Anomalous Dynamics of Water Confined in Protein–Protein and

Letter pubs.acs.org/JPCL

Anomalous Dynamics of Water Confined in Protein−Protein and Protein−DNA Interfaces Song-Ho Chong and Sihyun Ham* Department of Chemistry, Sookmyung Women’s University, Cheongpa-ro 47-gil 100, Yongsan-Ku, Seoul 04310, Korea S Supporting Information *

ABSTRACT: Confined water often exhibits anomalous properties not observable in the bulk phase. Although water in hydrophobic confinement has been the focus of intense investigation, the behavior of water confined between hydrophilic surfaces, which are more frequently found in biological systems, has not been fully explored. Here, we investigate using molecular dynamics simulations dynamical properties of the water confined in hydrophilic protein−protein and protein−DNA interfaces. We find that the interfacial water exhibits glassy slow relaxations even at 300 K. In particular, the rotational dynamics show a logarithmic decay that was observed in glass-forming liquids at deeply supercooled states. We argue that such slow water dynamics are indeed induced by the hydrophilic binding surfaces, which is in opposition to the picture that the hydration water slaves protein motions. Our results will significantly impact the view on the role of water in biomolecular interactions.

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interfacial water (Figure 1b), there appears an intermediate time region where the increase in the MSD is reduced compared to the bulk behavior. For the bridging water, which is a special type of the interfacial water forming concurrent hydrogen bonds with two proteins (Figure 1b), the MSD at the intermediate time is significantly flattened. This is reminiscent of the “cage effect” observed in supercooled water,19,20 where water molecules are trapped in a cage for a considerable time before starting the diffusion. Thus, the interfacial water exhibits a typical glassy character, and this shows up even at an ambient temperature. Another characteristic feature of the glassy dynamics is the non-Gaussian behavior at intermediate times where the cage effect sets in.21 The degree of non-Gaussianity is conventionally probed with the non-Gaussian parameter α2(t). This function takes a value of zero in the ballistic regime, starts to increase upon entering the intermediate regime, attains a maximum value there, which is higher in a more deeply supercooled state, and finally decreases to zero in the diffusion regime.21 The results for α2(t) shown in Figure 1d follow these general trends. For the bulk water, α2(t) remains close to zero since there is no cage effect at the ambient temperature. The maximum of α2(t) is somewhat developed for the surface water, and it is more significant for the interfacial water due to the onset of the cage effect. In particular, the peak height of α2(t) reaches ∼2.5 for the bridging water, which is comparable to the value observed for water at a deeply supercooled state.20 The interfacial water is thus glassy also in terms of the non-Gaussianity of the dynamics.

nderstanding the role played by water in mediating biomolecular interactions is among the central problems in physical chemistry.1−3 The best known water-mediated interaction is the hydrophobic interaction acting between apolar groups,4,5 which serves as the major driving factor for the protein folding.6 The resulting folded proteins typically possess such a tertiary structure in which hydrophobic groups are buried inside a core and hydrophilic groups are exposed to water. It therefore is not surprising that protein−protein and protein−DNA binding surfaces mainly comprise hydrophilic residues.7 Water is abundantly found in such hydrophilic biomolecular interfaces.8 However, although water in hydrophobic confinement has been the focus of intense study,9−12 in particular in connection with a possible dewetting-induced assembly,13−16 relatively less investigation has been carried out for the nature of the water molecules located between hydrophilic surfaces. Herein, we report results of molecular dynamics simulations (300 K and 1 bar) investigating the dynamics of the interfacial water in barnase−barstar17 and lac repressor−DNA18 complexes, which are among the most wellstudied protein−protein and protein−DNA systems. We analyze the mean-squared displacement (MSD), non-Gaussian parameter α2(t), and rotational correlators Cl(t) of the angularmomentum indices l = 1 and 2, which have been traditionally examined in the studies of liquid dynamics.19−23 Anomalous dynamical properties induced by the hydrophilic confinement shall be unveiled through the comparison with the behaviors of bulk water and noninterfacial surface water. We show in Figure 1c the MSD for the hydration water of the barnase−barstar complex (Figure 1a) in a log−log scale. All the curves display an initial t2 region describing the ballistic behavior. For the bulk water, this ballistic motion is followed by the diffusion dynamics proportional to t. For the surface and © XXXX American Chemical Society

Received: August 18, 2016 Accepted: September 23, 2016

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Figure 1. (a) Structure of the barnase−barstar complex. (b) Illustration of the noninterfacial surface water (spheres colored cyan represent water oxygen positions), interfacial water (orange), and bridging water forming concurrent hydrogen bonds (dotted lines) with two proteins. The average number of those waters ± standard deviation are reported in parentheses. (c−f) MSD of the center-of-mass position (c), non-Gaussian parameter α2(t) (d), rotational correlators C1(t) (e), and C2(t) (f) versus logarithmic time axis. In these panels, the curves for the bulk water (blue), noninterfacial surface water (cyan), interfacial water (orange), and bridging water (red) are displayed with different colors. The dashed lines in panel (c) represent a square (t2) and linear (t) time variation, whereas the dotted-dashed lines in (e) and (f) a logarithmic decay.

We next turn our attention to the rotational correlators C1(t) and C2(t) (Figure 1e,f). It is seen that the surface water displays a slower rotational dynamics than the bulk water and that the relaxation of the interfacial water is even slower. Notably, we observe a logarithmic decay at long times in the interfacial water. We find that this originates from the bridging water that exhibits a logarithmic decay over more than three decades in time (red curves in Figure 1e,f; see also Figure S1 comparing the bridging and nonbridging water dynamics). Although the logarithmic decay is not a universal glassy rotational relaxation (the universal relaxation is a stretched-exponential decay as indeed observed in supercooled water20), its presence has been

demonstrated for several glass-forming liquids at deeply supercooled states by optical-Kerr-effect measurements.24 This indicates that the interfacial water shows the glassy character also concerning the rotational dynamics. We also investigated the dynamics of the interfacial water in lac repressor−DNA complex18 (Figure 2a,b). We find quite similar results to those observed in the protein−protein complex: the MSD exhibits an intermediate region where the cage effect sets in (Figure 2c), α2(t) is highly non-Gaussian in this intermediate time region (Figure 2d), and the rotational dynamics is quite slow with the bridging water exhibiting the logarithmic decay (Figure 2e,f). However, unlike the case for 3968

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Figure 2. (a) Structure of the lac repressor−DNA complex. (b) Illustration of the noninterfacial surface water (spheres colored cyan represent water oxygen positions), interfacial water (orange), and the bridging water forming concurrent hydrogen bonds (dotted lines) with protein and DNA. The average number of those waters ± standard deviation are reported in parentheses. (c−f) MSD of the center-of-mass position (c), non-Gaussian parameter α2(t) (d), rotational correlators C1(t) (e), and C2(t) (f) versus logarithmic time axis. In these panels, the curves for the bulk water (blue), noninterfacial surface water (cyan), interfacial water (orange), and bridging water (red) are displayed with different colors. The dashed lines in panel (c) represent a square (t2) and linear (t) time variation, whereas the dotted-dashed lines in (e) and (f) a logarithmic decay.

the protein−protein complex, the rotational relaxation of the bridging water consists of two components each showing the logarithmic decay (red curves in Figure 2e,f). This reflects more chemically heterogeneous nature of the DNA surface consisting of a wide major groove, narrow minor groove, and charged backbone phosphate groups. For example, it was found in experiments25 and simulations26,27 that the dynamics of water are slower in the minor groove. Indeed, we observe that bridging water molecules belonging to the major and minor grooves display distinct relaxation times (Figure S2). The number of hydrogen bonds to protein or DNA is also found to significantly influence the rotational dynamics (see also Figure S2 and its caption). Furthermore, it has been recently demonstrated that the groove conformational fluctuations

serve as an additional source of the heterogeneous relaxations of the DNA hydration water.27 Hydration dynamics in RNA have also been shown to be heterogeneous, exhibiting a broad spectrum of relaxation times, depending on its local structures.28 Apart from such heterogeneous aspects, the interfacial water in the protein−DNA complex exhibits essentially the same glassy characters. What is the common feature in the protein−protein and protein−DNA interfaces that confers glassy characters on the interfacial water and that in particular gives rise to the logarithmic decay of the rotational dynamics? We notice in this regard the electrostatic complementarity of the biomolecular surfaces involved: the binding surface of barnase is positively charged, whereas that of barstar is negatively charged 3969

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Figure 3. (a,b) Surfaces are color coded by the electrostatic potential in which barnase and barstar (a) and lac repressor and DNA (b) are rotated so that the binding areas are facing the reader. (c,d) Probability distribution for cos θ between the water’s dipole vector and the electrostatic field at its position surrounding the barnase−barstar complex (c) and the lac repressor−DNA complex (d). In these panels, the probability distribution functions for the noninterfacial surface water (cyan), interfacial water (orange), and bridging water (red) are displayed with different colors.

(Figure 3a); the DNA binding domain of lac repressor is positively charged, whereas DNA is highly negatively charged because of the presence of the backbone phosphate (PO2−) groups (Figure 3b). This results in a strong electrostatic field that is exerted on the interfacial water. Although the role of electrostatic interactions can be probed by turning them off, as done in ref 29, we investigated their impact by examining to what extent water’s rotational degrees of freedom are affected. Indeed, we find that water’s dipole vector is highly oriented along the electrostatic field for the interfacial water, and this holds in particular for the bridging water (Figure 3c,d). The electrostatic interaction at the interface thus provides such a strong binding potential that significantly slows down the dynamics of the interfacial water. The logarithmic decay of the rotational dynamics can also be accounted for, at least qualitatively, in terms of the strong electrostatic field at the biomolecular interfaces by referring to the mode-coupling theory (MCT).30 Indeed, MCT predicts nearly logarithmic decay of rotational correlations when the reorientational dynamics is strongly hindered, and its schematic-model calculations successfully reproduced the optical-Kerr-effect spectra exhibiting the logarithmic relaxation.31 In supercooled liquids, the strong hindrance of

reorientational dynamics is brought about through the couplings to the slow density fluctuations, whereas in the systems studied here, this is due to the couplings to the strong electrostatic field. Despite such difference in detailed microscopic mechanisms, both the supercooled liquids and interfacial water are under the influence of the strong binding potentials that inhibit the rotational motions, and we consider that the MCT scenario provides a reasonable account of the appearance of the logarithmic decay in the interfacial water. The emergence of the glassy water dynamics and logarithmic decay at protein−protein and protein−DNA interfaces can thus be attributed to the presence of the strong electrostatic field, which in turn originates from the binding surface’s electrostatic complementarity. The electrostatic complementarity has been observed in numerous protein−protein binding surfaces.32 Furthermore, the DNA-binding domain of proteins predominantly comprises positively charged regions,33 which interact with DNA that is highly negatively charged. Therefore, the water molecules located at protein−protein and protein−DNA interfaces are generally expected to carry on glassy characters. Our results are in contrast to the glassy dynamics observed in water confined in a silica pore34 and a highly disordered calcium−silicate−hydrate35 at an ambient temperature, which 3970

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(6) Dill, K. Dominant Forces in Protein Folding. Biochemistry 1990, 29, 7133−7155. (7) Ansari, S.; Helms, V. Statistical Analysis of Predominantly Transient Protein−Protein Interfaces. Proteins: Struct., Funct., Genet. 2005, 61, 344−355. (8) Levy, Y.; Onuchic, J. N. Water Mediation in Protein Folding and Molecular Recognition. Annu. Rev. Biophys. Biomol. Struct. 2006, 35, 389−415. (9) Chandler, D. Interfaces and the Driving Force of Hydrophobic Assembly. Nature 2005, 437, 640−647. (10) Rasaiah, J. C.; Garde, S.; Hummer, G. Water in Nonpolar Confinement: From Nanotubes to Proteins and Beyond. Annu. Rev. Phys. Chem. 2008, 59, 713−740. (11) Berne, B. J.; Weeks, J. D.; Zhou, R. Dewetting and Hydrophobic Interaction in Physical and Biological Systems. Annu. Rev. Phys. Chem. 2009, 60, 85−103. (12) Giovambattista, N.; Rossky, P. J.; Debenedetti, P. G. Computational Studies of Pressure, Temperature, and Surface Effects on the Structure and Thermodynamics of Confined Water. Annu. Rev. Phys. Chem. 2012, 63, 179−200. (13) Lum, K.; Chandler, D.; Weeks, J. D. Hydrophobicity at Small and Large Length Scales. J. Phys. Chem. B 1999, 103, 4570−4577. (14) Liu, P.; Huang, X.; Zhou, R.; Berne, B. J. Observation of a Dewetting Transition in the Collapse of the Melittin Tetramer. Nature 2005, 437, 159−162. (15) Choudhury, N.; Pettitt, B. M. On the Mechanism of Hydrophobic Association of Nanoscopic Solutes. J. Am. Chem. Soc. 2005, 127, 3556−3567. (16) Giovambattista, N.; Lopez, C. F.; Rossky, P. J.; Debenedetti, P. G. Hydrophobicity of Protein Surfaces: Separating Geometry from Chemistry. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 2274−2279. (17) Buckle, A. M.; Schreiber, G.; Fersht, A. R. Protein−Protein Recognition: Crystal Structural Analysis of a Barnase−Barstar Complex at 2.0-Å Resolution. Biochemistry 1994, 33, 8878−8889. (18) Kalodimos, C. G.; Bonvin, A. M. J. J.; Salinas, R. K.; Wechselberger, R.; Boelens, R.; Kaptein, R. Plasticity in Protein− DNA Recognition: lac Repressor Interacts with its Natural Operator O1 through Alternative Conformations of its DNA-Binding Domain. EMBO J. 2002, 21, 2866−2876. (19) Gallo, P.; Sciortino, F.; Tartaglia, P.; Chen, S.-H. Slow Dynamics of Water Molecules in Supercooled States. Phys. Rev. Lett. 1996, 76, 2730−2733. (20) Sciortino, F.; Gallo, P.; Tartaglia, P.; Chen, S.-H. Supercooled Water and the Kinetic Glass Transition. Phys. Rev. E: Stat. Phys., Plasmas, Fluids, Relat. Interdiscip. Top. 1996, 54, 6331−6343. (21) Kob, W.; Donati, C.; Plimpton, S. J.; Poole, P. H.; Glotzer, S. C. Dynamical Heterogeneities in a Supercooled Lennard-Jones Liquid. Phys. Rev. Lett. 1997, 79, 2827−2830. (22) Chong, S.-H.; Kob, W. Coupling and Decoupling between Translational and Rotational Dynamics in a Supercooled Molecular Liquid. Phys. Rev. Lett. 2009, 102, 025702. (23) Mountain, R. D.; Thirumalai, D. Dynamical Aspects of Anisotropic Correlations in Supercooled Liquids. J. Chem. Phys. 1990, 92, 6116−6123. (24) Cang, H.; Novikov, V. N.; Fayer, M. D. Experimental Observation of a Nearly Logarithmic Decay of the Orientational Correlation Function in Supercooled Liquids on the Picosecond-toNanosecond Time Scales. Phys. Rev. Lett. 2003, 90, 197401. (25) Liepinsh, E.; Otting, G.; Wüthrich, K. NMR Observation of Individual Molecules of Hydration Water Bound to DNA Duplexes: Direct Evidence for a Spine of Hydration Water Present in Aqueous Solution. Nucleic Acids Res. 1992, 20, 6549−6553. (26) Saha, D.; Supekar, S.; Mukherjee, A. Distribution of Residence Time of Water around DNA Base Pairs: Governing Factors and the Origin of Heterogeneity. J. Phys. Chem. B 2015, 119, 11371−11381. (27) Duboué-Dijon, E.; Fogarty, A. C.; Hynes, J. T.; Laage, D. Dynamical Disorder in the DNA Hydration Shell. J. Am. Chem. Soc. 2016, 138, 7610−7620.

are driven mainly by the strong hydrophilic substrate−water interaction. Indeed, the protein−water interaction alone is not strong enough which typically results in only a 3−5 times slower surface water dynamics than in bulk,36 and the electrostatic effect is crucial to induce the glassy slow relaxations of the interfacial water. This is also contrary to the picture that the hydration water slaves protein motions, which is gained through the study of the so-called protein dynamical transition that is considered to be triggered by the hydration water.37,38 In fact, our results indicate that it is the biomolecular interfaces that induce the glassy interfacial water dynamics. Water is not just an environmental background but is an active and versatile entity essential for living systems, and this is accomplished by altering its characteristics depending on the milieus it is placed. The present work demonstrates and characterize such manifestations−glassy dynamics accompanying logarithmic rotational relaxations−that emerge when water is confined between hydrophilic biomolecular surfaces. Such sluggish water will definitely play a key structural role in determining the stability and specificity of biomolecular assembly. In addition, because of the dynamic nature of the anomalies induced by the hydrophilic confinement, it will be interesting to investigate how the water dynamics change during the protein−protein and protein−DNA binding processes, depending on the distance and relative orientation between two biomolecules and their conformational changes. Such investigation will clarify the detailed molecular mechanisms of biomolecular bindings including the static as well as dynamic role of the hydration water.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpclett.6b01858. Details on molecular dynamics simulations, bridging versus non-bridging water dynamics, bridging water dynamics at protein−DNA interface. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +82 2 710 9410. Fax: +82 2 2077 7321. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Samsung Science and Technology Foundation under Project Number SSTF-BA1401-13. REFERENCES

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