Edward Yu

Edward Yu

Professor - Physics & Astronomy

  • Structural and mechanistic aspects of membrane transport
  • X-ray crystallography of membrane proteins
  • Biophysics
A115 Physics Dept. of Physics & Astronomy Iowa State University Ames, IA 50011 Phone:  (515) 294-4955 Email:  ewyu@iastate.edu


B.S., Chemistry, Southern Illinois University, 1989 M.S., Physical Chemistry, Southern Illinois University, 1991 Ph.D., Physical Chemistry, Univ. of Michigan, 1997 Postdoctoral Fellow, UC Berkeley, 1998-2004

Research Interests

We currently focus on studying bacterial antibiotic resistance mediated by active multi-drug efflux transporters.  Our long-term goal is to elucidate the structures and fundamental mechanisms that give rise to multiple drug recognition and extrusion in these multi-drug transporters.  In addition, our laboratory addresses fundamental questions regarding the nature of multi-drug recognition and gene regulation in transcriptional regulators.  These regulators control the expression of multi-drug efflux transporters in bacteria by sensing the presence of a variety of structurally unrelated toxic compounds in the environment.  Our experimental approach mainly utilizes x-ray crystallography to elucidate toxic chemical binding and export by these membrane efflux transporters. (1)  Mechanism of antibiotic efflux in the MATE family of transporters Antibiotic efflux transporters are fundamental components in both intrinsic and acquired bacterial resistance to antimicrobials.  Neisseria gonorrhoeae, a bacterium that is responsible for causing the infectious disease gonorrhea, harbors multiple antibiotic efflux transporters from a number of different families.  In April 2007, the Centers for Diseases Control and Prevention (CDC) officially added Neisseria gonorrhoeae to the list of ‘Super Bugs’ that are now resistant to common antibiotics.  The primary target of this project is the N. gonorrhoeae NorM transmembrane efflux pump, which is a member of the most recently classified multidrug and toxic compound extrusion (MATE) family.  The structure and function of NorM is similar to YdhE from Escherichia coli.  To determine the capacity of these two transporters to confer multiple antimicrobial resistances, we previously cloned, expressed, and purified these two efflux proteins, and characterized their functions both in vivo and vitro.  In this study, we found that E. coli expressing norM and ydhE showed elevated (two-fold or greater) resistance to several antimicrobial agents, including fluoroquinolones, ethidium bromide, rhodamine 6G, acriflavin, proflavin, crystal violet, barberine, doxorubicin, novobiocin, enoxzcin, and tetraphenylphosphonium chloride. When expressed in E. coli, both transporters reduced ethidium bromide and norfloxacin accumulation through a mechanism requiring the proton-motive force and direct measurements of efflux confirmed that NorM behaves as a Na+-dependent transporter. The capacity of NorM and YdhE to recognize structurally divergent compounds was directly confirmed using steady-state fluorescence polarization assays.  These results revealed, for the first time, that these transporters bind antimicrobials with dissociation constants in the micromolar region.  We recently also crystallized the N. gonorrhoeae NorM multidrug efflux pump using hanging-drop vapor diffusion.  Despite these recent advances, no detailed structure-function information is available for the MATE family of transporters.  To fill these important gaps in our understanding of the active efflux systems in the MATE family, we thus plan to determine the X-ray structure of the N. gonorrhoeae NorM multidrug efflux pump, and identify important residues for multidrug recognition/extrusion in N. gonorrhoeae NorM and E. coli YdhE in this research project. (2)  How membrane proteins handle heavy metals Bacteria such as Escherichia coli have developed various mechanisms to overcome toxic environments that are unfavorable for their survival.  One important strategy that bacteria use to subvert toxic compounds, including heavy-metal ions, is the expression of membrane transporters that recognize and actively export these substances out of bacterial cells, thereby allowing the bugs to survive in extremely toxic conditions.  The project focuses on studying the E. coli CusABC efflux system that recognizes silver and copper ions and extrudes them out of the bacterial cell.  CusA is an inner membrane transporter, which belongs to the resistance-nodulation-division (RND) protein superfamily.  It consists of 1,047 amino acid residues. CusC, however, is a 457 amino acid polypeptide that forms an outer membrane channel in E. coli.  In conjunction with a 407 amino acid membrane fusion protein CusB, these proteins assemble as a tripartite efflux complex to mediate the extrusion of heavy-metal ions across both membranes of E. coli.  It has been proposed that CusB may act as an adaptor that interacts with CusA and CusC to form the CusABC complex.  The resulting CusABC complex (Figure 1) make direct contact with metal ions and selectively expel them out of the cell.  We recently cloned, expressed, and purified the full-length CusA, CusC, and CusB efflux proteins.  We will crystallize these proteins in detergent solutions using vapor-diffusion.  The crystal structures will provide us direct information about how these proteins handle heavy-metal ions.       Figure 1.  Model of the assembled tripartite CusABC efflux complex. (CusA, green; CusB, blue; CusC, red). From J. Eswaran et al., Curr. Opin. Struct. Biol., 2004, 14, 741.         (3)  Efflux pump mechanisms for drug recognition/extrusion Multi-drug efflux pumps interfere significantly with cancer chemotherapy and the treatment of bacterial infections, by recognizing a number of structurally unrelated toxic compounds and actively extruding them from cells.  One of our ongoing projects is to study the Escherichia coli AcrB transmembrane efflux pump, which shows the widest substrate specificity among all known multi-drug transporters.  We have determined the X-ray structures of AcrB in the presence of four structurally different toxic compounds (Figure 2).  The crystal structures illustrate that three ligand molecules bind simultaneously to the extremely large central cavity of 5000 cubic Angstroms, primarily by hydrophobic, aromatic stacking and van der Waals interactions.  Each ligand uses a slightly different subset of AcrB residues for binding.  The subsequent study of the efflux pump carried out by crystallizing a mutant AcrB with five structurally diverse ligands indicates that AcrB consists of two distinct binding sites.  These five ligands not only bind to various positions of the central cavity, but also to residues lining the deep external depression formed by the C-terminal periplasmic domain.  The structures also suggest that AcrB assembles as a trimer of three identical channels for the extrusion of drugs.  In the current phase, we are determining the structure and function of the AcrB transporter using X-ray crystallography and site-directed mutagenesis.  The planned work also focuses on co-crystallizing AcrB with the periplasmic membrane fusion protein AcrA, and determines the X-ray structure of the AcrAB co-crystal complex.           Figure 2. Structures of AcrB             (4)  Mechanisms of Antibiotic Efflux in Campylobacter Campylobacter jejuni is the leading bacterial cause of food-borne diarrhea in the USA as well as other developed countries.  It is also in the list of the NIAID Category B Priority Pathogens. C. jejuni is able to infect the host and colonize the intestinal tract.  To withstand the various deleterious environments, this microorganism contains 13 antibiotic efflux transporters that may be recruited to extrude antimicrobial compounds.  The primary contributor to antibiotic resistance in C. jejuni is the CmeABC antibiotic efflux transporter, which is tightly regulated by the transcriptional regulator CmeR.  We recently crystallized the CmeR regulator and have its x-ray structure determined (Figure 3).  The crystal structure have provided us valuable clues about how this regulator controls the production of the CmeABC antibiotic efflux transporter in C. jejuni.  We are currently co-crystallizing CmeR in the presence of bile acids and its target DNA.  We expect that these structures will give us novel insight into the mechanisms of multiple ligand recognition and gene regulation in CmeR.  The finding may also help to improve our knowledge of multi-drug resistance in Campylobacter.     Figure 3.  X-ray crystallographic structure of CmeR. Ribbon diagram of the CmeR dimer. The structure also shows a bound glycerol in the     antibiotic binding site of each molecule of CmeR.       (5)  Converting the AcrR regulator into a multiple chemical sensor This project addresses fundamental questions regarding the nature of multi-ligand recognition and gene regulation in transcriptional regulators.  The primary target is the Escherichia coli AcrR repressor that regulates the multidrug transporter AcrB.  The 215-residue AcrR consists of two domains, the C-terminal multi-ligand binding and the N-terminal DNA binding domains.  Upon binding a wide variety of structurally diverse ligands in the C-terminal region, it triggers conformational change at the N-terminal domain, prohibiting the binding of AcrR to its target DNA.  The net result is the over-expression of the AcrB transporter, which, in turn, promotes efflux from the cell, thus protecting it from toxic substances.  How can AcrR and other transcriptional repressors recognize a variety of toxic chemicals?  How do they respond to changing environmental conditions? An understanding of these repressors may allow us to uncover the mechanisms that these proteins use to bind multiple ligands, regulate gene expression, and transmit signal from one binding site to the other.  We have determined the x-ray crystallographic structure of AcrR (Figure 4), we will use this crystal structure to guide us to alter the function of AcrR and convert it into toxic chemical sensors.  In this project, we will convert AcrR to respond to chemicals that cannot be recognized by the regulator. Based on a simple Acr system network, we will develop a whole cell green fluorescence biosensor for multiple chemicals.  In addition, we will study the structural and functional relationships of the AcrR transcriptional regulator using X-ray crystallography and site-directed mutagenesis.         Figure 4. X-ray crystallographic structure for the AcrR regulator in the form of ribbon diagram.      

Selected Publications

1:  Bolla JR, Su CC, Yu EW. Biomolecular membrane protein crystallization. Philos Mag (Abingdon). 2012 Jul 1;92(19-21):2648-2661. PubMed PMID: 23869195; PubMed Central PMCID: PMC3712538. 2:  Delmar JA, Su CC, Yu EW. Structural mechanisms of heavy-metal extrusion by the Cus efflux system. Biometals. 2013 Aug;26(4):593-607. doi: 10.1007/s10534-013-9628-0. Epub 2013 May 9. PubMed PMID: 23657864; PubMed Central PMCID: PMC3732547. 3:  Bolla JR, Do SV, Long F, Dai L, Su CC, Lei HT, Chen X, Gerkey JE, Murphy DC, Rajashankar KR, Zhang Q, Yu EW. Structural and functional analysis of the transcriptional regulator Rv3066 of Mycobacterium tuberculosis. Nucleic Acids Res. 2012 Oct;40(18):9340-55. doi: 10.1093/nar/gks677. Epub 2012 Jul 19. PubMed PMID: 22821564; PubMed Central PMCID: PMC3467072. 4:  Su CC, Long F, Lei HT, Bolla JR, Do SV, Rajashankar KR, Yu EW. Charged amino acids (R83, E567, D617, E625, R669, and K678) of CusA are required for metal ion transport in the Cus efflux system. J Mol Biol. 2012 Sep 21;422(3):429-41. doi: 10.1016/j.jmb.2012.05.038. Epub 2012 Jun 6. PubMed PMID: 22683351; PubMed Central PMCID: PMC3423576. 5:  Long F, Su CC, Lei HT, Bolla JR, Do SV, Yu EW. Structure and mechanism of the tripartite CusCBA heavy-metal efflux complex. Philos Trans R Soc Lond B Biol Sci. 2012 Apr 19;367(1592):1047-58. doi: 10.1098/rstb.2011.0203. Review. PubMed PMID: 22411977; PubMed Central PMCID: PMC3297434. 6:  Routh MD, Zalucki Y, Su CC, Zhang Q, Shafer WM, Yu EW. Efflux pumps of the resistance-nodulation-division family: a perspective of their structure, function, and regulation in gram-negative bacteria. Adv Enzymol Relat Areas Mol Biol. 2011;77:109-46. Review. PubMed PMID: 21692368. 7:  Su CC, Long F, Zimmermann MT, Rajashankar KR, Jernigan RL, Yu EW. Crystal structure of the CusBA heavy-metal efflux complex of Escherichia coli. Nature. 2011 Feb 24;470(7335):558-62. doi: 10.1038/nature09743. PubMed PMID: 21350490; PubMed Central PMCID: PMC3078058. 8:  Lei HT, Shen Z, Surana P, Routh MD, Su CC, Zhang Q, Yu EW. Crystal structures of CmeR-bile acid complexes from Campylobacter jejuni. Protein Sci. 2011 Apr;20(4):712-23. doi: 10.1002/pro.602. PubMed PMID: 21328631; PubMed Central PMCID: PMC3081549. 9:  Su CC, Long F, Yu EW. The Cus efflux system removes toxic ions via a methionine shuttle. Protein Sci. 2011 Jan;20(1):6-18. doi: 10.1002/pro.532. Review. PubMed PMID: 20981744; PubMed Central PMCID: PMC3047057. 10:  Long F, Su CC, Zimmermann MT, Boyken SE, Rajashankar KR, Jernigan RL, Yu EW. Crystal structures of the CusA efflux pump suggest methionine-mediated metal transport. Nature. 2010 Sep 23;467(7314):484-8. doi: 10.1038/nature09395. PubMed PMID: 20865003; PubMed Central PMCID: PMC2946090. 11:  Su CC, Yang F, Long F, Reyon D, Routh MD, Kuo DW, Mokhtari AK, Van Ornam JD, Rabe KL, Hoy JA, Lee YJ, Rajashankar KR, Yu EW. Crystal structure of the membrane fusion protein CusB from Escherichia coli. J Mol Biol. 2009 Oct 23;393(2):342-55.  doi: 10.1016/j.jmb.2009.08.029. Epub 2009 Aug 18. PubMed PMID: 19695261; PubMed Central PMCID: PMC2792208. 12:  Routh MD, Su CC, Zhang Q, Yu EW. Structures of AcrR and CmeR: insight into the mechanisms of transcriptional repression and multi-drug recognition in the TetR family of regulators. Biochim Biophys Acta. 2009 May;1794(5):844-51. doi: 10.1016/j.bbapap.2008.12.001. Epub 2008 Dec 14. Review. PubMed PMID: 19130905; PubMed Central PMCID: PMC2729549. 13:  Gu R, Li M, Su CC, Long F, Routh MD, Yang F, McDermott G, Yu EW. Conformational change of the AcrR regulator reveals a possible mechanism of induction. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 Jul 1;64(Pt 7):584-8. doi: 10.1107/S1744309108016035. Epub 2008 Jun 11. PubMed PMID: 18607081; PubMed Central PMCID: PMC2443975. 14:  Long F, Rouquette-Loughlin C, Shafer WM, Yu EW. Functional cloning and characterization of the multidrug efflux pumps NorM from Neisseria gonorrhoeae and YdhE from Escherichia coli. Antimicrob Agents Chemother. 2008 Sep;52(9):3052-60. doi: 10.1128/AAC.00475-08. Epub 2008 Jun 30. PubMed PMID: 18591276; PubMed Central PMCID: PMC2533508. 15:  Su CC, Long F, McDermott G, Shafer WM, Yu EW. Crystallization and preliminary X-ray diffraction analysis of the multidrug efflux transporter NorM from Neisseria gonorrhoeae. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 Apr 1;64(Pt 4):289-92. doi: 10.1107/S1744309108006490. Epub 2008 Mar 21. PubMed PMID: 18391429; PubMed Central PMCID: PMC2374251. 16:  Li M, Gu R, Su CC, Routh MD, Harris KC, Jewell ES, McDermott G, Yu EW. Crystal structure of the transcriptional regulator AcrR from Escherichia coli. J Mol Biol. 2007 Nov 30;374(3):591-603. Epub 2007 Sep 29. PubMed PMID: 17950313; PubMed Central PMCID: PMC2254304. 17:  Su CC, Yu EW. Ligand-transporter interaction in the AcrB multidrug efflux pump determined by fluorescence polarization assay. FEBS Lett. 2007 Oct 16;581(25):4972-6. Epub 2007 Sep 25. Erratum in: FEBS Lett. 2007 Nov 27;581(28):5548. PubMed PMID: 17910961; PubMed Central PMCID: PMC2254335. 18:  Gu R, Su CC, Shi F, Li M, McDermott G, Zhang Q, Yu EW. Crystal structure of the transcriptional regulator CmeR from Campylobacter jejuni. J Mol Biol. 2007 Sep 21;372(3):583-93. Epub 2007 Jul 3. PubMed PMID: 17686491; PubMed Central PMCID: PMC2104645. 19:  Su CC, Rutherford DJ, Yu EW. Characterization of the multidrug efflux regulator AcrR from Escherichia coli. Biochem Biophys Res Commun. 2007 Sep 14;361(1):85-90. Epub 2007 Jul 17. PubMed PMID: 17644067; PubMed Central PMCID: PMC2104644. 20: Su CC, Shi F, Gu R, Li M, McDermott G, Yu EW, Zhang Q. Preliminary structural studies of the transcriptional regulator CmeR from Campylobacter jejuni. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2007 Jan 1;63(Pt 1):34-6. Epub 2006 Dec 16. PubMed PMID: 17183170; PubMed Central PMCID: PMC2330109.