IonâIon Repulsions and Charge-Shielding Effects Dominate the

Subscriber access provided by YORK UNIV

B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

Ion-Ion Repulsions and Charge Shielding Effects Dominate the Permeation Mechanism through the OmpF Porin Channel Juan Carlos Ahumada, Carlos Alemán, Jorge Soto-Delgado, and Juan Torras J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b09549 • Publication Date (Web): 05 Dec 2018 Downloaded from http://pubs.acs.org on December 6, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Ion-Ion Repulsions and Charge Shielding Effects Dominate the Permeation Mechanism through the OmpF Porin Channel Juan Carlos Ahumada,a,b Carlos Alemán,a,* Jorge Soto-Delgado,c and Juan Torras a,* a

Department of Chemical Engineering (EEBE) and Barcelona Research Center for Multiscale Science and Engineering, Universitat Politècnica de Catalunya, C/Eduard Maristany 10-14, 08019, Barcelona, Spain b

Departamento de Química, Universidad Técnica Federico Santa María, Casilla 110-V, Valparaíso, Chile

c

Departamento de Ciencias Químicas, Facultad de Ciencias Exactas, Universidad Andrés Bello, Quillota 980, Viña del Mar, Chile

Corresponding Authors * e-mail: [email protected] and [email protected]

ACS Paragon Plus Environment

1

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 31

ABSTRACT

OmpF is a wide channel bacterial porin frequently employed to study selective ionic translocation. The cationic preference of this porin is mainly determined by electrostatic forces between the translocated ion and the protein, the formation of ion pairs (e.g. K+···Cl−) being previously pointed as the main cause to favor the cationic transport through the constriction zone. Hybrid Quantum Mechanics / Molecular Mechanics – Molecular Dynamics (QM/MMMD) simulations, which have provided polarization-containing potentials of mean force (PMF) profiles for different permeation scenarios, reveal significant new insights related with the ion translocation mechanism. Results show that the permeation is dominated by electrostatic interactions, which in turn affect ion-protein interactions at the constriction zone. However, it is observed that ion flow is favored by ion-ion repulsions and, in a lesser extent, by chargeshielding effects, instead of the previously pointed ionic pair formation.


2

Page 3 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Introduction Ion channels of living organisms are pore-forming proteins (porins) located in cellular membranes that play a decisive role in communication and signaling processes within cellular communities.1-3 Outer membranes (OMs), which combine highly hydrophobic lipid bilayers with pore-forming proteins, play a dual role allowing the exchange of biological material for sustaining life and protecting from undesirable substances as a selective barrier. OMs are intimately related with human diseases since Gram-negative bacteria develop resistance to antibiotics due to modifications in their lipid or protein compositions.4 Moreover, such alterations affect the permeability and ion (cationic or anionic) selectivity of channels, which is a key function in many physiological and metabolic processes.5 Among the most extensively studied OMs are those of Gram-negative bacteria, Escherichia coli (E. coli) being the most used model system for the study of permeability and selectivity of ionic channels.6 OmpF is one of the major porins of E. coli. In this trimeric protein each monomer involves 340 amino acids and forms a 16-stranded anti-parallel β-barrel (Figure 1a).7 The antiparallel β-strands of the barrel are connected by eight short β-hairpins on the periplasmic side (T1-T8) and eight on the extracellular side (L1-L8). The inner barrel wall is about 30 Å high with elliptic section. One of the β-hairpins (L3) folds into the barrel, creating a constriction zone of 7 Å  11 Å at about half its height, restricting the accessibility (Figure 1a). The constriction zone of OmpF, which allows the passage of molecules with exclusion sized of 0.3: 6.4, 4.8 and 5.4 for K+, Na+ and Cl−, respectively) are the higher with values similar those CN reported in bulk water bulk (6.6, 5.2 and 6.0 for K+, Na+ and Cl−, respectively).17 In contrast, the CNs at the center of the PMF profiles are lower, the minimum value being located at ITC = −0.15 for K+, Na+ and Cl− ions (3.7, 2.6 and 2.0 for K+, Na+ and Cl−, respectively). Detailed inspection of the QM/MM-MD trajectories for such US window (ITC = −0.15) show partially


22

Page 23 of 31

desolvated ions embedded within the L3 channel loop, this scenario being more accentuated for the Na+ and Cl− ions than for K+. Obviously, the amount of ion···protein interactions at the constriction zone increases with decreasing CN values: K+ < Na+ < Cl−. 9.0

CN

7.5 6.0 4.5 3.0

K+ Na+ Cl-

(a)

1.5 0.0 -1.2

-0.8

-0.4

0

0.4

0.8

1.2

ITC

9.0

CN

7.5 6.0 4.5 3.0

K+ 2KA 2KB

(b)

1.5 0.0 -1.2

-0.8

-0.4

0

0.4

0.8

1.2

ITC

9.0 7.5

CN

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


6.0 4.5 3.0

KCl 2KACl 2KBCl

(c)

1.5 0.0 -1.2

-0.8

-0.4

0

0.4

0.8

1.2

ITC

Figure 7. Evolution of the water coordination number (CN) for pushed K+, Na+, and Cl− ions along ITC coordinate as derived from: (a) USK, USNa and USCl simulations; (b) USK, US2KA and US2KB simulations; and (c) USKCl, US2KACl and US2KBCl. Figure 7b indicates that ion-ion repulsion effects cause a slight increment in the averaged CN value of the translocated K+ ion (from CN= 5.9 for USK to CN= 6.1 and 6.0 for US2KA and US2KB, respectively). The CN at the maxima of the PMF, which is 5.4 for USK, decreases to CN= 3.2 for U2KB. Thus, the lowest permeation barrier (US2KB) coincides with the lowest CN value,


23


Page 24 of 31

corroborating that de-solvation is required to enhance the interactions with the residues at the walls of the protein pore. The addition of a fixed Cl− ion modifies the CN profile along the ITC translocation coordinate of K+. Thus, for USKCl the higher CN values correspond to the constriction zone (Figure 7c), the average value at such region being 6.0. However, when another fixed K+ ion is joining the simulation, besides the fixed Cl− ion, no significant changes in the coordination profile are observed (averaged CN = 6.2 and 5.7 for US2KACl and US2KBCl, respectively). Conclusions Hybrid QM/MM-MD simulations have been performed to obtain the PMFs for K+, Na+ and Cl− ions translocated through an OmpF channel using the US approach. Ion-ion repulsions, charge shielding effects and its synergetic combination, due to the presence of additional cations and anions within the channel, have been considered for simulating the K+ translocation. The simulations has been conducted in a fully water solvated system. The effect of the lipid bilayer is expected not change significantly the final ionic permeation mechanism since major interactions that might facilitate it permeation comes from ionic interactions with those ions that populate the channel. The selectivity derived from the PMF profiles, which is in agreement with experimental observations, shows a large coulombic component but modulated by the ion-protein interactions. Analysis of the interactions between different ions and the channel protein at the constriction zone points to more stabilizing interactions for Na+ than for K+ ion, which reduces the permeating barrier of the former. Besides, the Cl− ion exhibits a much stronger stabilizing interactions with the residues at the protein pore that makes more difficult to surpass the constriction zone. The facility to bind a Cl− ion in the constriction zone can help to reduce up to 32 % the permeation barrier of a K+ ion by charge-shielding effects. However, this effect is not


24

Page 25 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


as effective as the ion-ion repulsion, which reduces the barrier up to 40% when the additional cation is located between the extracellular region and the constriction zone of the channel. Indeed, the barrier almost disappears with values lower than usual thermal energy (86 % barrier reduction), leading to a diffusional phenomenon on the K+ translocation, when another cation is getting close to the constriction zone (~4.6 Å). The synergetic effect of both ionic repulsion and charge-shielding show a similar effect (88% barrier reduction). Although the cationic permeation mechanism presents a large coulombic component, important differences in ion-protein interactions at the constriction zone allow establishing differences. Thus, cation permeation is favored by ion-ion repulsions and, to a lesser extent, by charge-shielding effects.

ASSOCIATED CONTENT Supporting Information. Free energy transmembrane barriers, translocation free energy values, and complementary radial distribution functions (PDF).

AUTHOR INFORMATION The authors declare no competing financial interests.

ACKNOWLEDGMENT Authors acknowledge MINECO/FEDER (MAT2015-69367-R) and Agència de Gestió d'Ajuts Universitaris i de Recerca (2017SGR359) for financial support. J.C.A acknowledges a CONICYT Doctoral Fellowship (No. 21140238). Support for the research of C.A. was received


25


Page 26 of 31

through the prize “ICREA Academia” for excellence in research funded by the Generalitat de Catalunya.

REFERENCES 1.

Prindle, A.; Liu, J.; Asally, M.; Ly, S.; Garcia-Ojalvo, J.; Süel, G. M., Ion channels

enable electrical communication in bacterial communities. Nature 2015, 527, 59. 2.

Camilli, A.; Bassler, B. L., Bacterial Small-Molecule Signaling Pathways. Science 2006,

311 (5764), 1113-1116. 3.

Evans, W. H.; Martin, P. E. M., Gap junctions: structure and function. Mol. Membr. Biol.

2002, 19 (2), 121-136. 4.

Delcour, A. H., Outer membrane permeability and antibiotic resistance. Biochim.

Biophys. Acta, Proteins Proteomics 2009, 1794 (5), 808-816. 5.

Kass, R. S., The channelopathies: novel insights into molecular and genetic mechanisms

of human disease. J. Clin. Invest. 2005, 115 (8), 1986-1989. 6.

Aguilella, V. M.; Queralt-Martin, M.; Aguilella-Arzo, M.; Alcaraz, A., Insights on the

permeability of wide protein channels: measurement and interpretation of ion selectivity. Integrative Biology 2011, 3 (3), 159-172. 7.

Cowan, S. W.; Schirmer, T.; Rummel, G.; Steiert, M.; Ghosh, R.; Pauptit, R. A.;

Jansonius, J. N.; Rosenbusch, J. P., Crystal structures explain functional properties of two E. coli porins. Nature 1992, 358, 727. 8.

Dhakshnamoorthy, B.; Ziervogel, B. K.; Blachowicz, L.; Roux, B., A Structural Study of

Ion Permeation in OmpF Porin from Anomalous X-ray Diffraction and Molecular Dynamics Simulations. J. Am. Chem. Soc. 2013, 135 (44), 16561-16568.


26

Page 27 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


9.

Modi, N.; Benz, R.; Hancock, R. E. W.; Kleinekathöfer, U., Modeling the Ion Selectivity

of the Phosphate Specific Channel OprP. J. Phys. Chem. Lett. 2012, 3 (23), 3639-3645. 10.

Schirmer, T.; Phale, P. S., Brownian dynamics simulation of ion flow through porin

channels. J. Mol. Biol. 1999, 294 (5), 1159-1167. 11.

Pothula, K. R.; Solano, C. J. F.; Kleinekathöfer, U., Simulations of outer membrane

channels and their permeability. Biochim. Biophys. Acta, Biomembr. 2016, 1858 (7, Part B), 1760-1771. 12.

Im, W.; Roux, B. t., Ions and Counterions in a Biological Channel: A Molecular

Dynamics Simulation of OmpF Porin from Escherichia coli in an Explicit Membrane with 1M KCl Aqueous Salt Solution. J. Mol. Biol. 2002, 319 (5), 1177-1197. 13.

Chimerel, C.; Movileanu, L.; Pezeshki, S.; Winterhalter, M.; Kleinekathöfer, U.,

Transport at the nanoscale: temperature dependence of ion conductance. Eur. Biophys. J. 2008, 38 (1), 121. 14.

Pezeshki, S.; Chimerel, C.; Bessonov, A. N.; Winterhalter, M.; Kleinekathöfer, U.,

Understanding Ion Conductance on a Molecular Level: An All-Atom Modeling of the Bacterial Porin OmpF. Biophys. J. 2009, 97 (7), 1898-1906. 15.

Faraudo, J.; Calero, C.; Aguilella-Arzo, M., Ionic Partition and Transport in Multi-Ionic

Channels: A Molecular Dynamics Simulation Study of the OmpF Bacterial Porin. Biophys. J. 2010, 99 (7), 2107-2115. 16.

Cisneros, G. A.; Karttunen, M.; Ren, P.; Sagui, C., Classical Electrostatics for

Biomolecular Simulations. Chem. Rev. 2014, 114 (1), 779-814.


27


17.

Page 28 of 31

Bucher, D.; Guidoni, L.; Carloni, P.; Rothlisberger, U., Coordination Numbers of K+ and

Na+ Ions Inside the Selectivity Filter of the KcsA Potassium Channel: Insights from First Principles Molecular Dynamics. Biophys. J. 2010, 98 (10), L47-L49. 18.

Calandrini, V.; Dreyer, J.; Ippoliti, E.; Carloni, P., Hydration of chloride anions in the

NanC Porin from Escherichia coli: A comparative study by QM/MM and MD simulations. J. Chem. Phys. 2014, 141 (22), 22D521. 19.

Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L.,

Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 1983, 79 (2), 926-935. 20.

Becke, A. D., A new mixing of Hartree--Fock and local density-functional theories. J.

Chem. Phys. 1993, 98 (2), 1372-1377. 21.

Lee, C.; Yang, W.; Parr, R. G., Development of the Colle-Salvetti correlation-energy

formula into a functional of the electron density. Phys. Rev. B 1988, 37 (2), 785. 22.

Torras, J.; Seabra, G. d. M.; Deumens, E.; Trickey, S. B.; Roitberg, A. E., A versatile

AMBER-Gaussian QM/MM interface through PUPIL. J. Comput. Chem. 2008, 29 (10), 15641573. 23.

Torras, J.; Deumens, E.; Trickey, S. B., Software Integration in Multi-scale Simulations:

the PUPIL System. J. Comput. Aided Mater. Des. 2006, 13 (1-3), 201-212. 24.

Kefala, G.; Ahn, C.; Krupa, M.; Esquivies, L.; Maslennikov, I.; Kwiatkowski, W.; Choe,

S., Structures of the OmpF porin crystallized in the presence of foscholine-12. Protein Sci. 2010, 19 (5), 1117-1125.


28

Page 29 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


25.

Maier, J. A.; Martinez, C.; Kasavajhala, K.; Wickstrom, L.; Hauser, K. E.; Simmerling,

C., ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB. J. Chem. Theory Comput. 2015, 11 (8), 3696-3713. 26.

Case, D. A.; Betz, R. M.; Cerutti, D. S.; III, T. E. C.; Darden, T. A.; Duke, R. E.; Giese,

T. J.; Gohlke, H.; Goetz, A. W.; Homeyer, N., et al. AMBER 2016, University of California: San Francisco, 2016. 27.

Ryckaert, J.-P.; Ciccotti, G.; Berendsen, H. J. C., Numerical integration of the cartesian

equations of motion of a system with constraints: molecular dynamics of n-alkanes. J. Comput. Phys. 1977, 23 (3), 327-341. 28.

Torras, J.; He, Y.; Cao, C.; Muralidharan, K.; Deumens, E.; Cheng, H.-P.; Trickey, S. B.,

PUPIL: A systematic approach to software integration in multi-scale simulations. Comput. Phys. Commun. 2007, 177 (3), 265-279. 29.

Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman,

J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A., et al., Gaussian 09, Revision A.1. Gaussian, Inc.: Wallingford CT, 2009. 30.

Babin, V.; Roland, C.; Sagui, C., Adaptively biased molecular dynamics for free energy

calculations. J. Chem. Phys. 2008, 128 (13), 134101. 31.

Kumar, S.; Rosenberg, J. M.; Bouzida, D.; Swendsen, R. H.; Kollman, P. A., THE

weighted histogram analysis method for free-energy calculations on biomolecules. I. The method. J. Comput. Chem. 1992, 13 (8), 1011-1021. 32.

Saint, N.; Lou, K.-L.; Widmer, C.; Luckey, M.; Schirmer, T.; Rosenbusch, J. P.,

Structural and Functional Characterization of OmpF Porin Mutants Selected for Larger Pore Size. J. Biol. Chem. 1996, 271 (34), 20676-20680.


29


33.

Page 30 of 31

Phale, P. S.; Philippsen, A.; Widmer, C.; Phale, V. P.; Rosenbusch, J. P.; Schirmer, T.,

Role of Charged Residues at the OmpF Porin Channel Constriction Probed by Mutagenesis and Simulation. Biochemistry 2001, 40 (21), 6319-6325. 34.

Danelon, C.; Suenaga, A.; Winterhalter, M.; Yamato, I., Molecular origin of the cation

selectivity in OmpF porin: single channel conductances vs. free energy calculation. Biophys. Chem. 2003, 104 (3), 591-603. 35.

Berneche, S.; Roux, B., Energetics of ion conduction through the K+ channel. Nature

2001, 414 (6859), 73-77.


30

Page 31 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TOC Graphic


31

IonâIon Repulsions and Charge-Shielding Effects Dominate the

Recommend Documents

IonâIon Repulsions and Charge-Shielding Effects Dominate the