Metal Ion Capture Mechanism of a Copper Metallochaperone

Subscriber access provided by UNIV OF CAMBRIDGE

Article

Metal Ion Capture Mechanism of a Copper Metallochaperone Dhruva K. Chakravorty, Pengfei Li, Trang T. Tran, Craig A. Bayse, and Kenneth M. Merz Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.5b01217 • Publication Date (Web): 21 Dec 2015 Downloaded from http://pubs.acs.org on December 27, 2015

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 free 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 accessible to all readers and 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.

Biochemistry 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

Biochemistry

Metal Ion Capture Mechanism of a Copper Metallochaperone Dhruva K. Chakravorty,1,2,3,4* Pengfei Li,3,4 Trang T. Tran,2, †, Craig A. Bayse 5 and Kenneth M. Merz Jr.1,4* 1

Institute for Cyber Enabled Research, Michigan State University, 567 Wilson Road, East

Lansing, MI 48824 2

Department of Chemistry, University of New Orleans, 2000 Lakeshore Drive New Orleans, LA

70148 3

Department of Chemistry and Quantum Theory Project, University of Florida, Gainesville, FL

32611-8435 4

Department of Chemistry, Michigan State University, East Lansing, MI 48824

5

Department of Chemistry, Old Dominion University, Norfolk, VA

KEYWORDS (Word Style “BG_Keywords”). CusF, SilB, metallochaperone, metal ion force field, metal ion release, second-shell hydrogen bond, metal ion efflux, Cu(I) chemistry.

ACS Paragon Plus Environment

1

Biochemistry

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 A novel cation-π interaction between the bound Cu+ metal ion and Trp44 in the periplasmic Cu+/Ag+ metallochaperone Escherichia coli CusF protects Cu+ from the oxidative influence of the periplasm. In a popular model of metal ion transfer, a conformational change in the metal binding loop disrupts the cation-π interaction and moves Trp44 aside to provide access to the occluded metal ion-binding site (MBS) in an “open” conformation. In this study, our molecular dynamics (MD) simulations support this putative mechanism of metal ion transfer. We find that the apo protein transitions back and forth from the crystallographically observed “closed” state to the hypothesized open conformation over multiple microseconds. In agreement with nuclear magnetic resonance (NMR) data our simulations show that similar transitions are prohibited in Cu+•CusF, suggesting that the conformational transitions are gated by a metal ion mediated second shell hydrogen bonds between metal binding residue His36 and Asp37 of the metal binding loop region. Ab initio quantum mechanical calculations indicate that metal ion binding strengthens this interaction significantly, much like what is found in the case of other metalloproteins. The study builds towards a common evolutionary role of metal ion mediated second shell hydrogen bonds in metalloprotein structure and function.


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

Biochemistry

Abbreviations: MBS, metal binding site MD, molecular dynamics NMR, nuclear magnetic resonance MBL, metal binding loop PMF, potential of mean force DFT, density functional theory QM/MM, quantum mechanical/molecular mechanical PDB, protein data bank LJ, Lennard-Jones


3

Biochemistry

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 4 of 31

Section I: Introduction Understanding the nature of protein-protein interactions, along with the association between heavy metal ion resistance and the prevalence of antibiotic resistance in pathogenic bacteria, are issues of pharmaceutical interest. In the bacterial pathogen Escherichia coli, the cusCFBA operon encodes the CusC(F)BA proteins belonging to the resistance nodulation-cell division superfamily that provide Ag+ and Cu+ resistance.1, 2 In a prevailing hypothesis of metal ion efflux, the CusF metallochaperone (Figure 1) transports Cu+/Ag+ ions from the periplasm to the CusCBA metal ion transporter protein-complex, which expels the metal ion from the cell.3, 4 The mechanisms of metal ion capture by CusF, and its subsequent transfer to CusB, however, remains poorly understood. Recent experiments find that CusF and the N-terminal disordered region of CusB interact transiently when either is in the metal bound form, allowing for reversible metal ion transfer.5 Furthermore, metal ion binding quenches conformational dynamics in both proteins, suggesting a model of complexation in which the apo protein samples multiple conformational ensembles in search of a complementary conformation that will bind the quenched conformation of the metal ion bound protein.

As such, knowledge of the

conformational dynamics of CusF will address questions regarding the process of CusF:CusB complex formation, while providing insights into the role of metal ions in modulating, inducing or gating conformational changes that allow for protein-protein interactions.6 Crystal structures of apo and Cu+/Ag+-bound CusF find the protein in a similar conformation.7-9 The MBS consisting of His36, Met47 and Met49 residues is situated in a hydrophobic pocket. The bound metal ion forms a unique cation-π interaction with the aromatic rings on Trp44 that help shield it from the oxidative influence of the periplasm. A similar H(X7)W(X2)-M-X-M MBS motif has since been observed in the C-terminal metallochaperone


4

Page 5 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

Biochemistry

Figure 1. (a) Cartoon representation of the CusF metallochaperone (PDB code 2VB2) in the metal bound state. The metal binding loop extending from His36 to Met47 residues is shown in blue. The Cu+ metal ion is shown as a silver sphere, and its coordinating residues (Trp44, Met49, His36 and Met47) are depicted in stick notation. (b) Cartoon representation of apo-CusF and Cu+•CusF in the closed (PDB code: 2VB2) and observed open conformation in our MD simulations. Trp44 is depicted in stick notation. Observed transitions are shown with solid green arrows while the open to close transition in Cu+•CusF is hypothesized and is shown with a dashed green line.

domain of the membrane fusion protein SilB from Cupriavidus metallidurans CH34 (Figure SI.1) that shares a CusF like fold.10 In a previous study, we calculated the Cu+/Ag+-π interaction in CusF to be on the order of ~10 kcal/mol.11 The weak Trp44-Cu+ interaction in CusF likely


5

Biochemistry

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 6 of 31

plays a role in metal ion transfer between CusF and CusB, as metal ion transfer does not occur in the Trp44Met mutant CusF, in which coordination to Met44 replaces the cation-π interaction. In this study, we propose that in a likely mechanism of metal ion transfer, the Trp44 side-chain and metal-binding loop (MBL) swings away from Cu+ and the other metal binding residues creating an open state (see Figure 1 (b)) that will provide solvent or metal binding residues in CusB access to the Cu+ binding site. In support of this hypothesis, transverse relaxation rates from NMR studies of apo-CusF and the related C-terminal domain of apo-SilB suggest slow conformational motions in the MBL on the microsecond to millisecond time scales.9, 10 These data were, however, not described as providing evidence of a conformational change in CusF on a slower timescale and lie at odds with conformations of CusF and SilB proteins determined using crystallographic and NMR methods.9,10 Such conformational switching was not observed in the metal loaded state suggesting that the apo-protein may sample conformations that are not sampled by the metal ion bound form. Furthermore, an askance conformation in which the MBL moves aside has been observed in the crystal structure of Cu2+ bound Trp44Ala mutant form of CusF (PDB code: 3E6Z) in which a methionine residue from a neighboring protein completes the coordination of Cu2+, supports the existence of a short-lived open conformation.12

Section II: Methods In this study, we investigate the conformational dynamics of apo and Cu+- bound CusF and the related C-terminal domain of SilB on the time scale of microseconds to elucidate the role of conformational sampling in the mechanisms of metal ion capture and transfer.

The

AMBER12 suite of molecular dynamics and analysis programs was used to simulate apo- and Cu+ bound forms of wild type CusF and SilB metallochaperones and the Trp44Ala mutant CusF


6

Page 7 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

Biochemistry

using the ff99SB and ff99SBildn force fields.13, 14 Crystal structures of CusF in the apo, Ag+, Cu+ and Cu++ bound forms of the protein share the same conformation and metal binding site.7, 8 In order to accurately compare the conformational dynamics of the apo and metal bound forms of the protein and avoid biases arising from pre-organization at the metal binding site, simulations of Cu+-bound CusF were propagated from the 2VB2 crystal structure. The long period of MD sampling during the equilibration and production phases will further help remove artifacts owing to dependencies on the starting structure. The 2L55 NMR structure of the C terminal domain of apo-SilB was chosen as the starting point for our simulations of SilB.10 For purposes of brevity, the C terminal domain of apo-SilB is referred to as apo-SilB in the remained of the text. Charged amino acids were modeled in protonation states determined using the H++ protonation state calculation server. In the copper bound state, metal binding residues and the metal ion were modeled using our hybrid metal ion model that was developed previously.11 In this scheme, the Cu+-Met47, H36 and M49 interactions were treated with a “bonded” model representation, while the Cu+-Trp44 cation-π interaction was modeled as a parameterized non-bonded interaction.11 Though a variety of bonded, non-bonded, semi-bonded and polarizable force field models have been proposed to treat metal ions in biological systems,15-26 this hybrid force field afforded us the ability to study metal ion bound conformational states that lack the cation-π interaction.11 In an effort to model metal ion release from Cu+-CusF to solvent, we developed 12-6 Lennard-Jones (LJ) parameters to model Cu+ as a non-bonded model in our simulations. We have previously found that it is hard to simultaneously reproduce both, the hydration free energy and the ion-oxygen distance (IOD) of the metal ion in water simultaneously while employing the simplistic 12-6 LJ nonbonded model. As a result, we have often found metal ions to escape into solution from the


7

Biochemistry

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 8 of 31

metal binding motif. While we have overcome these issues by employing the 12-6-4 LennardJones parameters for non-bonded models of metal ions, here we exploit this tendency to simulate metal ion release from CusF.27, 28 Towards this, we developed a set of values for Cu+ in TIP4PEW water model that would escape into solution. Parameters for Cu+ (Rmin/2 at 1.156 Å and ε of 0.00050520 kcal/mol) were developed28 to match the experimentally observed values of hydration free energy of Cu+ in water (-125.5 kcal/mol) in lieu of coordination geometry.29, 30 Details of the process used to calculate metal ions parameters have been previously provided.28 Determining Cu+ coordination in Trp44Ala mutant of CusF using quantum mechanical /molecular mechanical calculations The nature of metal ion binding in the Trp44Ala mutant of CusF was investigated to identify the role of Trp44 and the cation-π interaction in CusF function. X-ray absorption spectroscopy experiments have found the metal ion bound in a tetrahedral coordination environment in the mutant8, 12 that is reminiscent of the metal ion-binding site observed in the Trp44Met mutant form of CusF. An effort to determine the crystal structure of the Cu+•W44ACusF resulted in oxidizing the copper ion. The resultant Cu2+ -bound crystal structure of CusF (PDB code: 3E6Z) found the metal binding loop in an askance conformation that allowed Met8 from a neighboring protein in the crystal structure to coordinate with the metal ion.12 It has been proposed that a water molecule or negatively charged ion may occupy the fourth coordination site at the metal binding site of Cu+•W44A-CusF. In an effort to provide a structural basis for metal ion coordination in W44A CusF, quantum mechanical molecular mechanical (QM/MM) calculations31 were performed on a solvated and energy minimized structure of W44A in which a copper ion was placed at the metal binding site. Details of these calculations have been provided previously.11 In brief, iterative QM/MM calculations were performed at the M06-


8

Page 9 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

Biochemistry

2X/LACVP3P+*/OPLS2005 level of theory in which the copper ion, His36, Met47, Met49 and all water molecules within 3 Å from the metal ion were included in the QM region.32 The remaining protein residues, solvent molecules and ions were treated as part of the MM region. A Cl− ion was introduced by replacing a water molecule that lay within the first solvation shell of the metal ion. Similar to our previous calculations performed on Cu+•W44M CusF, all protein residues and water molecules within 12 Å region from the QM region were allowed to optimize during the energy-minimization process while the remaining system was kept frozen.11 Each protein structure was solvated in a periodically replicated octahedral box of SPC/E water molecules that provided a 10 Å solvent envelope around every protein atom.33, 34 Explicit Cl- ions were added to net-neutralize the solvated system.35 The solvated protein was then energy-minimized and equilibrated using a well-defined procedure that has been described previously.6, 36, 37 In brief, the solvated protein was energy-minimized in a step-wise manner such that various parts of the system were included in the energy-minimization process in an incremental manner. All solvent molecules and counter ions were first energy-minimized while the protein structure was restrained using a strong harmonic potential. In the case of the Trp44Ala mutant form of CusF, Ala44 was also allowed to relax during this step as well.

In

subsequent stages of energy-minimization, the restraints were sequentially released from the side-chain hydrogen atoms, side-chain residues and backbone amide groups. In the final step, the entire system was energy-minimized with no restraints in place. The “relaxed” protein was next equilibrated using a simulated annealing-like approach in which the solvated system was gradually heated to 300 K over 200 ps of MD in the canonical ensemble (constant volume and constant temperature, NVT) while maintaining weak harmonic restraints on the protein structure.33 The protein was then equilibrated for 1 ns of equilibration at 300K in the isobaric


9

Biochemistry

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 10 of 31

and isothermal ensemble (NPT) without any restraints.33 MD trajectories were then propagated from the equilibrated structure in the isobaric and isothermal ensemble (NPT) at 300K. A time step of 2 fs was employed over the course of our simulations and frames were collected every 10,000 steps (20 ps).

Covalent bonds to hydrogen atoms were constrained using the SHAKE

algorithm while long-range electrostatic calculations were calculated using the particle mesh Ewald method.33 The system temperature was maintained using Langevin dynamics with a 2 ps-1 collision frequency. Two trajectories of apo-CusF were propagated for 7 μs, while trajectories of apo-CusF-HID, apo-SilB, W44A Cu+•CusF and W44A apo-CusF were simulated for 2.7 μs, 2.5 μs, 1.7 μs and 6.5 μs respectively.

A trajectory of metal bound CusF from the closed

conformation was propagated for 2.5 μs while a trajectory of Cu+•CusF was propagated for 1.45 μs from the open conformation. Ab initio interaction energy calculations Ab initio calculations were performed at the second order Møller–Plesset perturbation level of theory while employing the augmented Dunning correlation-consistent polarized aug-ccpVDZ basis sets38, 39 to calculate the basis set superposition energy-corrected interaction energies by using the implementation of the counterpoise method in the Gaussian09 electronic structure theory program.40 These calculations were performed on chemically representative model system geometries obtained from crystallographic coordinates. Prior to calculating the interaction energies, hydrogen atom positions were optimized by performing density functional theory (DFT) calculations using M06-2X functional and the 6-31+G* basis set. All heavy atoms were maintained in their crystal structure positions over the course of these calculations.41, 42 Two-dimensional potential of mean force calculations


10

Page 11 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

Biochemistry

Free energy differences between states were calculated using two-dimensional potential of mean force (PMF) calculations. Towards this, we performed a series of umbrella sampling calculations along two coordinates used to transition between the two states.

In order to

efficiently generate conformations for a detailed sampling study, PMF simulations were first performed to transition from state 1 to state 2 along the diagonal described in Figure SI.2. The total reaction pathway was divided into a number of windows such that values were incremented in each successive window. In this process, the final snapshot of the starting window was treated as the initial structure of the subsequent window. Conformations were sampled for 1 ns of MD utilizing a step size as 1 fs in each window and data points were stored after every 10 fs of sampling time. Conformations from the diagonal scan were used as starting structures for a detailed scan along both coordinates while maintaining the same overlap between adjacent windows. Unlike the initial scan, each window was equilibrated for 1 ns followed by 5 ns of production PMF MD.

The equilibrated structure from a window was used as a starting

conformation for neighboring windows. In order to adequately sample around minima, windows characterizing the minima were sampled for an additional 125 ns of biased MD. The data from the biased simulations was collected and unbiased using the weighted histogram analysis method (WHAM)43 to calculate a two-dimensional free energy profile.

Section III: Results and Discussion We performed over 27 µs of MD simulations to determine the proposed open conformation of CusF and capture the transitions between the crystallographically determined closed and open states. Normal mode calculations performed on the Bahar group server,16 suggest that apo-CusF may indeed visit the hypothesized open conformation. Access to such an


11

Biochemistry

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 12 of 31

open conformation in MD simulations will depend on Trp44 overcoming the tight packing within the hydrophobic pocket.

With a view towards overcoming these interactions and

accelerating the closed to open conformational change in CusF, we first performed MD simulations on the Trp44Ala mutant form of CusF on the time scale of hundreds of nanoseconds (see Figure 2).

Figure 2. RMSD profile of the Trp44Ala mutant apo-CusF, wild type apo-CusF and Cu+•CusF compared to the 2VB2 crystal structure of CusF in the closed conformation. The RMSD profile was calculated for Cα (peptide backbone carbon) atoms. The Trp44Ala mutant rapidly transitions to an open conformation while the wild type protein trajectories sample a closed conformation.


12

Page 13 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

Biochemistry

Figure 3. Snapshots from MD simulations of apo Trp44Ala CusF (red ribbon) showing the protein in (a) closed/askance, (b) open and (c) wide-open conformations. Val42, His35 and Leu81 residues are shown in ball and stick depiction. The angle described by the Cα backbone carbon atoms of Val42-His35-Leu81 are found to be between 0-85, 85-110 and 110-180 degrees in these states respectively. Using the angle formed between Val42-His35-Leu81 Cα backbone carbon atoms (see Figure 3) as a metric to distinguish between conformations, we find that the Trp44Ala mutant form of CusF samples conformations corresponding to the crystallographicallly observed askance conformation and novel open conformations en-route to a “wide-open” conformation. After sampling the wide-open conformation, the protein returns to its native closed conformational state observed in the crystal structures. In a similar manner, wild-type CusF gradually transitions from the closed conformation to an open conformation and finally to a wide-open conformation. In this conformation, the metal binding site is more solvent accessible (Figure SI.4). The protein then returned to the closed conformation over 7 μs of MD (Figure 4


13

Biochemistry

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 14 of 31

and Movie SI.1). Similar results were obtained from a replicate simulation of apo-CusF and from simulations of the C-terminal metallochaperone fragment of apo-SilB. Over the course of these simulations, we further find the base of the β-barrel domain in CusF samples conformations that squeezed and widen the base of the protein (Figure SI.5). A heat map based on an inclusive metric that includes the putative CusB binding region, the MBL and the mobile loop region

Figure 4. (A) Cartoon depiction of CusF showing the triangular area described by Ala40-Gln75Lys58 and Asn43-Leu78-Lys31 residues. (B) Heat map of apo-CusF using the surface area of the triangles as a metric. The protein largely samples three conformations whose normalized population distributions are shown in (C). State 1 refers to (D) the closed conformation observed in crystal structures, State 2 refers to (E) the open conformation and State 3 refers to (F) the wide-open conformation. (G) Transitions between the conformations over 7 us of MD are shown with blue spheres representing State 1, red spheres representing State 2 and green spheres representing State 3.


14

Page 15 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

Biochemistry

described by residues Gln75 and Leu78 (see Figures 4.A and SI.3) finds the protein transition between the closed and wide-open conformations via the askance or open-conformations. These results provide a rationale for hitherto unexplained transverse relaxation rates observed in NMR studies of apo-CusF and the related C-terminal domain of apo-SilB, which suggested slow conformational motions in the MBL on the microsecond to millisecond time scales. Our simulations emphasize the role of conformational dynamics in metallochaperone function and elaborate on the conformations of CusF and SilB proteins determined from crystallographic and NMR methods. In a likely mechanism for Cu+-bound CusF to transition from the closed to the open conformation, the protective Cu+ cation-π interaction with Trp44 will be disrupted. In this scenario, water molecules or negatively charged ions may interact with the bound Cu+ metal ion to complete its coordination sphere in the open conformation. In support of this hypothesis, our QM/MM optimization calculations on the metal binding site of Cu+•W44A-CusF confirmed that a water molecule or a Cl- ion may complete the coordination environment of the bound Cu+ ion. Unlike our simulations of apo-CusF, we find that Cu+-bound CusF does not undergo similar conformational changes and remains trapped in the closed crystallographic conformation over multiple microseconds of MD (see Figure SI.6). These results agree closely with NMR-derived conformational dynamics data, which suggest that the slow conformational motions in the metal binding loop are quenched upon metal ion binding.9, 10 We next attempted to identify the determinants of the gating mechanism that allows for conformational changes in apo-CusF but prevents them in Cu+•CusF. While our QM/MM calculations suggest that metal ion coordination will be satisfied in the open conformation, our MD simulations indicate that other effects may be at play. In Cu+•CusF, the metal binding


15

Biochemistry

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 16 of 31

residues remained locked in a single conformation, suggesting that organization of the metal binding residues may likely stabilize interactions that lock the protein in the closed conformation. In support of this hypothesis, trajectories of apo-CusF found the MBS to be preorganized for metal ion binding in the closed conformation, while being disorganized in the open

Figure 5. Role of the His36-Asp37 hydrogen bond interaction in conformational switching observed in apo-CusF. (A) Cartoon representation of the metal binding loop region of CusF showing His36, Asp37 and Met47 residues in stick depiction. The hydrogen bonds between His36-Asp37 and His36-Met47 are shown with black lines. (B) These hydrogen bond are absent in the wide-open state. (C) A plot of the His36(N)-Asp37(O) hydrogen bond (heavy atom) distance over the course the simulation. The larger hydrogen bond distances indicate that the hydrogen bond is weaker in the open conformation and is absent in the wide-open conformations observed in our simulations. and wide-open conformations (see Figure SI.7). In order to clarify the role of organization at the MBS in CusF conformational dynamics, we performed simulations of Cu+•CusF starting from an open-conformation adopted from our simulations of apo-CusF. In these simulations, the metal binding residues were held in their metal bound conformation, while the remaining residues in


16

Page 17 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

Biochemistry

the MBL remained in a disorganized state. Unlike in the case of apo-CusF, the protein samples the open conformation over multiple microseconds and fails to return to the closed conformation. These simulations suggest that residues in the MBL were unable to re-form a critical interaction around the MBS owing to the constraints imposed by the metal ion. Taken together, these results indicate that the orientation of the Cu+ binding residues play a pivotal role in the conformational switching in CusF. Crystal structures of Cu+•CusF and apo-CusF and the C-terminal fragment of apo-SilB find a network of hydrogen bonds that connect the metal binding residue His36 to other residues in the MBL.7, 8 The Nε-H atom on the side-chain of His36 interacts with the backbone carbonyl group of the adjacent Asp37 residue. The His36 backbone amide group also forms a hydrogen bond with the backbone amide group of Met47 (see Figure 5), anchoring the two ends of the MBL. The hydrogen bond distances observed in crystal structure of apo and metal bound CusF are presented in Table SI.1. Our simulations of apo-CusF reveal that while these hydrogen bonds are present in the closed conformation, they break prior to the transition to the open conformations and once again reform prior to the protein returning to its closed conformation. Furthermore, an analysis of correlated motions suggests that residues in the MBL move in a correlated manner owing to the network of hydrogen bonds (see Figure 6). Overall, our calculations suggest that disrupting the His36-Asp37 hydrogen bond in apo-CusF starts a “zipper-like” cascade that breaks the hydrogen bond and van der Waals interactions between the residues comprising the loop. This in turn allows solvent entry and forces the protein to access the wide-open conformation. These interactions then re-form in a sequence similar to “zipping up”, re-creating the hydrophobic pocket and returning the protein to the observed closed conformation. In contrast, in Cu+/Ag+•CusF, His36 and Met47 remained locked in their metal


17

Biochemistry

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 18 of 31

binding conformations, helping preserve the His36-Asp37 and His36-Met47 hydrogen bonds at the base of the metal binding loop. This effect likely prevents the protein from switching between conformational states in our simulations of Cu+•CusF.

Figure 6. Analysis of correlated motions of backbone Cα carbon atoms from our simulations of apo-CusF. The correlation ranges from +1 for perfect correlation when residues move in the same direction to -1 for perfect anti-correlation in which residues move away from each other. MBL residues are anti-correlated with residues forming the β-barrel structure that are closest to it but are correlated to residues at the base of the β-barrel structure that have been implicated in interacting with CusB by experiments. In addition to locking His36 in its metal bound conformation, electronic effects resulting from metal ion binding can further stabilize the second shell His36-Asp37 interaction. Previous studies have investigated the role of second shell metal ion mediated hydrogen bond networks in protein structure and function.44-46 A previous DFT study found that hydrogen bond between the metal binding ligand, His67 and Asp49 bond was influenced by the zinc ion in active site of the


18

Page 19 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

Biochemistry

horse liver alcohol dehyrdrogenase enzyme.47 A Cu+ mediated second shell hydrogen bond has been implicated in the allosteric mechanism of the copper metallosensor protein Mycobacterium tuberculosis CsoR along with various zinc-binding ArsR/SmtB family transcriptional repressors and the MarR transcriptional factor AdcR.22, 48 Indeed, in a recent study, we discovered that metal ion binding strengthens second shell hydrogen bonds at allosterically active metal binding sites in transcriptional repressor proteins.22 These hydrogen bonds in turn dictate the extent of conformational sampling in these proteins. A similar effect was, however, not observed at sites that were not allosterically active, suggesting a possible conserved role for metal ion mediated hydrogen bonds in metal-sensing transcriptional repressor proteins. In addition to transcriptional repressor proteins, similar hydrogen bonds have been found to be critical for Cu+ exchange involving the metallochaperone HAH1 and other cupredoxins.49-51 Towards quantifying the influence of metal ion on the second shell His36-Asp37 hydrogen bond interaction in CusF, we calculated the interaction energies between the metal bound cluster and Asp37 for metal bound and apo conformations of CusF from crystal structure geometries (see Figure 7 and Table 1).

Figure 7. Chemically representative model systems used to calculate the inter-fragment interaction energies for the metal loaded state between the metal bound cluster consisting of the


19

Biochemistry

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 20 of 31

Cu+ ion, His36, Trp44, Met47 and Met 49 (Fragment 1) and Asp37 (Fragment 2). Apo-state interaction energies between the two fragments were calculated in the absence of the metal ion. The effect of metal ion binding on this interaction was calculated by subtracting the interaction energy values calculated for the apo cluster (M47•M49•H36 interaction energy with Asp37) from the interaction energy calculated for the metal bound state (Cu•M47•M49•H36 interaction energy with Asp37).

We find the interaction between the His36 and Asp37

containing fragments increases by ~7 kcal/mol in the metal bound state as compared to apo-CusF (see Table 1). Such a significant change in interaction energy between the fragments suggests that metal ion binding indeed helps stabilize this interaction.

Figure 8. RMSD of trajectory of apo-CusF-HID with respect to the closed conformation of CusF. The protein transitions between the open and closed conformations on the time scale of hundreds of nanoseconds much like our simulations of Trp44Ala CusF.


20

Page 21 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

Biochemistry

In an effort to understand the impact of the His36-Asp37 interaction on the time-scales of conformational transitions in apo-CusF, we eliminated the His36-Asp37 hydrogen bond by altering the protonation state of His36 from His-NεH to His-NδH, a protonation state not possible in metal bound CusF. For purposes of clarity, we refer to this protonation state model of apo-CusF as apo-CusF-HID in subsequent sections of the text.

Unlike apo-CusF, our

simulations find apo-CusF-HID to rapidly transition from the closed to open states on the time scale of hundreds of nanoseconds (see Figure 8). Simulations performed on the SilB fragment with these protonation states found similar transitions were possible at these time scales as well as shown in Figure SI.8. These transitions were faster than those observed in our simulations of apo-CusF for multiple trajectories, suggesting that the His36-Asp37 hydrogen bond may indeed play a role in dictating the conformational dynamics of apo-CusF. In a putative mechanism of metal ion transfer between Cu+•CusF and apo-CusB, it is likely that the interactions between the proteins drive Cu+•CusF to sample the open conformation. In an attempt to estimate the free energy difference between the closed and open conformations of Cu+•CusF, we performed two-dimensional potential of mean force (PMF) calculations. Our simulations of apo and metal-bound CusF suggest that the distance between Cu+ ion and the CZ3 atom of Trp44 and the protein backbone Φ dihedral angle described by the backbone atoms of Glu36 and Met47 provided a useful metric to distinguish between the closed and open state of the metallochaperone. While in a closed conformation, we found the Cu+CZ3(Trp44) distance to be 2.33 Å and the dihedral angle to be -147.53° (see Figure 9). In the wide-open conformation observed in our simulations, the corresponding Cu+-CZ3(Trp44) distance increases to 17.42 Å while the dihedral angle is now 47.61°. Towards building an


21

Biochemistry

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 22 of 31

understanding for this process, we performed two-dimensional PMF simulations using an umbrella-sampling approach that helped transition the metal bound protein from the closed to the open conformation by sampling along these coordinates.33 For our initial scan, the total reaction coordinate pathway was divided into 39 windows with an increment of 0.5 Å and 6.48° in each successive window along the distance and dihedral coordinates respectively. A harmonic potential with a force constant of 10 kcal mol-1Å-2 was applied to the Cu+-CZ3 distance while a force constant of 500 kcal mol-1 Rad-2 was applied to the dihedral angle in these calculations. The final conformation from these calculations along the diagonal had Cu+-Trp44 distance of 21.33 Å and Φ dihedral with a value of 98.72°. For the final scan, we sampled an area covering 39 windows along the Cu+-Trp44 distance, and 44 windows across the Glu46-Met47 dihedral angle, adding up to 1716 windows and over 9 μs of PMF-MD sampling.

Figure 9. The Cu-Trp44 and Glu46-Met47 dihedral coordinates (black lines) for our twodimensional PMF MD simulations shown in (a) closed and (b) open structure of CusF.


22

Page 23 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

Biochemistry

While these PMF calculations are limited in terms of the conformational changes owing to the choice of coordinates, they provide us with a lower estimate of an 8 kcal/mol energetic penalty for the protein to undergo this transition. In support of our findings, these calculations found that the His36-Asp37 hydrogen bond breaks en-route to the open conformational state (see Figure SI.9). In contrast, the His36-Met47 hydrogen bonds are broken later, in the process of transitioning from the open to a wide-open conformation. Finally, in an attempt to simulate metal ion release, we replaced the hybrid-bonded-model description of Cu+ in our simulations of Cu+•CusF with a non-bonded model of the Cu+ ion that was parameterized to escape into solution.15 In these simulations, we find that Cu+•CusF rapidly transitions to an open or askance conformation in the process of metal ion release and then returns to its closed conformation (see Figure SI.10). While these simulations are qualitative and cannot be relied upon at this time to give time-scale information, they do provide support for the general hypothesis that the open conformation plays an important role in metal ion release in CusF function.

Section IV: Conclusions In this study, we have provided atomistic details of the conformational dynamics in CusF and the metallochaperone portion of SilB that take place on the microsecond time scale. We propose that the function of these structurally similar copper metallachaperones depends on a previously unobserved novel conformational state that provides direct access to the metal binding site. While the apo protein is able to transition back forth between these states, the copper bound protein is unable to switch between conformational states, providing a rationale for previously unexplained experimental data.9,

10

Our calculations find that while the cation-π

interaction between Cu+ and Trp44 plays an important role in protecting the metal ion, a Cu+-


23

Biochemistry

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 24 of 31

mediated second shell coordination His36-Asp37 hydrogen bond likely controls the conformational switching in these proteins. These results may be validated by unnatural amino acid substitution experiments that target this specific hydrogen bond interaction.22 It is likely that interactions between the CusF and CusB proteins, allow CusF to overcome the energetic penalty required to transition to this open conformation. In this study, we find further support for the conserved evolutionary role of second coordination shell hydrogen bonds at metal binding sites that may be exploited in strategies targeting the metal ion homeostasis machinery in bacterial pathogens.22, 52

ASSOCIATED CONTENT Supporting Information. Tables, figures, hydrogen bond statistics, Gaussian input files and Movie SI.1 that shows a 7 μs MD trajectory of apo-CusF transition from the closed to the wideopen conformation and return to the closed conformation. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *email: [email protected] (KMM) and [email protected] (DKC); Tel: (517) 355-9715 Fax: (517) 353-7248

Present Addresses † TTT - Department of Chemistry, University of Florida, Gainesville, FL 32611-8435 Author Contributions


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

Biochemistry

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding Sources National Institutes of Health (K.M.M. GM044974) National Science Foundation and the Louisiana Board of Regents (D.K.C NSF-EPSCoR LASiGMA EPS-1003897 and LEQSF-EPS (2014)-PFUND-388). ACKNOWLEDGMENT We thank Dr. M. Nihan Ucisik and Ms. Sarah Gordon for helpful discussions. We gratefully acknowledge the National Institutes of Health (K.M.M. GM044974) and the National Science Foundation (D.K.C NSF-EPSCoR LASiGMA EPS-1003897) for funding and thank high performance computing at the University of Florida and the Louisiana Optical Network Initiative for computational support.


25

Biochemistry

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 26 of 31

References: [1] Ma, Z., Jacobsen, F. E., and Giedroc, D. P. (2009) Coordination chemistry of bacterial metal transport and sensing, Chem. Rev. 109, 4644-4681. [2] Su, C. C., Long, F., Zimmermann, M. T., Rajashankar, K. R., Jernigan, R. L., and Yu, E. W. (2011) Crystal structure of the CusBA heavy-metal efflux complex of Escherichia coli, Nature 470, 558-562. [3] Kim, E. H., Nies, D. H., McEvoy, M. M., and Rensing, C. (2011) Switch or Funnel: How RND-Type Transport Systems Control Periplasmic Metal Homeostasis, J. Bacteriol. 193, 2381-2387. [4] Chacon, K. N., Mealman, T. D., McEvoy, M. M., and Blackburn, N. J. (2014) Tracking metal ions through a Cu/Ag efflux pump assigns the functional roles of the periplasmic proteins, Proc. Natl. Acad. Sci. U. S. A. 111, 15373-15378. [5] Mealman, T. D., Zhou, M., Affandi, T., Chacon, K. N., Aranguren, M. E., Blackburn, N. J., Wysocki, V. H., and McEvoy, M. M. (2012) N-terminal region of CusB is sufficient for metal binding and metal transfer with the metallochaperone CusF, Biochemistry 51, 6767-6775. [6] Ucisik, M. N., Chakravorty, D. K., and Merz, K. M., Jr. (2015) Models for the Metal Transfer Complex of the N-Terminal Region of CusB and CusF, Biochemistry 54, 4226-4235. [7] Xue, Y., Davis, A. V., Balakrishnan, G., Stasser, J. P., Staehlin, B. M., Focia, P., Spiro, T. G., Penner-Hahn, J. E., and O'Halloran, T. V. (2008) Cu(I) recognition via cation-pi and methionine interactions in CusF, Nat. Chem. Biol. 4, 107-109. [8] Loftin, I. R., Franke, S., Blackburn, N. J., and Mcevoy, M. M. (2007) Unusual Cu(I)/Ag(I) coordination of Escherichia coli CusF as revealed by atomic resolution crystallography and X-ray absorption spectroscopy, Prot. Sci. 16, 2287-2293. [9] Mealman, T. D., Bagai, I., Singh, P., Goodlett, D. R., Rensing, C., Zhou, H. J., Wysocki, V. H., and McEvoy, M. M. (2011) Interactions between CusF and CusB Identified by NMR Spectroscopy and Chemical Cross-Linking Coupled to Mass Spectrometry, Biochemistry 50, 2559-2566. [10] Bersch, B., Derfoufi, K. M., De Angelis, F., Auquier, V., Ekende, E. N., Mergeay, M., Ruysschaert, J. M., and Vandenbussche, G. (2011) Structural and metal binding characterization of the C-terminal metallochaperone domain of membrane fusion protein SilB from Cupriavidus metallidurans CH34, Biochemistry 50, 2194-2204. [11] Chakravorty, D. K., Wang, B., Ucisik, M. N., and Merz, K. M., Jr. (2011) Insight into the cation-pi interaction at the metal binding site of the copper metallochaperone CusF, J. Am. Chem. Soc. 133, 19330-19333. [12] Loftin, I. R., Blackburn, N. J., and McEvoy, M. M. (2009) Tryptophan Cu(I)-pi interaction fine-tunes the metal binding properties of the bacterial metallochaperone CusF, J. Biol. Inorg. Chem. 14, 905-912. [13] Case, D. A., Darden, T. A., T.E. Cheatham, I., Simmerling, C. L., Wang, J., Duke, R. E., Luo, R., Walker, R. C., Zhang, W., Merz, K. M., Roberts, B., Wang, B., Hayik, S., Roitberg, A., Seabra, G., Kolossvai, I., Wong, K. F., Paesani, F., Vanicek, J., Liu, J., Wu, X., Brozell, S. R., Steinbrecher, T., Gohlke, H., Cai, Q., Ye, X., Wang, J., Hsieh, M.-J., Cui, G., Roe, D. R., Mathews, D. H., Seetin, M. G., Sagui, C., Babin, V., Luchko, T., Gusarov, S., Kovalenko, A., and Kollman, P. A. (2010) AMBER 11, University of California, San Francisco.


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

Biochemistry

[14] Lindorff-Larsen, K., Piana, S., Palmo, K., Maragakis, P., Klepeis, J. L., Dror, R. O., and Shaw, D. E. (2010) Improved side-chain torsion potentials for the Amber ff99SB protein force field, Prot.: Struc., Func. Bioinfo. 78, 1950-1958. [15] Li, P., Roberts, B. P., Chakravorty, D. K., and Merz, K. M., Jr. (2013) Rational Design of Particle Mesh Ewald Compatible Lennard-Jones Parameters for +2 Metal Cations in Explicit Solvent, J. Chem. Theory Comput. 9, 2733-2748. [16] Chennubhotla, C., and Bahar, I. (2007) Signal propagation in proteins and relation to equilibrium fluctuations, Plos Comput. Biol. 3, 1716-1726. [17] Ucisik, M. N., Chakravorty, D. K., and Merz, K. M., Jr. (2013) Structure and Dynamics of the N-Terminal Domain of the Cu(I) Binding Protein CusB, Biochemistry 52, 6911-6923. [18] Chakravorty, D. K., Wang, B., Lee, C. W., Giedroc, D. P., and Merz, K. M. (2011) Simulations of Allosteric Motions in the Zinc Sensor CzrA, J. Am. Chem. Soc. 134, 33673376. [19] Chakravorty, D. K., Wang, B., Lee, C. W., Guerra, A. J., Giedroc, D. P., and Merz, K. M., Jr. (2013) Solution NMR refinement of a metal ion bound protein using metal ion inclusive restrained molecular dynamics methods, J. Biomol. NMR 56, 125-137. [20] Chakravorty, D. K., and Merz, K. M., Jr. (2015) Role of substrate dynamics in protein prenylation reactions, Acc. Chem. Res. 48, 439-448. [21] Chakravorty, D. K., and Merz, K. M., Jr. (2014) Studying allosteric regulation in metal sensor proteins using computational methods, Adv. Prot. Chem. Struc. Biol. 96, 181-218. [22] Chakravorty, D. K., Parker, T. M., Guerra, A. J., Sherrill, C. D., Giedroc, D. P., and Merz, K. M., Jr. (2013) Energetics of zinc-mediated interactions in the allosteric pathways of metal sensor proteins, J. Am. Chem. Soc. 135, 30-33. [23] Fuchs, J. F., Nedev, H., Poger, D., Ferrand, M., Brenner, V., Dognon, J. P., and Crouzy, S. (2006) New model potentials for sulfur-copper(I) and sulfur-mercury(II) interactions in proteins: from ab initio to molecular dynamics, J. Comput. Chem. 27, 837-856. [24] Dalosto, S. D. (2007) Computer simulation of the interaction of Cu(I) with cys residues at the binding site of the yeast metallochaperone Cu(I)-Atx1, J. Phys. Chem. B 111, 29322940. [25] Rodriguez-Granillo, A., and Wittung-Stafshede, P. (2008) Structure and dynamics of Cu(I) binding in copper chaperones Atox1 and CopZ: a computer simulation study, J. Phys. Chem. B 112, 4583-4593. [26] Op't Holt, B. T., and Merz, K. M., Jr. (2007) Insights into Cu(I) exchange in HAH1 using quantum mechanical and molecular simulations, Biochemistry 46, 8816-8826. [27] Li, P., and Merz, K. M., Jr. (2014) Taking into Account the Ion-induced Dipole Interaction in the Nonbonded Model of Ions, J. Chem. Theory Comput. 10, 289-297. [28] Li, P., Song, L. F., and Merz, K. M., Jr. (2015) Systematic Parameterization of Monovalent Ions Employing the Nonbonded Model, J. Chem. Theory Comput. 11, 1645-1657. [29] Burda, J. V., Pavelka, M., and Šimánek, M. (2004) Theoretical model of copper Cu (I)/Cu (II) hydration. DFT and ab initio quantum chemical study, J. Molec. Struc.: THEOCHEM 683, 183-193. [30] Marcus, Y. (1991) Thermodyanmics of solvation of ions. Part 5 - Gibbs free energy of hydration at 298.15 K., J. Chem. Soc., Faraday Trans. 87, 5. [31] Senn, H. M., and Thiel, W. (2009) QM/MM Methods for Biomolecular Systems, Angew. Chem. Int. Edit. 48, 1198-1229.


27

Biochemistry

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 28 of 31

[32] Truhlar, D. G., Zhao, Y., and Schultz, N. E. (2006) Design of density functionals by combining the method of constraint satisfaction with parametrization for thermochemistry, thermochemical kinetics, and noncovalent interactions, J. Chem. Theo. Comput. 2, 364-382. [33] Allen, M. P., and Tildesley, D. J. (1987) Computer Simulations of Liquids, Clarendon Press, Oxford. [34] Jorgensen, W. L., and Tirado-Rives, J. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 6665-6670. [35] Joung, I. S., and Cheatham, T. E., 3rd. (2008) Determination of alkali and halide monovalent ion parameters for use in explicitly solvated biomolecular simulations, J. Phy. Chem. B 112, 9020-9041. [36] Bayden, A. S., Gomez, E. F., Audie, J., Chakravorty, D. K., and Diller, D. J. (2015) A combined cheminformatic and bioinformatic approach to address the proteolytic stability challenge in peptide-based drug discovery, Biopolymers ASAP article. [37] Carter, J. D., Mathias, J. D., Gomez, E. F., Ran, Y., Xu, F., Galiano, L., Tran, N. Q., D'Amore, P. W., Wright, C. S., Chakravorty, D. K., and Fanucci, G. E. (2014) Characterizing solution surface loop conformational flexibility of the GM2 activator protein, J. Phys. Chem. B 118, 10607-10617. [38] Dunning, T. H. (1989) Gaussian-Basis Sets for Use in Correlated Molecular Calculations .1. The Atoms Boron through Neon and Hydrogen, J. Chem. Phys. 90, 1007-1023. [39] Balabanov, N. B., and Peterson, K. A. (2005) Systematically convergent basis sets for transition metals. I. All-electron correlation consistent basis sets for the 3d elements ScZn, J. Chem. Phys. 123, 64107-64122. [40] Frisch, M. J., Trucks, G. W., Schlegel, H. B., Scuseria, G. E., Robb, M. A., Cheeseman, J. R., Montgomery, J. A. J., Vreven, T., Kudin, K. N., Burant, J. C., Millam, J. M., Iyengar, S. S., Tomasi, J., Barone, V., Mennucci, B., Cossi, M., Scalmani, G., Rega, N., Petersson, G. A., Nakatsuji, H., Hada, M., Ehara, M., Toyota, K., Fukuda, R., Hasegawa, J., Ishida, M., Nakajima, T., Honda, Y., Kitao, O., Nakai, H., Klene, M., Li, X., Knox, J. E., Hratchian, H. P., Cross, J. B., Bakken, V., Adamao, C., Jaramillo, J., Gomperts, R., Stratmann, R. E., Yazyev, O., Austin, A. J., Cammi, R., Pomelli, C., Ochterski, J. W., Ayala, P. Y., Morokuma, K., Voth, G. A., Salvador, P., Dannenberg, J. J., Zakrzewski, V. G., Dapprich, S., Daniels, A. D., Strain, M. C., Farkas, O., Malick, D. K., Rabuck, A. D., Raghavachari, K., Foresman, J. B., Ortiz, J. V., Cui, Q., Baboul, A. G., Clifford, S., Cioslowski, J., Stefanov, B. B., Liu, G., Liashenko, A., Piskorz, P., Komaromi, I., Martin, R. L., Fox, D. J., Keith, T., Al-Laham, M. A., Peng, C. Y., Nanayakkara, A., Challacombe, M., Gill, P. M. W., Johnson, B., Chen, W., Wong, M. W., Gonzalez, C., and Pople, J. (2009) Gaussian 09, A.01 ed., Gaussian, Inc., Wallingford, CT. [41] Cramer, C. J., and Truhlar, D. G. (2009) Density functional theory for transition metals and transition metal chemistry, Phy. Chem. Chem. Phys. 11, 10757-10816. [42] Ditchfie.R, Hehre, W. J., and Pople, J. A. (1971) Self-Consistent Molecular-Orbital Methods .9. Extended Gaussian-Type Basis for Molecular-Orbital Studies of Organic Molecules, J. Chem. Phys. 54, 724-728. [43] Kumar, S., Bouzida, D., Swendsen, R. H., Kollman, P. A., and Rosenberg, J. M. (1992) The Weighted Histogram Analysis Method for Free-Energy Calculations on Biomolecules .1. The Method, J. Comput. Chem. 13, 1011-1021.


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

Biochemistry

[44] Gutten, O., and Rulisek, L. (2015) How simple is too simple? Computational perspective on importance of second-shell environment for metal-ion selectivity, Phys. Chem. Chem. Phys. 17, 14393-14404. [45] Stone, E. M., Chantranupong, L., and Georgiou, G. (2010) The second-shell metal ligands of human arginase affect coordination of the nucleophile and substrate, Biochemistry 49, 10582-10588. [46] Dudev, T., Lin, Y. L., Dudev, M., and Lim, C. (2003) First-second shell interactions in metal binding sites in proteins: a PDB survey and DFT/CDM calculations, J. Am. Chem. Soc. 125, 3168-3180. [47] Gervasio, F. L., Schettino, V., Mangani, S., Krack, M., Carloni, P., and Parrinello, M. (2003) Influence of outer-shell metal ligands on the structural and electronic properties of horse liver alcohol dehydrogenase zinc active site, J. Phys. Chem. B 107, 6886-6892. [48] Ma, Z., Cowart, D. M., Ward, B. P., Arnold, R. J., DiMarchi, R. D., Zhang, L. M., George, G. N., Scott, R. A., and Giedroc, D. P. (2009) Unnatural Amino Acid Substitution as a Probe of the Allosteric Coupling Pathway in a Mycobacterial Cu(I) Sensor, J. Am. Chem. Soc. 131, 18044-18045. [49] Badarau, A., and Dennison, C. (2011) Copper trafficking mechanism of CXXC-containing domains: insight from the pH-dependence of their Cu(I) affinities, J. Am. Chem. Soc 133, 2983-2988. [50] Marshall, N. M., Garner, D. K., Wilson, T. D., Gao, Y. G., Robinson, H., Nilges, M. J., and Lu, Y. (2009) Rationally tuning the reduction potential of a single cupredoxin beyond the natural range, Nature 462, 113-116. [51] Wernimont, A. K., Huffman, D. L., Lamb, A. L., O'Halloran, T. V., and Rosenzweig, A. C. (2000) Structural basis for copper transfer by the metallochaperone for the Menkes/Wilson disease proteins, Nat. Struct. Biol. 7, 766-771. [52] Chakravorty, D. K. and Merz K. M. Jr. (2014) Studying Allosteric Regulation in Metal Sensor Proteins Using Computational Methods, in Biomolec. Model. Sim.: Adv. Prot. Chem. Struc. Biol. (Karabencheva-Christova, T., Ed.) 6-SEP-2014 ed., p 38, Academic Press, United Kingdom.


29

Biochemistry

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 30 of 31

TABLES

CusF PDB structure

Super Molecule

Fragment 1

Cu+•2VB2a

-0.3469

-0. 3182

Ag+•2VB3a

-0.2013

Ag+•2QCPa

Fragment 2

Interaction Energy (Eh)

Interaction Energy (kcal/mol)

-0. 2870

-0.0245

-15.42

-0.1726

-0.2870

-0.0250

-15.71

-0.1974

-0.1687

-0.2870

-0.0241

-15.15

apo-2VB2a

-0.1829

-0.1542

-0.2870

-0.0128

-8.06

apo-2VB3a

-0.1869

-0.1582

-0.2870

-0.0132

-8.33

apo-1ZEQa

-0.1829

-0.1542

-0.2870

-0.0138

-8.71

apo-1ZEQb

-0.1830

-0.1543

-0.2871

-0.0129

-8.14

apo-2QCPa

-0.1829

-0.1542

-0.2870

-0.0116

-7.33

apo-2QCPb

-0.1829

-0.1542

-0.2871

-0.0105

-6.59

4

(x10 Eh)

4

(x10 Eh)

3

(x10 Eh)

Table 1. Change in interaction energy values as a result of metal ion binding. Interaction energy values calculated between the metal bound cluster (fragment 1) and Asp 37 (fragment 2) for the apo and metal bound states of CusF. Metal binding strengthens the interaction between the two fragments by ~8 kcal/mol. a. b.

Interaction energies were calculated at the MP2/aug-cc-pVDZ level of theory Interaction energies were calculated at the MP2/6-311++G(d,p) level of theory


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

Biochemistry

Table of Contents Graphic


31

Metal Ion Capture Mechanism of a Copper Metallochaperone

Recommend Documents