Trapping intermediates in metal transfer reactions of the CusCBAF export pump of Escherichia coli

Chacón, Kelly N.; Perkins, Jonathan; Mathe, Zachary; Alwan, Katherine; Ho, Ethan N.; Ucisik, Melek N.; Merz, Kenneth M.; Blackburn, Ninian J.

doi:10.1038/s42003-018-0181-9

Trapping intermediates in metal transfer reactions of the CusCBAF export pump of Escherichia coli

Journal Article · Tue Nov 13 23:00:00 EST 2018 · Communications Biology

DOI:https://doi.org/10.1038/s42003-018-0181-9· OSTI ID:1624530

^[1]; Perkins, Jonathan ^[2]; Mathe, Zachary ^[2]; Alwan, Katherine ^[3]; Ho, Ethan N. ^[2]; Ucisik, Melek N. ^[4]; ^[5]; Blackburn, Ninian J. ^[3]

Reed College, Portland, OR (United States); DOE/OSTI
Reed College, Portland, OR (United States)
Oregon Health & Science University, Portland, OR (United States)
Baylor College of Medicine, Houston, TX (United States)
Michigan State Univ., East Lansing, MI (United States)

Escherichia coli CusCBAF represents an important class of bacterial efflux pump exhibiting selectivity towards Cu(I) and Ag(I). The complex is comprised of three proteins: the CusA transmembrane pump, the CusB soluble adaptor protein, and the CusC outer-membrane pore, and additionally requires the periplasmic metallochaperone CusF. Here we used spectroscopic and kinetic tools to probe the mechanism of copper transfer between CusF and CusB using selenomethionine labeling of the metal-binding Met residues coupled to RFQ-XAS at the Se and Cu edges. The results indicate fast formation of a protein–protein complex followed by slower intra-complex metal transfer. An intermediate coordinated by ligands from each protein forms in 100 ms. Stopped-flow fluorescence of the capping CusF-W44 tryptophan that is quenched by metal transfer also supports this mechanism. The rate constants validate a process in which shared-ligand complex formation assists protein association, providing a driving force that raises the rate into the diffusion-limited regime.

View Accepted Manuscript (DOE)

Research Organization:: SLAC National Accelerator Laboratory, Menlo Park, CA (United States). Stanford Synchrotron Radiation Lightsource (SSRL)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES); USDOE Office of Science (SC), Biological and Environmental Research (BER); National Institutes of Health (NIH); National Institute of General Medical Sciences (NIGMS); Murdock Trust Natural Sciences

Grant/Contract Number:: AC02-76SF00515

OSTI ID:: 1624530

Journal Information:: Communications Biology, Journal Name: Communications Biology Journal Issue: 1 Vol. 1; ISSN 2399-3642

Publisher:: Springer NatureCopyright Statement

Country of Publication:: United States

Language:: English

References (52)

Structural mechanisms of heavy-metal extrusion by the Cus efflux system Delmar, Jared A.; Su, Chih-Chia; Yu, Edward W. BioMetals, Vol. 26, Issue 4 https://doi.org/10.1007/s10534-013-9628-0	journal	May 2013
EPR Spectroscopy Targets Structural Changes in the E. coli Membrane Fusion CusB upon Cu(I) Binding Meir, Aviv; Abdelhai, Ahmad; Moskovitz, Yoni Biophysical Journal, Vol. 112, Issue 12 https://doi.org/10.1016/j.bpj.2017.05.013	journal	June 2017
Crystal Structure of the Membrane Fusion Protein CusB from Escherichia coli Su, Chih-Chia; Yang, Feng; Long, Feng Journal of Molecular Biology, Vol. 393, Issue 2 https://doi.org/10.1016/j.jmb.2009.08.029	journal	October 2009
Charged Amino Acids (R83, E567, D617, E625, R669, and K678) of CusA Are Required for Metal Ion Transport in the Cus Efflux System Su, Chih-Chia; Long, Feng; Lei, Hsiang-Ting Journal of Molecular Biology, Vol. 422, Issue 3 https://doi.org/10.1016/j.jmb.2012.05.038	journal	September 2012
Modeling protein association mechanisms and kinetics Zhou, Huan-Xiang; Bates, Paul A. Current Opinion in Structural Biology, Vol. 23, Issue 6 https://doi.org/10.1016/j.sbi.2013.06.014	journal	December 2013
Models for the Metal Transfer Complex of the N-Terminal Region of CusB and CusF Ucisik, Melek N.; Chakravorty, Dhruva K.; Merz, Kenneth M. Biochemistry, Vol. 54, Issue 27 https://doi.org/10.1021/acs.biochem.5b00195	journal	June 2015
Spectroscopy of carbon monoxide-hemocyanins. Phosphorescence of the binuclear carbonylated copper centers Finazzi-Agro, Alessandro; Zolla, Lello; Flamigni, Lucia Biochemistry, Vol. 21, Issue 2 https://doi.org/10.1021/bi00531a032	journal	January 1982
Periplasmic Metal-Resistance Protein CusF Exhibits High Affinity and Specificity for Both Cu ^I and Ag ^I ^† Kittleson, Joshua T.; Loftin, Isabell R.; Hausrath, Andrew C. Biochemistry, Vol. 45, Issue 37 https://doi.org/10.1021/bi0612622	journal	September 2006
Interactions between CusF and CusB Identified by NMR Spectroscopy and Chemical Cross-Linking Coupled to Mass Spectrometry Mealman, Tiffany D.; Bagai, Ireena; Singh, Pragya Biochemistry, Vol. 50, Issue 13 https://doi.org/10.1021/bi102012j	journal	April 2011
Structure and Dynamics of the N-Terminal Domain of the Cu(I) Binding Protein CusB Ucisik, Melek N.; Chakravorty, Dhruva K.; Merz, Kenneth M. Biochemistry, Vol. 52, Issue 39 https://doi.org/10.1021/bi400606b	journal	September 2013
A Selenocysteine Variant of the Human Copper Chaperone for Superoxide Dismutase. A Se-XAS Probe of Cluster Composition at the Domain 3−Domain 3 Dimer Interface ^† Barry, Amanda N.; Blackburn, Ninian J. Biochemistry, Vol. 47, Issue 17 https://doi.org/10.1021/bi8001049	journal	April 2008
Selenocysteine Positional Variants Reveal Contributions to Copper Binding from Cysteine Residues in Domains 2 and 3 of Human Copper Chaperone for Superoxide Dismutase ^† Barry, Amanda N.; Clark, Kevin M.; Otoikhian, Adenike Biochemistry, Vol. 47, Issue 49 https://doi.org/10.1021/bi801438g	journal	December 2008
Direct Metal Transfer between Periplasmic Proteins Identifies a Bacterial Copper Chaperone ^† Bagai, Ireena; Rensing, Christopher; Blackburn, Ninian J. Biochemistry, Vol. 47, Issue 44 https://doi.org/10.1021/bi801638m	journal	November 2008
Fundamental Aspects of Protein−Protein Association Kinetics Schreiber, G.; Haran, G.; Zhou, H. -X. Chemical Reviews, Vol. 109, Issue 3 https://doi.org/10.1021/cr800373w	journal	March 2009
Absorption, emission, and photophysical properties of copper(I) pyrazole complexes and their carbonyl adducts Sorrell, Thomas N.; Borovik, A. S. Inorganic Chemistry, Vol. 26, Issue 12 https://doi.org/10.1021/ic00259a029	journal	June 1987
Luminescence behavior of copper(I)-imidazole complexes. A spectroscopic model for the carbonyl derivative of hemocyanin Sorrell, Thomas N.; Borovik, A. S. Journal of the American Chemical Society, Vol. 108, Issue 9 https://doi.org/10.1021/ja00269a075	journal	April 1986
Dynamic Multibody Protein Interactions Suggest Versatile Pathways for Copper Trafficking Keller, Aaron M.; Benítez, Jaime J.; Klarin, Derek Journal of the American Chemical Society, Vol. 134, Issue 21 https://doi.org/10.1021/ja3018835	journal	May 2012
Stable Cu(II) and Cu(I) Mononuclear Intermediates in the Assembly of the CuA Center of Thermus thermophilus Cytochrome Oxidase Chacón, Kelly N.; Blackburn, Ninian J. Journal of the American Chemical Society, Vol. 134, Issue 39 https://doi.org/10.1021/ja307276z	journal	September 2012
Probing Transient Copper Chaperone−Wilson Disease Protein Interactions at the Single-Molecule Level with Nanovesicle Trapping Benítez, Jaime J.; Keller, Aaron M.; Ochieng, Patrick Journal of the American Chemical Society, Vol. 130, Issue 8 https://doi.org/10.1021/ja7107867	journal	February 2008
Crystal structure of the CusBA heavy-metal efflux complex of Escherichia coli Su, Chih-Chia; Long, Feng; Zimmermann, Michael T. Nature, Vol. 470, Issue 7335 https://doi.org/10.1038/nature09743	journal	February 2011
The Atx1-Ccc2 complex is a metal-mediated protein-protein interaction Banci, Lucia; Bertini, Ivano; Cantini, Francesca Nature Chemical Biology, Vol. 2, Issue 7 https://doi.org/10.1038/nchembio797	journal	May 2006
Chaperone-mediated copper handling in the periplasm Kim, Eun-Hae; Rensing, Christopher; McEvoy, Megan M. Natural Product Reports, Vol. 27, Issue 5 https://doi.org/10.1039/b906681k	journal	January 2010
The assembled structure of a complete tripartite bacterial multidrug efflux pump Symmons, Martyn F.; Bokma, Evert; Koronakis, Eva Proceedings of the National Academy of Sciences, Vol. 106, Issue 17 https://doi.org/10.1073/pnas.0900693106	journal	April 2009
Tracking metal ions through a Cu/Ag efflux pump assigns the functional roles of the periplasmic proteins Chacon, K. N.; Mealman, T. D.; McEvoy, M. M. Proceedings of the National Academy of Sciences, Vol. 111, Issue 43 https://doi.org/10.1073/pnas.1411475111	journal	October 2014
Adaptor protein mediates dynamic pump assembly for bacterial metal efflux Santiago, Ace George; Chen, Tai-Yen; Genova, Lauren A. Proceedings of the National Academy of Sciences, Vol. 114, Issue 26 https://doi.org/10.1073/pnas.1704729114	journal	June 2017
Energetics of Copper Trafficking between the Atx1 Metallochaperone and the Intracellular Copper Transporter, Ccc2 Huffman, David L.; O'Halloran, Thomas V. Journal of Biological Chemistry, Vol. 275, Issue 25 https://doi.org/10.1074/jbc.c000172200	journal	April 2000
Substrate-linked Conformational Change in the Periplasmic Component of a Cu(I)/Ag(I) Efflux System Bagai, Ireena; Liu, Wenbo; Rensing, Christopher Journal of Biological Chemistry, Vol. 282, Issue 49 https://doi.org/10.1074/jbc.m703937200	journal	September 2007
A rapid, exact curved-wave theory for EXAFS calculations Gurman, S. J.; Binsted, N.; Ross, I. Journal of Physics C: Solid State Physics, Vol. 17, Issue 1 https://doi.org/10.1088/0022-3719/17/1/019	journal	January 1984
Structure and mechanism of the tripartite CusCBA heavy-metal efflux complex Long, Feng; Su, Chih-Chia; Lei, Hsiang-Ting Philosophical Transactions of the Royal Society B: Biological Sciences, Vol. 367, Issue 1592 https://doi.org/10.1098/rstb.2011.0203	journal	April 2012
The product of the ybdE gene of the Escherichia coli chromosome is involved in detoxification of silver ions Franke, Sylvia; Grass, Gregor; Nies, Dietrich H. Microbiology, Vol. 147, Issue 4 https://doi.org/10.1099/00221287-147-4-965	journal	April 2001
State-of-the-Art Analysis of Whole X-ray Absorption Spectra Binsted, N.; Hasnain, S. S. Journal of Synchrotron Radiation, Vol. 3, Issue 4 https://doi.org/10.1107/s0909049596005651	journal	July 1996
The Cus efflux system removes toxic ions via a methionine shuttle: The Cus Efflux System Su, Chih-Chia; Long, Feng; Yu, Edward W. Protein Science, Vol. 20, Issue 1 https://doi.org/10.1002/pro.532	journal	November 2010
Program DYNAFIT for the Analysis of Enzyme Kinetic Data: Application to HIV Proteinase Kuzmič, Petr Analytical Biochemistry, Vol. 237, Issue 2 https://doi.org/10.1006/abio.1996.0238	journal	June 1996
Metal Ion Capture Mechanism of a Copper Metallochaperone Chakravorty, Dhruva K.; Li, Pengfei; Tran, Trang T. Biochemistry, Vol. 55, Issue 3 https://doi.org/10.1021/acs.biochem.5b01217	journal	January 2016
Copper Transport and Trafficking at the Host–Bacterial Pathogen Interface Fu, Yue; Chang, Feng-Ming James; Giedroc, David P. Accounts of Chemical Research, Vol. 47, Issue 12 https://doi.org/10.1021/ar500300n	journal	October 2014
A Novel Copper-Binding Fold for the Periplasmic Copper Resistance Protein CusF ^† ^, ^‡ Loftin, Isabell R.; Franke, Sylvia; Roberts, Sue A. Biochemistry, Vol. 44, Issue 31 https://doi.org/10.1021/bi050827b	journal	August 2005
N-Terminal Region of CusB Is Sufficient for Metal Binding and Metal Transfer with the Metallochaperone CusF Mealman, Tiffany D.; Zhou, Mowei; Affandi, Trisiani Biochemistry, Vol. 51, Issue 34 https://doi.org/10.1021/bi300596a	journal	August 2012
Single-Molecule Dynamics and Mechanisms of Metalloregulators and Metallochaperones Chen, Peng; Keller, Aaron M.; Joshi, Chandra P. Biochemistry, Vol. 52, Issue 41 https://doi.org/10.1021/bi400597v	journal	October 2013
Insights into Cu(I) Exchange in HAH1 Using Quantum Mechanical and Molecular Simulations ^† Op't Hol, Bryan T.; Merz, Kenneth M. Biochemistry, Vol. 46, Issue 30 https://doi.org/10.1021/bi7007195	journal	July 2007
Insight into the Cation−π Interaction at the Metal Binding Site of the Copper Metallochaperone CusF Chakravorty, Dhruva K.; Wang, Bing; Ucisik, Melek N. Journal of the American Chemical Society, Vol. 133, Issue 48 https://doi.org/10.1021/ja208662z	journal	December 2011
Probing Transient Copper Chaperone−Wilson Disease Protein Interactions at the Single-Molecule Level with Nanovesicle Trapping Benítez, Jaime J.; Keller, Aaron M.; Ochieng, Patrick Journal of the American Chemical Society, Vol. 131, Issue 2 https://doi.org/10.1021/ja809445z	journal	December 2008
Crystal structures of the CusA efflux pump suggest methionine-mediated metal transport Long, Feng; Su, Chih-Chia; Zimmermann, Michael T. Nature, Vol. 467, Issue 7314 https://doi.org/10.1038/nature09395	journal	September 2010
Cu(I) recognition via cation-π and methionine interactions in CusF Xue, Yi; Davis, Anna V.; Balakrishnan, Gurusamy Nature Chemical Biology, Vol. 4, Issue 2 https://doi.org/10.1038/nchembio.2007.57	journal	December 2007
EPR spectroscopy identifies Met and Lys residues that are essential for the interaction between the CusB N-terminal domain and metallochaperone CusF Meir, Aviv; Natan, Adi; Moskovitz, Yoni Metallomics, Vol. 7, Issue 7 https://doi.org/10.1039/C5MT00053J	journal	January 2015
Energetics of Copper Trafficking between the Atx1 Metallochaperone and the Intracellular Copper Transporter, Ccc2 Huffman, David L.; O'Halloran, Thomas V. Journal of Biological Chemistry, Vol. 275, Issue 25 https://doi.org/10.1074/jbc.C000172200	journal	April 2000
Unification of the Copper(I) Binding Affinities of the Metallo-chaperones Atx1, Atox1, and Related Proteins: DETECTION PROBES AND AFFINITY STANDARDS Xiao, Zhiguang; Brose, Jens; Schimo, Sonja Journal of Biological Chemistry, Vol. 286, Issue 13 https://doi.org/10.1074/jbc.M110.213074	journal	January 2011
Mechanism of ATPase-mediated Cu ⁺ Export and Delivery to Periplasmic Chaperones : THE INTERACTION OF ESCHERICHIA COLI CopA AND CusF Padilla-Benavides, Teresita; George Thompson, Alayna M.; McEvoy, Megan M. Journal of Biological Chemistry, Vol. 289, Issue 30 https://doi.org/10.1074/jbc.M114.577668	journal	June 2014
Substrate-linked Conformational Change in the Periplasmic Component of a Cu(I)/Ag(I) Efflux System Bagai, Ireena; Liu, Wenbo; Rensing, Christopher Journal of Biological Chemistry, Vol. 282, Issue 49 https://doi.org/10.1074/jbc.M703937200	journal	September 2007
State-of-the-Art Analysis of Whole X-ray Absorption Spectra Binsted, N.; Hasnain, S. S. Journal of Synchrotron Radiation, Vol. 3, Issue 4 https://doi.org/10.1107/S0909049596005651	journal	July 1996
Unusual Cu(I)/Ag(I) coordination of Escherichia coli CusF as revealed by atomic resolution crystallography and X-ray absorption spectroscopy Loftin, Isabell R.; Franke, Sylvia; Blackburn, Ninian J. Protein Science, Vol. 16, Issue 10 https://doi.org/10.1110/ps.073021307	journal	October 2007
Molecular Analysis of the Copper-Transporting Efflux System CusCFBA of Escherichia coli Franke, S.; Grass, G.; Rensing, C. Journal of Bacteriology, Vol. 185, Issue 13 https://doi.org/10.1128/JB.185.13.3804-3812.2003	journal	July 2003
Crystal Structure of Escherichia coli CusC, the Outer Membrane Component of a Heavy Metal Efflux Pump Kulathila, Rithika; Kulathila, Ragini; Indic, Mridhu PLoS ONE, Vol. 6, Issue 1 https://doi.org/10.1371/journal.pone.0015610	journal	January 2011

Cited By (1)

Crystal structures of AztD provide mechanistic insights into direct zinc transfer between proteins Neupane, Durga Prasad; Fullam, Stephanie Hope; Chacón, Kelly Natalia Communications Biology, Vol. 2, Issue 1 https://doi.org/10.1038/s42003-019-0542-z	journal	August 2019

Similar Records

Tracking metal ions through a Cu/Ag efflux pump assigns the functional roles of the periplasmic proteins

Journal Article · Mon Oct 13 00:00:00 EDT 2014 · Proceedings of the National Academy of Sciences of the United States of America · OSTI ID:1349641

Crystal Structure of the Membrane Fusion Protein CusB from Escherichia coli

Journal Article · Mon Mar 29 00:00:00 EDT 2010 · J. Mol. Biol. · OSTI ID:1006036

Direct Metal Transfer Between Periplasmic Proteins Identifies a Bacterial Copper Chaperone

Journal Article · Mon May 11 00:00:00 EDT 2009 · Biochem. 47:11408,2008 · OSTI ID:958625

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
Biological fluorescence
Metalloproteins
Metals
Pathogens

Trapping intermediates in metal transfer reactions of the CusCBAF export pump of Escherichia coli

Citation Formats

References (52)

Cited By (1)

Similar Records

Related Subjects