Hydration studies on the archaeal protein Sso7d using NMR measurements and MD simulations
© Bernini et al; licensee BioMed Central Ltd. 2011
Received: 14 April 2011
Accepted: 21 October 2011
Published: 21 October 2011
How proteins approach surrounding molecules is fundamental to our understanding of the specific interactions that occur at the surface of proteins. The enhanced surface accessibility of small molecules such as organic solvents and paramagnetic probes to protein binding sites has been observed; however, the molecular basis of this finding has not been fully established. Recently, it has been suggested that hydration dynamics play a predominant role in controlling the distribution of hot spots on surface of proteins.
In the present study, the hydration of the archaeal multifunctional protein Sso7d from Solfolobus solfataricus was investigated using a combination of computational and experimental data derived from molecular dynamics simulations and ePHOGSY NMR spectroscopy.
We obtained a convergent protein hydration landscape that indicated how the shape and stability of the Sso7d hydration shell could modulate the function of the protein. The DNA binding domain overlaps with the protein region involved in chaperon activity and this domain is hydrated only in a very small central region. This localized hydration seems to favor intermolecular approaches from a large variety of ligands. Conversely, high water density was found in surface regions of the protein where the ATP binding site is located, suggesting that surface water molecules play a role in protecting the protein from unspecific interactions.
It is very unlikely that proteins interact randomly with their molecular environment. Water, the most ubiquitous and abundant molecule of life, certainly plays a major role in controlling intermolecular interactions among biomolecules. To understand the interactions in specific protein surface regions that trigger biological functions requires an accurate delineation of protein hydration dynamics.
Protein hydration at the atomic level can be investigated using a variety of independent techniques such as molecular dynamics (MD) simulations [1–4], high resolution X-ray crystallography [5, 6] and NMR spectroscopy. Nuclear magnetic relaxation dispersion (MRD) studies seem to be particularly suited to determining the number of water molecules that are tightly bound to proteins [7, 8], but cannot provide information at atomic resolution per se. In principle, a detailed spatial distribution of water molecules that exhibit long residence times can be defined from hydration NMR experiments based on cross-relaxation , by analyzing the water-protein Overhauser effect (NOEwp) arising from selective water excitation. Pulsed field gradients, generated by standard hardware, are included in ePHOGSY-type sequences  limiting typical artifacts due to selective pulses . General developments of high resolution hydration NMR experiments have been critically reviewed  and signals observed in ePHOGSY-type spectra have been predicted also from chemical exchange or relayed Overhauser effects. Indeed, as suggested in the pioneering work of Wüthrich and co-workers , relayed water-protein Overhauser effects (NOE(wp)s) cannot be measured separately from NOEwps . The NOE(wp)s expected from all the amino acids bearing exchangeable hydrogens on their side chains are also propagated to hydrogens that are spatially close to the exchangeable side-chain hydrogen atoms. Because the amino acid side chains that bear the exchangeable hydrogen atoms are also the ones which are most frequently involved in the binding of water molecules , the interpretation of ePHOGSY signals in terms of protein hydration seems to be limited to only very few cases [9, 15]. It has been suggested  that a partial solution to the latter problem could be to compare the hydration patterns obtained from NMR studies and from MD simulations. A convergence of the computationally and experimentally derived hydration data could provide mutual validation.
MD simulations have indicated that strong hydration sites are not found in surface regions where protein hot spots are present . This result is of primary relevance to predictions of the functional properties of protein surfaces. ePHOGSY NMR studies have experimentally confirmed the absence of strong hydration sites at protein active sites . Furthermore, as recently reviewed , a tight correlation exists between the absence of MD derived high water density sites and surface regions highly accessible to paramagnetic probes [16, 19].
The 63 aminoacids long DNA binding protein, Sso7d, from the extreme thermophilic crenarchaeon Sulfolobus solfataricus supports multiple and structurally well-defined activities [20, 21] making it a good model protein for analyzing the relationship between solvent dynamics, surface accessibility and biological function. Because Sso7d binds different substrates, such as nucleic acids, ATP and misfolded/unfolded proteins [22, 23], as a model, it offers a unique opportunity to investigate how the different ligand molecules can adopt their own approach to bind to their targets on the protein surface. Sso7d surface accessibility has previously been studied using paramagnetic probes that preferentially accessed the specific surface regions where interactions with DNA and misfolded/unfolded proteins occurred . The finding that some surface-exposed regions of Sso7d were equally inaccessible to TEMPOL and Gd(III)(DTPA-BMA) has been ascribed to the presence of tightly bound water molecules .
The hydration of Sso7d and the homologous protein Sac7d from Sulfolobus acidocaldarius has been analyzed from crystal structures of DNA-protein complexes [25, 26]. In these complexes, four water molecules that were differently arranged in a diamond shape were found at the interface between Sso7d/Sac7d and the bound DNA. The four water molecules were apparently acting as a molecular filler to optimize protein-DNA binding. A series of conserved water molecules, including those that were involved in the Sso7d ATPase activity, were also observed. The structural stability of Sso7d , prevented major conformational changes of the protein backbone within the MD simulation timescale and allowed protein hydration investigations to be carried out using the MD Hydration Site (MDHS) approach [1, 4]. In the present work, Sso7d hydration was studied using MD simulations and ePHOGSY NMR spectroscopy to identify correlations between solvent dynamics and the functional features encoded in the protein surface.
Molecular Dynamics simulation
The MD results indicate that: (i) the simulation procedures that were used are reliable, because, independently from the reference structure used for the MD run, the Sso7d surface presents similar MDHS distributions and similar τ values; and (ii) because all the water molecules that were conserved in the crystal structures overlap with the MDHS, the hydration site predictions are accurate (see Figure 3).
ePHOGSY NMR measurements
The hydration dynamics of Sso7d has been investigated through 100 ns MD simulations and ePHOGSY NMR studies. As reported in Figure 2, the MDHS were unevenly distributed on the protein surface, indicating that the Sso7d hydration shell exhibited different local stabilities. A complex network of different contributions that include local flexibility and hydrophobicity in the protein structure determined the modes of molecular traffic of water and other molecules around the protein surface.
By comparing computational and experimental data on the dynamic aspects of Sso7d hydration, several interesting features became apparent. Correlations between the crystallographic water molecules and the MDHS were found only for water molecules that were in conserved positions. Furthermore, even though the signals observed in the Sso7d ePHOGSY spectra could not be unambiguously assigned to NOEwp or NOE(wp), the close correlation between the corresponding hydrogens and nearby MDHS was noticeable (Figure 3 and 7). From these findings it can be suggested that signals in ePHOGSY or equivalent spectra are, at least in a qualitative way, diagnostic of the hydration state of the protein. Conversely, the absence of the spectral signals over extended regions of the protein surface strongly supports the presence of a protein hot spot.
The hydration landscape of Sso7d consistently offered by X-ray diffractometry, NMR and MD simulations indicates that, in the protein surface region where binding to DNA and misfolded/unfolded proteins occurs [22, 23], a diamond-shaped cluster of four water molecules  is anchored to the Phe32-carbonyl located in the center of the binding surface via the water molecule 1001 (see Figure 8). The MDHS close to the Ser31 and Arg43 side chains clearly pointed to the positions of water molecules 1001 and 1006, in agreement with the observed ePHOGSY signals from the β and σ hydrogen atoms of, respectively, the Ser31 and Arg43 side chains in the 4-5 β-strand pair (see Figure 8). Moreover, the Phe32 region in the three stranded β-sheet was the only residue that showing relevant water residence times (see Figure 5).
In the Sso7d-GTGATCGC complex [PDB:1C8C], the backbone amide group of Lys28 and the N3 of G15 from the mismatched T-G base-pair are H-bond bridged via water 1128. A water molecule was found in a similar position for the complex with canonical base pairing . An MDHS, consistently found near the 26-28 protein loop, stabilized the water molecule that interacted with the base-pair (see Figure 8). In the middle of the protein/DNA interaction surface, Trp24-Nε1 and Ser31-Oγ exhibited medium sized ePHOGSY effects that were confirmed by the presence of a nearby MDHS. Comparative analysis of X-ray, NMR and MD results clearly delineated a binding sub-site in this protein region that was partially protected by water molecules that were easily displaced; thus favoring a large variety of interactions. In the DNA-bound structure, the MDHS was replaced by the N2 and N3 atoms of the G3 aromatic ring, indicating that hydrogen bonding could also be established by the amino acid side chains that support the Sso7d chaperone activity.
It was very interesting to compare the present dynamic hydration profile with our previous investigation of Sso7d surface accessibility using TEMPOL and Gd(III)(DTPA-BMA) paramagnetic probes . The anomalous small paramagnetic perturbations observed for the surface exposed Sso7d P-loop region  spanning residues 35-41, can be now interpreted in terms of high solvent density (many MDHS are found in this region) which would have prevented free local diffusion of the two probes (see Figure 3). The MD evidence is strengthened by the presence of four conserved water molecules in the region and by ePHOGSY signals from the backbone amide groups of Gly37, Gly39, Lys40 and Thr41. Furthermore, the lack of an ePHOGSY signal and the MDHS shield for the apical Gly38 residue explains the observed strong paramagnetic attenuation in the presence of Gd(III)(DTPA-BMA).
From a similar analysis, it was apparent how Sso7d loop I, including Tyr8, Lys9, Gly10 and Glu11 residues, was free from MDHS and conserved water molecules (see Figure 3). At the same time, loop I contained the NH group of Lys9 which appeared among the most TEMPOL and Gd(III)(DTPA-BMA) accessible backbone amides .
Combined analyses of MD and NMR results, particularly when data from ePHOGSY spectra and perturbation profiles induced by soluble paramagnetic probes are available, can provide a detailed view of protein surface dynamics. Surface accessibility and hydration of proteins appear strongly coupled, suggesting that the investigation procedure described here represents a new powerful strategy for protein hot spot mapping. It should be noted the dynamic character of the obtained data can offer an unique perspective for delineating transient sites where disruptors of protein-protein interactions can bind.
Three-dimensional structures, derived from NMR or X-ray experiments, of DNA-binding protein 7d from Sulfolobus solfataricus, Sso7d [UniProt:P39476] are available from the Protein Data Bank  both in the free form and in complexes with different DNA fragments [PDB:1BF4, PDB:1BNZ, PDB:1C8C, PDB:1JIC, PDB:1SSO]. In the present work, the numbering of the amino acid residues is from the crystal structures, with the first alanine in position 2.
Conserved water identification
To identify the conserved positions of water molecules in the crystal structures, the method proposed by Knight and coworkers was employed . For each structure, all water molecules within 0.36 nm of a nitrogen, oxygen or sulfur atom of the Sso7d protein were included in the search. After structurally aligning the heavy atoms in the proteins, water molecules from the different structures that fell within a common sphere of 0.2 nm radius and that were found in at least 2 out of 3 structures, were considered as conserved ones.
ns MD simulations were performed with explicit solvent using the lowest energy NMR structure of Sso7d [PDB:1JIC]  and the crystal structure of a Sso7d-DNA complex [PDB:1C8C] as the starting structures after removal of DNA and water molecules. The GROMACS package  with the AMBER force fields  were used for the MD run of solvated structure in a cubic box of equilibrated TIP3P water molecules . The initial shortest distance between the protein and the box boundaries was set to 1.0 nm and chloride ions were added to achieve global electric neutrality. Afterwards, the energy of the system was minimized with 900 steps of conjugate gradients. To achieve good equilibration prior to the production MD run, the system was subjected to a short (20 ps) MD run during which the atoms of the macromolecule were restrained to their position and only the solvent were allowed to move. The protein-water system was simulated in the NPT ensemble at constant temperature (300 K) and pressure (1 atm); a weak coupling to external heat and pressure baths was applied (relaxation times were 0.1 ps and 0.5 ps, respectively). All covalent bonds were constrained using the LINCS algorithm and non-bonded interactions were computed using the PME method  with a grid spacing of 0.12 nm for electrostatic contribution and with a 0.9 nm cut-off for the van der Waals contribution. An integration time step of 2 fs was used and trajectory snapshots were saved every 0.2 ps.
Because the backbone RMSD leveled off after an equilibration period of 100 ps, the subsequent analysis of the MD trajectories was carried out from 100 ps onward.
Water density function and residence time calculations
Solvent density maps, with maxima defined as molecular dynamics hydration sites (MDHS) [1, 4], were calculated from the atomic coordinates of the explicit waters in the simulation. The space surrounding the protein was divided into two shells: the first extended to a distance of 0.6 nm from the protein surface and included the protein hydration sites; the second represented the bulk solvent and extended from 0.6 nm to 0.8 nm from the protein surface. The water positions were computed in a 3D grid (step-size 0.05 nm). For each frame, the protein was superimposed onto a reference structure to eliminate the effects of translations and rotations. A 3D iso-contour plot of the resulting water density was obtained using the PyMOL software package (http://www.pymol.org), see Figure 2. Solvent density map correlation was carried out using MapMan software . The average residence times for the water molecules were computed for both MD trajectories according to the method described in . A residence times profile was obtained by binning values into 100 ps bins for the range 0-1000 ps and in a single bin for 1000 ps onward.
NMR samples were prepared by dissolving Sso7d in H2O/D2O (90:10 v/v) to make 1.2 mM protein solutions which were adjusted to pH 4.5 by the addition of small amounts of HCl or KOH. Procedures for protein expression and purification have been reported elsewhere .
NMR measurements were carried out at 300 K using a Bruker DRX 600 spectrometer to reproduce the experimental conditions of the original structural studies . Data processing and spectral analysis were performed using the, XWinNMR (Bruker BioSpin GmbH, Germany) and Sparky  software respectively. 1D ePHOGSY spectra with NOE and ROE were obtained by running 1,024 scans over 8,192 data points and 2D ePHOGSY-TOCSY spectra with NOE (see Figure 6) were obtained using 256 increments and 320 scans over 2,048 data points. In all the ePHOGSY experiments, a spectral width of 8,096 Hz was used, the mixing time to build up the intermolecular Overhauser effects was 202 ms, and the TOCSY mixing time was 75 ms. The water-selective 180° Gaussian pulse in all the ePHOGSY sequences was achieved according to Dalvit  with a duration of 25 ms. Water suppression was achieved following the scheme of Hwang . Observed chemical shifts were congruent with those from BioMagResBank (http://www.bmrb.wisc.edu) for the backbone1H and15N resonances for Sso7d [BMRB entry:5909].
enhanced Protein Hydration Observed through Gradient SpectroscopY
Heteronuclear Single Quantum Coherence
Nuclear Magnetic Resonance
nuclear Overhauser effect
root mean square deviation
rotating-frame Overhauser effect
Total Correlation SpectroscopY.
We thank the Istituto Toscano Tumori (ITT), Italy, for financial support. We also thank Matteo Bernini for picture development and Prof. Lucia Zetta for helpful discussion.
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