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Collaborative Research Between the Center for Computational Sciences and other Colleges and Departments Across the University

Determination of the structure and mechanisms of regulation of mosquito Met protein in the juvenile hormone signaling pathway of Aedes aegypti (in collaboration with Reddy Palli, Department of Entomology, College of Agriculture)

Juvenile hormone III (JHIII) is a component of one of the insect cell signaling pathways that coordinates a number of development-related functions within the insect. These specific functions not only vary from tissue to tissue but are dependent on the stage of development of the insect. That is, the mechanisms of action of JHIII exhibit both functional and temporal separation. One of the key functions of JHIII is to maintain the insect in its juvenile stage of development to prevent the insect from premature entry into adulthood.

JHIII functions by binding to a target protein within the insect cell. The binding of this hormone initiates a series of yet unidentified structural changes in the protein resulting in its translocation into the nucleus and initiating transcription of specific development-related genes. Discovery of a ligand that mimics JHIII but has a higher binding affinity for its target protein has potential use as an effective insecticide against certain insects. One such insect is Aedes aegypti, the mosquito that carries malaria.

The goals of our research are several-fold:

determine the unknown 3-dimensional structure of the full-length JHIII-binding protein (a.k.a. methoprene-tolerant (Met) protein) in A. aegypti;
identify one or more ligands that bind to mosquito Met with higher binding affinity than pyriproxifen, an analogue of JHIII exhibiting a higher binding affinity than JHIII
identify the conformational change(s) induced within the full-length mosquito Met protein subsequent to binding of JHIII. In absence of JHIII, Met exists in the cell as a homodimer. The conformational change(s) induced by JHIII binding results in the

dissociation of the homodimeric protein and formation of a heterodimeric protein

identify the binding site(s) on mosquito Met where steroid receptor coactivator (SRC – putatively identified as the protein that forms a heterodimer with Met upon binding of JHIII by Met)
identify potential binding sites on Met for additional ligands and/or peptides that may play a role in regulating the binding of JHIII to Met as well as regulating the activity of the Met-SRC hoeterodimer.
identify those ligands/peptides that bind to the above identified sites on mosquito Met that may play a role in the regulation of the signal transduction pathway involving juvenile hormone. Of particular interest are ligands that act as allosteric regulators of Met or the Met-SRC complex. Subsequent identification of ligands that interfere with normal regulation of Met in situ may offer a second approach to the development of new insecticides for use in the eradication of malaria.

Results to date:

Determined 3-dimensional solvated structure of PAS-B domain (JH III binding domain) of Aedes aegypti methoprene-tolerant (Met) protein by molecular dynamics simulation (in Amber).
Determined 3-dimensional solvatedstructure of full-length mosquito Met protein containing 977 aminoacid residues. The initial structural determination determined using the online protein structure server I_TASSER (http://zhanglab.ccmb.med.umich.edu/ITASSER/) followed by molecular dynamics simulation in Amber). This result represents the first 3D structure of the full-length A. aegypti Met protein generated (Figure 1).

Figure 1: 3D structure of mosquito Met protein

Identified a number of putative ligand binding sites classified as druggable on the full-length Met protein using LigBuilder and Molegro software suites along with a set of pharmacophores specific for each of these sites.
Identified potential phosphorylation sites and putative post-translational modification sites (glycosylation, amidation, and myristoylation) on the full-length Met protein.
Identified the extensive disordered regions of the full-length Met protein and a putative flexible arm within the protein that may be involved in DNA binding and activation of gene transition subsequent to the transport of the Met-SRC complex into the cell nucleus.
Identified a number of specific ligands that bind to a number of the identified binding sites on the full-length Met protein, including sites that exhibit binding of ATP, CoenzymeA, CoenzymeQ, dihydropteroate, and palmitic acid. Identified specific amino acid residues in the Met protein that exhibit hydrogen bonding with ATP. Experiments are currently being carried out in the laboratory in which residues at these sites are being mutated in order to disrupt H-bonding to see how this disruption in protein-ligand interactions affects Met protein function.
Carried out virtual molecular docking studies to identify putative amino acid residues on the full-length Met protein at the surface of the PAS-B domain that are involved in the H-bonding interactions between Met and SRC proteins to form the heterodimeric Met-SRC protein complex. Experiments are currently being carried out in the laboratory in which residues at these sites are being mutated in order to disrupt H-bonding to see how this disruption in protein-protein interactions affects Met-SRC heterodimer formation and stability and Met protein function.

Current investigations in progress:

Molecular dynamics simulation analysis of homodimerization of mosquito Met and heterodimerization of Met and SRC proteins induced by the binding of JHIII and JHIII analogues.
MD simulation of the interactions of the Met-SRC protein complex with the cell nuclear membrane during transport into the cell nucleus.
MD simulation of the interaction of Met-SRC with the promoter region of the genes whose transcription is induced Met-SRC.

Bioinformatics of Cimex lectularius: the Bed Bug Genome Project (in collaboration with Reddy Palli, Department of Entomology, College of Agriculture)

The global resurgence of bed bugs has created one of the most serious pest challenges in a generation. Several research laboratories all over the world started working on various aspects of bed bug biology, physiology, behavior, chemical communication, reproduction, vector competence, insecticide resistance, endosymbionts and population genetics. Currently very little information is currently available on genes and proteins of bed bugs, but availability of genome sequence of this insect will help in all areas of bed bug research and may lead to utilization of innovative, efficient and high throughput approaches to conduct research needed to solve this problem.

The bed bug genome project is an international collaboration based at the University of Kentucky that will provide insights into many areas of bed bug research such as behavior, reproduction, disease vectoring ability (or the lack thereof), and insecticide resistance. Project collaborators include University of Kentucky, North Carolina State University, Ohio State University, University of Illinois, Virginia Tech, and University of Tuebingen, Germany.

Upcoming research project in the bedbug genome project:

MD investigations of the Na+-channel in the bedbug and the mechanism whereby the Na+-channel confers pesticide resistance in the bedbug.

Identification of ligand binding sites in choline acetyltransferase (ChAT) and computational de novo design of drugs capable of reversing the effect of congenital mutations in ChAT associated with a certain type of the inherited neuromuscular disorder congenital myasthenic syndrome (in collaboration with David Rodgers, Center for Structural Biology, Department of Molecular and Cellular Biochemistry, College of Medicine)

Acetylcholine is a neurotransmitter that is involved in a number of neurologic functions. Certain neurological-associated diseases (e.g. congenital neuromuscular disease and Alzheimer disease) may be due, in part, to alterations in certain cholinergic neurons. The synthesis of acetylcholine from acetyl-CoA and choline within the terminal end of the nerve is catalyzed by the enzyme choline acetyltransferase (ChAT). Twenty-four point mutations in ChAT have been identified in patients with inherited disorders affecting the neuromuscular junction (congenital myasthenic syndromes).

This research involved the identification of potential binding sites for ligands on this enzyme and the computational de novo synthesis of candidate drugs capable of reversing the effect of these mutations in vivo.

Results to date:

• Determined the 3-dimensional solvated structure of ChAT
• Identified a number of key internal and surface ligand-binding sites on ChAT.
• Completed de novo synthesis of pharmacophores corresponding to each of the identifed key ligand-binding sites.
• Identified a candidate drug consistent with the pharmacophore structure at one of the key ligand-binding sites employing virtual molecular docking analysis.

Molecular docking of angiotensin to neprilysin and molecular dynamics of neprilysin (in collaboration with David Rodgers, Center for Structural Biology, Department of Molecular and Cellular Biochemistry, College of Medicine)

Neprilysin (enkphalinase) is a zinc-dependent metallo-endopeptidase that is widely expressed in mammalian tissues, including neural synapses in the central nervous system. This enzyme cleaves a number of physiological substrates. As a consequence, it has been suggested that modulation of this enzyme’s specificity toward specific substrates might enhance its use as a potentiall therapeutic agent for the treatment of certain neurological diseases such as Alzheimer’s disease. It also has activity associated with other systemic processes. Inhibition of neprilysin acitivity may also provide a mechanism for treatment of pain and hypertension.

Angiotensin is a peptide hormone that causes vasoconstriction and a subsequent increase in blood pressure following its conversion from angiotensin I to angiotensin II and is a target for the treatment of antihypertension. One suggested approach for the treatment of congestive heart failure and chronic hypertension inhibition of the formation of angiotensin II by endopeptidase cleavage.

• Investigation of the binding of angiotensin to neprilysin by virtual molecular docking analysis.

Current work in progress:

Atom typing of a tetrahedral zinc (Zn) atom bonded to four specific amino acid residues. Although the atom type for this particular Zn atom is required to run molecular dynamics simulations of certain Zn-containing proteins, no atom type for this specific Zn atom currently exists. This data will initially be used to run MD simulations of neprilysin complexed with angiotensin to investigate the binding of angiotensin by neprilysin. The atom type data will also be made available for the entire computational biology research community.

Development and implementation of an Imaging Technology wiki to enhance collaborations between researchers and clinicians in the College of Medicine (in collaboration with the Dept of Radiology and the Center for Clinical and Translational Science (CCTS), College of Medicine)

• Kickoff meeting with Imaging Technology group to discuss how to create an environment for effective collaborations between researchers and clinicians utilizing medical imaging technology.

Current work in process:
Preliminary development of a medical imaging technology Wiki for the purpose of promoting and enhancing collaboration between researchers and clinicians within CCTS and other departments within UKMC and the UK Health Care System involved in Imaging Technology.

Structure and molecular dynamics of the inner membrane drug efflux transporter protein AcrB: Comparison of wild-type AcrB and proteins carrying R780A and R780A / M774K mutations (in collaboration with Yeinan Wei, Department of Chemistry)

Multidrug efflux pumps play an important role both in the drug resistance of bacteria and the development of the resistance of cancer cells to chemotherapeutic drugs. A major multidrug efflux system in Gram-negative bacteria consists of a complex of three proteins, one of which is the inner membrane transporter protein AcrB. Using site-directed mutagenesis, one particular arginine residue, R780, was found to be a key residue involved in AcrB function and protein complex stability. Replacement of this arginine by the amino acid alanine (R780A) was found to decrease AcrB stability resulting in the inactivity of AcrB. The replacement of methionine 774 by lysine (a positively-charged amino acid like arginine) acted as a repressor mutation restoring the drug efflux activity of the double mutant AcrB close to the level of the wild-type (WT) R780 protein.

To investigate if the R780A mutation induces a local structural rearrangement of AcrB resulting in the observed decrease in protein stability and loss of drug efflux activity, the 3-dimensional structures of the WT R780 and mutant R780A proteins were determined by molecular dynamics (MD) simulation in Amber. Comparison of the structural conformations of the two proteins showed that a region of the protein remote from residue 780 undergoes a disordered coil to beta sheet conformational change in the mutant protein, which may account for the loss in AcrB activity (Figures 2 and 3).

Fig 2. 3D structure of WT R780 AcrB

Fig 3. 3D structure of R780A mutant AcrB

Current work in progress:

Additional MD simulation studies are currently being carried out to determine if the disordered coil conformation is restored in the R780A / M774K double mutant. Additional studies employing virtual molecular docking and MD simulation are also being carried out to determine if the R780A mutation results in disruption of the interaction(s) between AcrB and the other two proteins (AcrA and TolC) that compose the trimeric drug efflux pump complex.

Molecular dynamics and combined quantum mechanics/molecular mechanics (QM/MM) simulation of flavodoxin (in collaboration with Anne-Frances Miller, Department of Chemistry)

Flavodoxins are electron-transfer proteins that contain the prosthetic group flavin ononucleotide. In the bacterium Escherichia coli, flavodoxin is reduced by the FAD-containing protein NADPH:ferredoxin (flavodoxin) oxidoreductase. flavodoxins serve as electron donors in the reductive activation of anaerobic ribonucleotide reductase, biotin synthase, pyruvate formate lyase, and cobalamin-dependent methionine synthase. Although three-dimensional structures are known for many of these proteins and domains, very little is known about the structural aspects of their interactions and the dynamics of electron-tranfer.

Current work in progress:

MD simulation and QM/MM computational analysis on the binding of flavin mononucleotide (FMN) to apo-flavodoxin in order to generate accurate NMR parameters computationally.

Publications

Sheetz, Michael
Publications

2015

Eun Suk Song, Mehmet Ozbil, Tingting Zhang, Michael Sheetz, David Lee, Danny Tran, Sheng Li, Rajeev Prabhakar, Louis B. Hersh and David W. Rodgers. The Polyanion Activation Sites of Insulin Degrading Enzyme (submitted to PLOS ONE, 2015)

2014

Antonis Anriotis, Michael Sheetz, Ernst Richter, and Madhu Menon, "Band alignment and optical absorption in Ga(Sb)N alloys,” J. Physics: Condens Matter 26 055013 (2014).
Rachel Angers, Jeffrey Christansen, Amy V. Nalls, Hae-Eun Kang, Nora Hunter, Ed Hoover, Candace K. Mathiason, Michael Sheetz, and Glenn C. Telling, “Structural effects of PrP polymorphisms on intra- and inter-species prion transmission,” Proc. Natl. Acad. Sci. (submitted 2014)

2013

Linliang Yu, Wei Lu, Cui Ye, Zhaoshuai Wang, Meng Zhong, Qian Chai, Michael Sheetz, and Yinan Wei. (2013) Role of a Conserved Residue R780 in Escherichia coli Multidrug Transporter AcrB. Biochemistry 52, 6790−6796.
Antonis Andriotis, Michael Sheetz, Ernst Richter, and Madhu Menon. (2013) Band alignment and optical absorption in Ga(Sb)N alloys. J. Physics: Condens Matter 26 055013 (2014)
A. N. Andriotis, G. Mpourmpakis, S. Lisenkov, R. M. Sheetz and M. Menon, “U-calculation of the LSDA+U functional using the hybrid B3LYP and HSE functionals", Phys. Status Solidi B 250, 356 (2013).

2012

A. N. Andriotis, E. Richter, S. Lisenkov, R. M. Sheetz and M. Menon, “Electronic structure, optical properties and electronic conductivity of SiC nanowires", Journal of Computational and Theoretical Nanoscience (JCTN) 9, 2008 (2012).

2011

R. M. Sheetz, E. Richter, A. N. Andriotis, S. Lisenkov, C. Pendyala, M. K. Sunkara and M. Menon, “Visible light absorption and large band gap bowing in dilute alloys of gallium nitride with antimony", Phys. Rev. B84 075304 (2011).
S. Lisenkov, A. N. Andriotis, R. M. Sheetz and M. Menon, “Effects of co-doping on the ferromagnetic enhancement in ZnO", Phys. Rev. B83, 235203 (2011).

2010

A. N. Andriotis, R. M. Sheetz and M. Menon, \LSDA+U approximation: An ecient calculation of the U-values", Phys. Rev. B 81 245103 (2010).
A. N. Andriotis, R. M. Sheetz and M. Menon, \Defect-induced defect-mediated magnetism in ZnO and carbon-based materials", J. Phys.: Condens. Matter 22 , 334210 (2010)
A. N. Andriotis, R. M. Sheetz, E. Richter and M. Menon, \Structural, electronic, magnetic, and transport properties of carbon-fullerene-based polymers", The Oxford Handbook of Nanoscience and Technology, edited by A. V. Narlikar and Y. Y. Fu, Vol. II: Materials, Chapter 21, page 745 (2010).

2009

R. M. Sheetz, I. Ponomareva, E. Richter, A. N. Andriotis and M. Menon, “Defectinduced optical absorption in the visible in ZnO nanowire", Phys. Rev. B80, 195314 (2009).

2008

A. N. Andriotis, R. M. Sheetz, N. Lathiotakis and M. Menon, “Tailoring the induced magnetism in carbon-based and non-traditional inorganic nanomaterials", Int. J. Nanotechnol., Vol. 6, Nos. 1/2 164 (2008).
A.N. Andriotis, R.M. Sheetz, E. Richter, and M. Memon "Band alignment and optical absorption in Ga(Sb)N alloys", J. Phys.: Condens. Matter 26 055013 (2014).
A. N. Andriotis, G. Mpourmpakis, S. Lisenkov, R. M. Sheetz and M. Menon, U-calculation of the LSDA+U functional using the hybrid B3LYP and HSE functionals , Phys. Status Solidi B 250, 356 (2013).

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