Case Study of Hydrogen Bonding in a Hydrophobic Cavity - American

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A Case Study of Hydrogen Bonding in a Hydrophobic Cavity Yi-Chen Chen, Chao-Sheng Cheng, Siu-Cin Tjong, Hsien-Sheng Yin, and Shih-Che Sue J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp5097053 • Publication Date (Web): 20 Nov 2014 Downloaded from http://pubs.acs.org on November 26, 2014

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The Journal of Physical Chemistry

A Case Study of Hydrogen Bonding in a Hydrophobic Cavity Yi-Chen Chen1, Chao-Sheng Cheng1, Siu-Cin Tjong1,2, Hsien-Sheng Yin1,* and Shih-Che Sue1,*

1

Institute of Bioinformatics and Structural Biology and Department of Life Science,

National Tsing Hua University, Hsinchu 30013, Taiwan 2

Department of Chemistry and Frontier Research Center on Fundamental and Applied

Sciences of Matters, National Tsing Hua University, Hsinchu 30013, Taiwan *

Corresponding author

*

Address for correspondence

Shih-Che Sue, Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 30013, Taiwan, Tel: (886)-3-5742025; Fax: (886)-3-5715934; E-mail: [email protected]

Hsien-Sheng Yin, Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 30013, Taiwan, Tel: (886)-3-5742469; Fax: (886)-3-5715934; E-mail: [email protected]

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ACS Paragon Plus Environment


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Abstract

Protein internal hydrogen bonds and hydrophobicity determine protein folding and structure stabilization, and the introduction of a hydrogen bond has been believed to represent a better interaction for consolidating protein structure. We observed an alternative example for chicken IL-1β. The native IL-1β contains a hydrogen bond between the Y157 side chain OηH and I133 backbone CO, whereby the substitution from Tyr to Phe abolishes the connection and the mutant without the hydrogen bond is more stable. An attempt to explain the energetic view of the presence of the hydrogen bond fails when only considering the nearly identical X-ray structures. Here, we resolve the mechanism by monitoring the protein backbone dynamics and interior hydrogen bond network. IL-1β contains a hydrophobic cavity in the protein interior, and Y157 is one of the surrounding residues. The Y157 OηH group introduces an unfavorable energy in the hydrophobic cavity, therefore, sequestering itself by forming a hydrogen bond with the proximate residue I133. The hydrogen bonding confines Y157 orientation but exerts a force to disrupt the hydrogen bond network surrounding the cavity. The effect propagates over the entire protein and reduces the stability, as reflected in the protein backbone dynamics observed by an NMR H/D exchange experiment. We describe the particular case in which a hydrogen bond does not necessarily confer enhanced protein stability while the disruption of hydrophobicity must be integrally considered.

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Introduction

Interleukin-1 (IL-1) family comprises a group of cytokines that act as key players in the regulation of inflammatory processes,1-2 with 11 members in the same family including agonists, antagonists and receptor.3 Among these members, IL-1β has been linked to a wild variety of diseases, and its activation depends on interaction with its receptor and the receptor accessory protein.4-7 IL-1βs have been extensively studied in mammals,8-12 and the related structures have been solved and show great sequence identity and structural similarity.13 Recently, the chicken IL-1β structure has been proven to adopt a conserved structural fold in spite of the distantly related sequences.14 The crystal structures of IL-1βs across different species are composed of 12-14 β-strands and 1-2 short α-helices, with the β-strands enclosing the β-barrel to form a β-trefoil scaffold (Fig. 1A and B).15-16 The IL-1β structures consistently contain a central non-polar cavity.11 The existence of the interior cavity is a structural character for IL-1βs, and the consistency suggests importance. Currently, few protein cases containing cavities have been reported. In the identified cavities, the interior can be empty or occupied by solvent, which depends on cavity hydrophobicity, size and solvent accessibility.17-20 The cavities in IL-1βs are tightly packed and entirely hydrophobic.21 In human IL-1β, the non-polar cavity is stabilized by the hydrophobic residues of eight aliphatic and four aromatic residues;22 the cavity in chicken IL-1β is surrounded by I14, F22, L30, L46, L69, M78, L89, F110, F123, I133, V143 and Y157 (Fig. 1C).11 To understand the consequence of hydrophobic residues in regulating protein stability, a number of mutants have been constructed for human and chicken IL-1βs by introducing distinct residues to alter the hydrophobicity of the cavity.11, 23 The corresponding structure and energetic consequence of the mutations was evaluated showing that the residues around the hydrophobic cavity play important roles in regulating protein stability.23 However, there is no clear evidence for a correlation between hydrophobicity and protein stability.23 The specific residue Y157 in chicken IL-1β is interesting for protein stability because a structurally similar residue, Phe, is located at the corresponding position (F146) in human IL-1β. To understand the effect of the hydrophobic cavity, we mutated Tyr to Phe 3



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at position 157 (Y157F) in chicken IL-1β and determined the X-ray structure.11 The Xray diffraction results showed a nearly identical structure, including the cavity, though the root-mean-square derivation (RMSD) value of all backbone Cαs was approximately 0.35 Å compared to the wild type. However, the Y157F mutant surprisingly showed greater protein stability, with a 10-degree higher melting temperature (Tm).11 The introduced mutation was found to have no effect on either the cavity size or side-chain orientation (Fig. 1C).11 The only visible difference is the Tyr side chain hydroxyl group (-OηH) and a possibly existing hydrogen bond between the -OH group and I133 backbone carbonyl oxygen (CO). The mutant Y157F lacks this bonding. However, we failed to demonstrate this in the X-ray structure because the protons are invisible in the coordinates. Although the presence of the hydrogen bond seems to be a main source for the destabilization effect in native chicken IL-1β, further investigation is required to reveal the mechanism. If this notion can be proven, i.e., that the presence of the hydrogen bond appears to weaken proximal protein packing and reduce the overall protein stability, this could be an interesting case of a hydrogen bond less contributing in protein stabilization. In addition, Tyr contains a hydrophilic group at the end of the side chain, and the introduction of a hydroxyl group (-OH) in the hydrophobic cavity will be energetically unfavorable. Therefore, studying this replacement will provide a better understanding of the correlation of hydrophobicity and hydrogen bonding in a hydrophobic cavity. The introduction of a hydrogen bond between fragments has been believed to represent more interactions to consolidate protein structure. The presence of hydrogen bonds has been usually linked to the enhancement of structural stability. However, the identical X-ray structures failed to illustrate the reason why the removal of a hydrogen bond instead enhanced the stability of chicken IL-1β. To resolve this apparent contradiction, we first identified the existence of the hydrogen bond between the Y157 OηH group and I133 CO by NMR. Based on NMR chemical shift comparison and an H/D exchange experiment, we profiled the mechanism destabilizing native chicken IL-1β. The net effect is derived from the summation of the protein interior hydrophobicity and hydrogen bond network. This research therefore provides the idea that to introduce a hydrogen bond does not necessarily correlate with a stability enhancement in 4


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macromolecular folding.

Figure 1. The structures and sequences of IL-1βs. (A) Ribbon representation of the structural comparison: chicken IL-1β wild type (blue, PDB: 2WRY), chicken IL-1β mutant Y157F (magenta, PDB: 3NJ5) and human IL-1β (green, PDB: 9ILB). The residue Y157 of chicken IL-1β is indicated in the structure. (B) The sequences of human and chicken IL-1βs are aligned to compare their secondary structures. (C) The determined chicken IL-1β structures show identical cavities (yellow mesh surface) surrounded by hydrophobic residues: wild type (blue) and mutant Y157F (magenta).

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Materials and Methods

Cloning, expression and purification. The DNA sequences of wild-type chicken IL-1β and the mutant Y157F were amplified by PCR and ligated into the pET-6H vector. The final products contain 10 additional residues (M(H)6AMA) at the N-terminus, including a hexa-His tag for purification. E. coli strain BL21(DE3) (Invitrogen) was used for protein expression. The transformation was performed by the heat-shock method, and the transformed cells were cultured in 1 L M9 minimal medium supplemented with 100 µg/ml ampicillin with vigorous shaking (~ 200 rpm) at 37 °C. When the OD600 reached 0.6, the cells were induced with 0.5 mM IPTG for 20 hours at 18 °C. The cells were harvested by centrifugation (6000 x g, 20 min at 4 °C) and resuspended in binding buffer (25 mM Tris-HCl pH 8.0, 100 mM NaCl and 40 mM imidazole). The cells was disrupted by sonication, and the cell debris was removed by centrifugation (30,000 x g, 20 min at 4°C). The supernatant was applied onto a column packed with 10 ml Ni-charged IMAC resin (GE Healthcare) equilibrated with binding buffer. Nonspecific binding proteins were removed by washing buffer (25 mM Tris-HCl pH 8.0, 100 mM NaCl and 50 mM imidazole), and the bound His-tagged IL-1β proteins were eluted with elution buffer (25 mM Tris-HCl pH 8.0, 100 mM NaCl and 200 mM imidazole). To improve the purity, the IL-1β protein was concentrated, exchanged into FPLC buffer (25 mM Tris-HCl pH 7.4, 100 mM NaCl, 1 mM EDTA and 0.7 ml/L β-mercaptoethanol) and further purified using a Superdex 75 size exclusion column with an AKTA Explorer (GE Healthcare). The purity of the IL-1βs was check by SDS-PAGE, and the protein concentrations were determined by the Bio-Rad protein assay. To prepare uniformly

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proteins for the NMR study, M9 medium was supplemented with

N or 15

15

N,

13

C-labeled

NH4Cl (1 g/L) and

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C-glucose (2 g/L) as the sole nitrogen and carbon sources. The final proteins were

prepared in NMR buffer (10 mM Tris pH 6, 50 mM NaCl, 1 mM EDTA and 1 mM DTT) at 7 mg/ml for wild type and 10 mg/ml for Y157F.

Circular dichroism (CD) measurements. CD spectra were obtained using an Aviv 202 spectropolarimeter (Aviv Biomedical Inc., Lakewood, NJ). Samples of 10-20 µM in 10 mM potassium phosphate buffer (pH 7.4) were subjected to CD measurement at 25 °C. 6


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The range of spectrum was recorded from 195 to 260 nm, with a resolution of 1 nm. The thermal stabilities were monitored by changes in ellipticity at 217 nm, with a gradient temperature from 4 °C to 96 °C, a 1 °C temperature increment and a heating rate of 1 °C/min. Temperature-induced denaturation was used to determine the effects of mutations on protein stability.24 Proteins undergo an unfolding process between the two states, and the enthalpy (∆H) and entropy (∆S) differences were estimated from the temperature dependence of the population these two states using the van’t Hoff relationship: ln K = - ∆H/RT + ∆S/R. The equilibrium constant K is determined by using the relationship K = [F]/[U], where [F] and [U] represent the concentrations of the folded and unfolded states at each given temperature. A linear plot of ln K versus 1/T yields a slope of - ∆H/R and a y-intercept of ∆S/R.

NMR backbone experiments. Backbone assignments for wild-type and mutant chicken IL1β were performed using

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N,

13

C-double labeled IL-1β. 2D 1H-15N HSQC and 3D

experiments of HNCA, HN(CO)CA, HN(CA)CB, HN(COCA)CB and HNCO spectra were acquired using a Bruker AV600 spectrometer at 298K. All 2D and 3D spectra datasets were processed by NMRPipe25 and analyzed using Sparky.26 1H NMR chemical shifts were referenced to the 1H resonance frequency of 2.2-dimethy-2-siapentane-5sulfate (DSS) at 0 ppm. All resonances in the

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N-HSQC spectra of the wild type and

mutant Y157F were identified. The structure difference between the two IL-1βs was extracted from the composited chemical shift difference of NH and N, as calculated by considering the relation ∆δN+H = [(∆δ2NH+∆δ2N/25)]1/2, where ∆δNH and ∆δN are the chemical shift differences for NH and 15N, respectively.27-28 Because the chemical shifts of Cα and Cβ correlate to protein secondary structure29-31, the local secondary structures of IL-1β were predicted by measuring (∆Cα - ∆Cβ) (ppm), where ∆C is the difference between the observed chemical shifts and the values in random coil32. Residual dipolar coupling (RDC) of backbone

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N-1H (DNH) was measured on a 0.5 mM protein sample

aligned in 12 mg/ml pf1 filamentous phage. The DNH of each residue was measured from the difference of 1JNH splitting in the isotropic and oriented environments by analyzing 2D IPAP (inphase/anti-phase) 1H-15N HSQC spectra.33

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NMR H/D exchange. H/D exchange was initiated by rapid buffer exchange from H2O into 100% D2O using a PD MiniTrap G-25 column (GE Healthcare). The D2O-replaced sample was subjected to a series of 1H-15N HSQC spectra that were recorded every 30 min using a Bruker AV600 spectrometer, with a total exchange period of 31 hours. The first experiments were started within 15 min after buffer exchange. Protection factors (PFs) of individual residues were determined by calculating kin/kex, where kex is the exchange rate constant obtained by fitting a single-exponential function to the intensities of amides in the series of HSQC spectra and Kint is the intrinsic exchange rate constant obtained using the program SPHERE, which corrects for temperature and pH effects.34

Molecular dynamics (MD) simulations. Gromacs 3.3 package was used to perform MD simulations of the wild-type IL-1β and the Y157F mutant. The missing hydrogen atoms in the two X-ray coordinates were added using the VMD 1.9 charm 22 protein force field. The starting structures were individually immersed into a rectangular box with equilibrating single-point charge water molecules, and the water molecules in the crystal structures were retained. To achieve electro-neutrality, two water molecules were randomly replaced by chloride ions. The MD simulations began with the steepest descent energy minimization and were subsequently performed using decreasing positional restraints to equilibrate the systems. After equilibration, both systems were simulated for 20 nsec in the isobaric-isothermal ensemble (the NpT ensemble) using periodic boundary conditions. The temperature was maintained at 300 K, and the pressure was controlled using Berendsen's weak-coupling algorithm set at a pressure of one atmosphere. The integration time step was 2 fsec, and the corresponding coordinates were collected every 0.5 psec during the entire simulation period.

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Results

Thermal stabilities of IL-1βs. We used CD spectrometry to monitor thermal stability of wild-type IL-1β and the mutant Y157F. The two proteins showed similar CD curves but with very different thermal-induced denaturation curves (Fig. 2A). We monitored the change in ellipticity at 217 nm, a characteristic wavelength for β-sheets. The wild-type protein has a lower thermal stability than Y157F, with melting temperatures (Tm) of 48 °C and 58 °C for the wild type and mutant, respectively. The Y157F mutation results in a 10-degree increase in the melting temperature, confirming the better conformational stability of Y157F.11 The thermodynamic parameters obtained from a simple two-state fit of the far-UV CD data are shown in Fig. 2A, and we analyzed the thermal denaturation curves by van't Hoff analysis to obtain the enthalpy and entropy changes during the unfolding process (Fig. 2B). We obtained the folding equilibrium constant K for various temperature points and plotted ln K as a function of 1/T. We preserved the region allowed to fit linearly in the final analysis. The linear plots of ln K versus 1/T yield a slope of ∆H/R and a y-intercept of ∆S/R, where ∆H and ∆S represent enthalpy and entropy changes. We calculated free energy difference (∆G) of the unfolding process at any temperature by the determined ∆H and ∆S (Table 1). The percentages of differences in enthalpy and entropy are 19.2 % (33.3 kcal/mol) and 16.7 % (0.09 kcal/mol•K), respectively, where the ∆G between native IL-1β and Y157F is 8.23 kcal/mol at 25 °C. The determined value matches to a previous study of human IL-1β, in which the F146Y mutant showed a 9 kcal/mol difference in free energy.23 In general, ∆H corresponds to the binding energy (dispersion forces, electrostatic interaction, van der Waals potentials and hydrogen bonding), whereas entropy is a thermodynamic quantity interpreted as the degree of disorder or randomness in a system. The Y157F mutation led to equal contributions from enthalpy and entropy. Therefore, we expect to observe differences occurring in the protein structure as well as in the dynamics.

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Figure 2. Thermal stability of recombinant chicken IL-1βs. (A) The thermal denaturation curves of wild type (WT) and Y157F monitored by far-UV CD at 217 nm. (B) The corresponding van’t Hoff plots.

Backbone chemical shift variations. We employed NMR to differentiate structural differences. We completed backbone assignments of NH, 15N, 13Cα, 13Cβ and 13CO of the two proteins and compared the 1H-15N HSQC spectra (Fig. 3A). The spectra revealed similar but non-identical resonance distributions. Because nucleus chemical shifts are sensitive to the local chemical environment, it is promising to monitor the chemical shifts of the protein backbone to reveal structural differences. We first examined the HSQCs and calculated the composited chemical shift difference of ∆δN+H (Fig. 3B). The profile of ∆δN+H shows noticeable shifts in the following regions: I14-F22, H34, L35, T121-E124, G130-L137 and Q148-K158, where C134 on β11 is the most significant variation with a perturbation greater than 1 ppm. In contrast, the mutation site only demonstrated mild variation (Fig. 3B). Second, we examined chemical shift differences in the backbone carbonyl carbons (∆δCO), and the experiments showed significant variations in the regions of I14-C21, Q29-L35, T120-F123, F132-Q138, G144-Q151 and N153-K158, with I133 and Cys134 being the most significant variations (Fig. 3B). We noticed that the directions of the variations are opposite for the two residues, with I133 having an upfield shift and C143 having a downfield shift in Y157F. We depict the corresponding HNCO

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strip plots of I133, C134 and Y157/F157 in Fig. 3C; again, residue 157 lacks the perturbation. Third, we measured chemical shift variations of ∆δCα and ∆δCβ (Fig. 3B). Compared to ∆δN+H and ∆δCO, the variations in ∆δCα and ∆δCβ were relatively small, and the differences were generally less than 0.5 ppm.

Figure 3. Chemical shift differences between chicken IL-1β wild type (WT) and mutant Y157F. (A) 1H-15N HSQC spectra of WT (red) and Y157F (blue). (B) Chemical shift differences between WT and Y157F: (from up to down) ∆δN+H=[(∆δ2N/25+∆δ2NH)]1/2 from HSQC, where ∆δ2N and ∆δ2NH are the chemical-shift differences for N and NH, respectively; ∆δCO, ∆δCα and ∆δCβ from the HNCO, HNCA and HNCACB spectra, representing the chemical-shift differences for 13CO, 13Cα and 13Cβ. The mutation site is indicated in red. (C) The strip plots of the corresponding HNCO spectra of residues I133, C134 and Y157/F157: WT (red), Y157F (blue).

Secondary structure of IL-1βs. The chemical shifts of

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Cα and

13

Cβ are sensitive to

protein secondary structure, and the difference between the observed 13C chemical shifts 11



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and the random coil values can represent protein secondary structural tendency.32 The parameter ∆δCα - ∆δCβ can be used as an index for secondary structure, whereby positive and negative values represent α-helix and β-sheet structures, respectively. We estimated the value of ∆δCα - ∆δCβ by averaging three consecutive residues centered at a particular residue and plotted the values of ∆δCα - ∆δCβ for wild-type IL-1β and Y157F as a function of residue number, as shown in Fig. 4. The secondary structure estimation well corresponds to the X-ray crystal structure; wild-type IL-1β and Y157F showed identical secondary structure profiles. However, we observed small but noticeable variations of ∆(∆δCα - ∆δCβ) at F132-T135, V143 and A155, which are located in or near β11, β13 and β14, respectively (Fig. 4, bottom). The β-sheet tendency is enhanced slightly in the region of β11 and β13 in Y157F. The most significant variation, although less than 1 ppm, was centered at residue C134.

Figure 4. Secondary structure of chicken IL-1βs. Secondary structure estimated by the chemical shift parameter (∆δCα - ∆δCβ): (from top to bottom) wild type (WT), mutant Y157F and the difference, ∆(∆δCα - ∆δCβ). The secondary structures determined by X-ray are indicated above for comparison.

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Residual dipolar coupling (RDC) values. We obtained one bond 1H-15N RDC values (DNH) in the presence of the alignment medium, pf1 filamentous phage. The proteins were partially aligned in solution and led to an incomplete averaging of anisotropic dipolar couplings. The DNH value of each residue from the difference of dipolar coupling in the isotropic and phage-aligned environments was measured to compare the spatial orientation of each 1H-15N bond vector in native IL-1β and Y157F (Fig. 5A). The two proteins have very similar values, indicating their similar tertiary structures. However, although they are generally correlated with each other either in loops or secondary structures, the values are not fully identical (Fig. 5B). We detected significant differences (> 5 Hz) in the residues R52, V67, Q90, I100, F108, T146 and Y/F157, which are not specifically localized in any particular region. Therefore, the Y157F mutation caused subtle tertiary structural changes with dispersive distribution in the IL-1β structure.

Figure 5. The 1H-15N residual dipolar coupling (RDC), DNH, measured in pf1 phage medium. (A) Sequence variation of chicken WT (above) and Y157F (below). (B) Correlations between WT and Y157F: residues in the secondary structure (black circles) and loop (grey circles). 13



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NMR hydrogen-deuterium (H/D) exchange experiment. NMR H/D exchange monitors the exchange process of a protein amide proton replaced by a deuterium atom, and the exchange rate measured by NMR reflects site-specific information of solvent accessibility and hydrogen bond formation.35 This information is also correlated with local and global flexibility/stability of a polypeptide backbone.36-37 H/D exchange was initiated by rapidly replacing the two IL-1βs into D2O, and a series of HSQC spectra were acquired at different intervals of D2O exchange time. After a 31-hour exchange, 38 residues of the wild-type IL-1β were still available for detection in HSQC, whereas 74 residues remained for the Y157F mutant (Fig. 6). These residues were mapped onto the structures, and most residues are distributed in the core region (Fig. 6). We analyzed the H/D exchange data in terms of protection factors (PFs).34 Slow H/D exchange and high PF correspond to a better protection for an amide. The calculated log(PF)s for wild type and Y157F were compared with the secondary structure elements and the corresponding PF differences, ∆log(PF) (Fig. 7A). Many ∆log(PF)s are positive, indicating the better protection of Y157F amides. The protected amides are mainly distributed in the central βsheet region of β-sheet 1-5 and 10-14, surrounding the hydrophobic cavity. Only five residues, E86, F110, Y111, F128 and I133, showed noticeable negative ∆log(PF)s indicating better protections in the wild type. As the regions in α-helix, β6, β7 and β8, are less protected in both structures, we could not detect the amides during the H/D exchange experiments. We indicated the residues with better protections on the structures, as shown in Fig. 7B. The replacement of F157 stabilizes the proximate β11 and β13 as well as the other regions surrounding the cavity.

Molecular dynamics (MD) simulation. To gain further insight into the potential nature of the structure and dynamics of the IL-1βs, molecular dynamics simulations of the two molecules were performed. The two IL-1βs adopted compact conformations and demonstrated nearly the same potential energies (Fig. 8A). However, the wild type showed larger structural fluctuations, as reflected in its higher RMSD values (Fig. 8B). The results correspond to the lower Tm observed in the CD measurements. We measured the representative distances between the Y157/F157 Cζ and I133 backbone carbonyl oxygen. The trajectory profiles of the distances in the two proteins were generally similar, 14


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with comparable values, with both exhibiting certain variations, varying from 5 Å to 3 Å (Fig. 8C). This finding indicates structural fluctuations occurring in the local area but without substantial conformational change and also indicates the integrity of the hydrophobic cavity during the simulation. Based on the distance and bond orientation, we identified hydrogen bond formation between the Y157 OηH and I133 backbone carbonyl oxygen in the wide type. At the beginning of simulation, the hydroxyl hydrogen was placed with a random orientation that could freely rotate and search for an acceptor. The hydrogen bond formed with the engaged I133 when the OηH group rotated to the proper orientation. The hydrogen bond was formed and broken alternately during the entire simulation and was detected in 60 to 70 % of the simulation time (Fig. 8D, black stripes). During the period with hydrogen bond formation, the average distance between the -OηH and O is 2.3 ± 0.3 Å. The averaged Cζ-OηH – O angel is 123 o ± 5 o, and the OηH – O=C angel is 104 o ± 6 o.

Figure 6. H/D exchange experiment of chicken IL-1βs. The overlay of 1H,15N-HSQC spectra of wild type (red) and Y157F (blue) after incubation in D2O solution for 31 hours at 25 °C. Y157F shows more residues that are resistance to H/D exchange, and the residues are labeled in the HSQC. The protected amides are mapped onto the corresponding structures.

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Figure 7. Protection factors (PFs) of chicken IL-1βs. (A) The protection factors of backbone amides are derived from 1H-15N HSQC H/D exchange experiments at 25 °C. Profiles show the log(PF) of each amide group of WT (red circles) and Y157F (blue bars). The PF difference, ∆log(PF), and the secondary structure are compared in the top panels. (B) The residues with higher PFs in WT are mapped on the ribbon representations (left), and the higher PFs in Y157F are mapped onto its structure (right). The residues are colored according to the magnitude of ∆log(PF): red, > 3; orange, 2-3; green, 1-2; blue, 0.5-1. Uncolored residues show no significant ∆log(PF) differences.

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Figure 8. Molecular dynamics simulation of chicken IL-1βs. The integration time step was 2 fsec, and a total 20 nsec simulation time was performed by sampling the trajectory every 0.5 psec. (A) Potential energy, (B) RMSD of Cα, (C) the distance between Y157/F157 Cζ to I133 CO and (D) hydrogen bond between residues Y157 OηH and I133 CO in native IL-1β.

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Table 1. Thermal dynamics parameters of IL-1βs

∆H

∆S

∆G25 °Ca

Tm

(kcal/mol)

(kcal/mol•K)

(kcal/mol)

(°C)

Wild type

173.6

0.54

12.33

48.1

Y157F

206.9

0.63

20.56

58.1

a

∆G25 °C is the free energy difference of unfolding of the proteins during heat-induced

thermal denaturation.

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Discussion

We studied chicken IL-1β and a specific mutant, Y157F, to understand the consequence of hydrogen bonding in the interior of a hydrophobic cavity. The two proteins, with the same X-ray structures, demonstrated different properties in protein stability, with the mutant Y157F being much more stable.11 However, it remained unclear how the mutation caused the stability enhancement. Here, we profile the mechanism by analyzing the NMR chemical shift differences (∆δN+H and ∆δCO), the exchange rates of amides (PFs) and the backbone

15

N-1H RDCs. The wild type actually contains one

additional hydrogen bond between Y157 and I133. This visible difference influences the protein stability as well as the protein structure. The 1H-15N HSQC spectra of the wild type and Y157F displayed residues with chemical shift differences (Fig. 3A), reflecting structural changes. The most perturbed residues are C134 in HSQC (∆δN+H > 1 ppm) and I133 in HNCO (∆δCO > 2 ppm) (Fig. 3B). These two residues, together with other perturbed residues, constitute the site of structural perturbation. They are spatially distributed near the element β11 (W131-C134). The backbone of Y/F157, however, showed very mild perturbation. We suspected a hydrogen bond between the Y157 hydroxyl group and I133 backbone carbonyl oxygen, with the mutant Y157F lacking the connection. We inspected chemical shifts by NMR and found that the chemical shift of the backbone carbonyl (δCO) is sensitive to hydrogen bonding, with δCO moving linearly downfield with a strengthened hydrogen bond.38-40 Such a downfield shift predominantly arises from the large downfield shift of δ22 and is less related to δ11 and δ33, in which δ11, δ22 and δ33 are the components of chemical shift tensor.38-39 Thus, the chemical shift difference, ∆δCO, reveals the variable strength of the hydrogen bond. In Fig. 3C, ∆δCO shows a very significant upfield shift in I133 after Y157 is replaced (Fig. 3C). The direction of ∆δCO corresponds to the abolishment of hydrogen bonding. Furthermore, we also observed a downfield shift of C134 CO in Y157F (Fig. 3C), which represents a strengthened hydrogen bond with its hydrogen bond donor, the G144 backbone NH. We depict the relative locations of these residues in Fig. 9, and C134 and G144 are located at the paired anti-parallel β sheet, β11 and β13, respectively. In the mutant, without the interaction of the Y157 OηH, the structural element β11 moves 19



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close to the paired β-sheet, β13, and away from the cavity.

Figure 9. A model demonstrating the interaction network centered around the residues of Y157 (left) and F157 (right). The corresponding PFs of the individual residues are labeled. The hydrogen bonds between the residues are indicated by dashed lines.

We confirmed this conclusion by MD simulation. Y157 is stably located inside the hydrophobic cavity, where the aromatic ring packs on the cavity surface with a low degree of movement (Fig. 8C). The distance between the Y157 side chain hydroxyl oxygen and I133 backbone carbonyl oxygen is close enough to allow hydrogen bonding. MD simulation supports the presence of the hydrogen bond, but the bonding appears to be semi-stable. The hydrogen bond modulates the interaction in the local structure, and the absence of side chain hydroxyl group releases the bonding. The detection of backbone amide H/D exchange provides more direct site-specific information correlated with the local backbone flexibility. We have identified a significant number of residues that exhibited higher degrees of PFs in Y157F (Fig. 7). The higher PFs suggested amide groups with better protection. We noticed that the PFs of C134 (β11) and G144 (β13) had an order of magnitude larger than the comparable 20


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residues in the wild type (Fig. 7A and 9). This result corresponds to the observation for ∆δCO, in which the hydrogen bonds between C134 and G144 are strengthened in Y157F, and also corresponds to the enhancement of the β-sheet tendency in the local secondary structures of β11 and β13 (Fig. 4). Without the association between Y157 and I133, the anti-parallel β sheet of β11 and β13 actually becomes more compact, and the stabilization propagates to the neighboring structure along the cavity surface. Therefore, we observed that the stabilized residues distribute to the secondary structural elements next to the hydrophobic cavity (Fig. 7B). Interestingly, there were few residues better protected in native IL-1β. Residue I133 is particularly important because it is directly involved in the association of Y157. The I133 NH correlates to the hydrogen bond connection with the F123 backbone CO. Y157 and F123 are located at the same side of the cavity, and the association of the Y157 OηH and I133 CO actually exerts a force that assists the hydrogen bonding between I133 NH and F123 CO and creates a better interaction between the two residues (Fig. 9). In the chicken IL-1β model, it might be presumed that the better stability of the wild type is due to the additional hydrogen bond. However, this assumption did not match the real situation. To properly address the question, we consider the hydrophobic scales. The free energy difference of distributing Tyr and Phe residues from water to a non-polar phase is approximately 1-3 kcal•mol-1 depending on the water accessibility of the non-polar phase.41-42 The result defines the Tyr side-chain hydrophobicity as driving the equilibrium to be unfavorable for insertion. We expect that the introduction of the hydroxyl group will disrupt the hydrophobicity. Because the hydrophobicity is critical for maintaining the cavity fold, the hydroxyl group of Y157 brings an energy disturbance. To overcome this, the formation of the hydrogen bond to I133 CO intrinsically prevents the polar -OH group from disrupting the integrity of the cavity hydrophobicity. The presence of the hydrogen bond sequesters the hydrophilic groups, therefore reducing the energy penalty. The hydrogen bond strength of OH – O is approximately 1-5 kcal•mol-1, depending on the bond length and angle.43-44 If one considers the hydrophobic effect with unfavorable energy (∆G > 0), the role of the hydrogen bond (∆G < 0) compensates for the loss of partitioning of the Tyr residue into the hydrophobic cavity. However, the hydrogen bond formation pulls the two β-sheets of β11 and β13 apart and disrupts the 21



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hydrogen bond network. The new introduced hydrogen bond does not appear to be constructive, and the effect propagates throughout the entire molecule. The overall protein stability is reduced afterward. The size of the non-polar cavity of IL-1β is big enough to accommodate 1-2 water molecules.19, discussed.9,

21

The extent to that water is present in the non-polar cavity has been

19, 21, 45

NMR study has reported the existence of disordered water in the

cavity and the dynamic water cannot be visualized in X-ray structure.45 However, the more recent X-ray crystallographic and computer simulation studies reported that water molecule couldn’t stably stay within the cavity due to the small cavity volume and the high hydrophobicity.21,

46

The current study actually supports the later idea that the

occupancy of the cavity by water is very low. If the cavity contains water, the residue Y157 OηH enables to select water as a hydrogen bond acceptor and the bonding with I133 would not be necessarily required. In the case, the water molecule could stabilize Y157 in a hydrophobic environment and meanwhile, protein stability could be maintained. This is not consistent with our observation. Thus, the non-polar cavity in IL1β should be solvent free. In addition, the protein contains dynamics with both open and closed statuses. The dynamics allows solvent to penetrate into the protein interior transiently. If the hydrogen bond is destructive and semi-stable, the native structure with Y157 might have better penetration for solvent to diffuse into the protein interior and disrupt the local structure, which subsequently causes a faster H/D exchange with the labile protons. Alternatively, the elimination of the hydrogen bond in Y157F eases the cavity and slows down the “breathing”. It explains the slow H/D exchange rate occurred in Y157F mutant. Protein internal hydrogen bonds contribute to protein folding. In addition, protein interior hydrophobicity is a determining factor in driving protein folding and structure stabilization. Despite the generality of the concepts, attempts to determine the energetic picture of the presence of hydrogen bond formation in a protein interior environment have sometimes given conflicting ideas. The introduction of a hydrogen bond between fragments has been believed to represent more interactions to consolidate protein structure; thus, the presence of hydrogen bonds is usually linked to the enhancement of structural stability. However, hydrogen bond formation requires a hydrogen bond donor 22


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and acceptor, with both groups having great polarities, and free energy is required to compensate for the introduction of a hydrogen bond into a hydrophobic environment. In addition, not all hydrogen bonds are constructive in fitting into the hydrogen bond network to enhance protein stability. Here, we detail the mechanism of mutational effects in chicken IL-1β. We describe a well-defined case for which the result can more precisely define the role of hydrogen bonds. Although the benefit of protein folding could be partially derived from hydrogen bond formation, the energy loss of the partitioning of hydrophilic groups into the non-polar environment is significant. The hydrogen bond could be destructive and disrupts protein stability and structure. Based on this aspect, the presence of hydrogen bonds in the protein interior is described by an alterative insight: the overall effect of hydrogen bonds is to keep protein folded correctly, not to enhance protein stability and the formation of hydrogen bonds is to compensate for the energy loss of hydrophobicity disruption. Considering that the hydrophobic effect is still the major source driving protein folding, the introduction of a hydrogen bond does not necessarily confer protein stability enhancement in all cases.

23



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Author Information Corresponding author: [email protected]; [email protected]

Acknowledgements

The authors are grateful to the Taiwan National Science Council (NSC) for financial support, to the National Synchrotron Radiation Research Center for protein crystallography and CD measurement, to the NMR facility at National Tsing Hua University and the Core Facility for Protein Structural Analysis at Academia Sinica for NMR measurements.

Abbreviations IL-1β, interleukin-1β; CD, circular dichroism; HSQC, heteronuclear single-quantum correlation spectroscopy; NMR, nuclear magnetic resonance; RDC, residual dipolar coupling; PF, protection factor

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Case Study of Hydrogen Bonding in a Hydrophobic Cavity - American

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