryotic transmembrane signal transduction system at high resolution
H. Zhao, L. Tang*
Department of Molecular Biosciences, University of Kansas, 1200 Sunnyside Avenue, Lawrence, KS 66045

Living organisms have evolved the ability to sense and adapt to environmental change and stress, which is crucial to survival. Prokaryotes and some archaea and eukaryotes fulfill this task by utilizing two-component regulatory system (TCS) to sense external or internal signals and implement appropriate responses through regulation of gene expression. TCSs play pivotal roles in adaptation, development and pathogenesis. A paradigmatic TCS consists of two proteins: a multi-domain, multi-function sensor histidine kinase (HK), and a response regulator (RR) that receives the signal from the sensor kinase and trigger downstream responses, acting as a transcription factor in many cases. Molecular mechanisms underlying TCS signal transduction are not well understood. Many HKs are multidomain, transmembrane proteins that transduce signals from periplasmic compartments into cytoplasm by signal propagation along multiple domains. YycFG is an essential TCS and is specific to low G+C Gram-positive bacteria including a group of leading causative agents of human infections that cause diseases ranging from pneumonia to meningitis, endocarditis and bacteremia. Treatment of these infections has been complicated by rapid emergence of antibiotic-resistance. Some of these low G+C Gram-positive pathogens, such as Bacillus anthracis and Clostridium botulinum, are classified as potential bioterror agents. Using the YycFG TCS as a model, we have launched systematic investigation on molecular basis of TCS transmembrane signal transduction using high resolution structural approaches. X-ray crystallography and electron cryo-microscopy are employed to tackle three critical aspects in TCS signaling, i.e., transmembrane signal propagation, HK:RR specificity and gene regulation by RR.

This project is supported by the NIH COBRE award P20RR17708 and by Department of Molecular Biosciences, University of Kansas.

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D6. Molecular Dynamics Studies of Gating Mechanism of MscL as a Function of Helix Tilt Angle
Ritesh Kumar and Wonpil Im
Department of Molecular Biosciences and Center for Bioinformatics, University of Kansas, Lawrence KS

MscL, the mechanosensitive channel of large conductance, forms a homo-pentameric pore in which each subunit contains two transmembrane (TM) domains (TM1 and TM2) involved in the channel gating, and a C-terminal cytoplasmic helical domain. The crystal structure (PDB ID:2OAR) represents a closed state of the channel, showing a funnel shaped pore with a large opening on the periplasmic side and the narrowest point near the cytoplasm. Assuming that TM helix tilting due to membrane tension drives MscL gating, molecular dynamics simulations have been performed by applying the recently developed helix tilt restraint potential to the TM helices of MscL in a DMPC lipid bilayer. An initial structure of the MscL/DMPC complex without C-terminal helix (Gln110-Asn125) was generated using Membrane Builder in the CHARMM-GUI website (http://www.charmm-gui.org). CT?P (constant temperature, surface tension, and pressure) dynamics was used to allow the system size along the XY axes to vary during the simulation. The P21 image transformation was used to allow the number of lipid molecules in the top and bottom leaflets to vary during the simulations. Molecular dynamics simulations have been performed by tilting TM1 and TM2 helices and the tilt angles of TM1 helices was gradually varied from 35° (PDB ID:2OAR) to 65° by 1° and those of TM2 from 33° (PDB ID:2OAR) to 63° by 1°, together. Detailed information on the change of the pore size, molecular interactions, and pressure profile as a function of helix tilt, and thus the possible gating mechanisms of MscL at the atomic level will be presented.

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D7. Molecular Dynamics Studies of Protegrin-1 in Different Membrane Bilayers
Huan Rui, Jinhyuk Lee and Wonpil Im*
Department of Molecular Biosciences and Center for Bioinformatics, University of Kansas, 2030 Becker Drive, Lawrence, KS 66047

Abstract: Protegrin-1 (PG-1) is known as an antimicrobial peptide that plays an important role in the innate immune system. It specifically targets the invading pathogen membrane and kills the pathogen by disturbing its membrane and releasing the cell content. It has been revealed recently that PG-1 adapts different oligomer states in different membrane bilayers. The solid-state NMR experiment suggests PG-1 oligomer pore formation in POPC (palmitoyl-oleylphosphatidylcholine) bilayer and PG-1 dimer is crucial for the pore formation. It is still not clear, however, what is the driving force of the oligomerization. Therefore, it is important to study the behavior of PG-1 monomer and dimer in different membranes. Here we performed extended molecular dynamics simulations (40 ns) of all-atom PG-1 monomer and dimer in DLPC (dilauroylphosphatidylcholine) and POPC membrane. All the atoms in the system are treated explicitly. During the simulation, the β-hairpin structure of the peptide remains stable in each system, but hydrogen bond patterns might vary in different lipid bilayers. Peptide orientation changes are observed in both of the bilayers. We also saw the local membrane thinning effect in both of the membrane bilayers.

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D8. Testing our understanding of divergent mitochondrial and microsomal cytochrome b5 biophysical properties via “core swap” mutants
Sudharsan Parthasarathy², Krzysztof Kuczera^1,2, David. R. Benson^1,2
¹Depts. of Chemistry and ² Molecular Biosciences, University of Kansas

Fully functional proteins occur in a wide variety of environmental conditions. Within organisms there exists isoforms of proteins that evolved specifically to perform their function in specific sub-cellular locations. Two such isoforms of mammalian cytochrome b5 (Microsomal b5 [Mc b5] and Mitochondrial b5 [OM b5]) remarkably different biophysical properties in spite of similar sequence and structure. Our most recent efforts have been directed toward generating and studying a b5 variant that combines the most stabilizing regions of OM b5 (core 1) and Mc b5 (core 2). We have obtained a 1.7 Å crystal structure of the mutant which shows that the packing interactions observed in rOM and bMc b5 are conserved in the corresponding regions of hybrid b5. Despite the fact that hybrid b5 required only 12 mutations in Mc b5, its stability and hemin-binding properties are virtually identical to those of OM b5. In addition, the redox potential of hybrid b5 is shifted in the direction of that exhibited by OM b5. This provides additional key support to our hypothesis that nature has utilized evolutionary divergence of Mc and OM b5 stability as a means of optimizing the redox properties of the two isoforms for their specialized sub-cellular roles.

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Higuchi Biosciences Center
University of Kansas
2099 Constant Avenue
Lawrence, KS 66047-2535
785-864-5183
hbc@ku.edu