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The role of side chain conformational flexibility in surface recognition by Tenebrio molitor antifreeze protein

Canadian Institutes of Health Research (CIHR) Group in Protein Structure and Function, Department of Biochemistry, and Protein Engineering Network of Centres of Excellence, University of Alberta, Edmonton, Alberta T6G 2H7, Canada

Reprint requests to: Brian D. Sykes, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada; e-mail: brian.sykes{at}ualberta.ca; fax: (780) 492-0886.

(RECEIVED April 7, 2003; ACCEPTED April 9, 2003)

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.0369503.

	Abstract

TOP Abstract Introduction Results Discussion Materials and methods References

 
Two-dimensional nuclear magnetic resonance spectroscopy was used to investigate the flexibility of the threonine side chains in the ß-helical Tenebrio molitor antifreeze protein (TmAFP) at low temperatures. From measurement of the ³J_ß ¹H-¹H scalar coupling constants, the ₁ angles and preferred rotamer populations can be calculated. It was determined that the threonines on the ice-binding face of the protein adopt a preferred rotameric conformation at near freezing temperatures, whereas the threonines not on the ice-binding face sample many rotameric states. This suggests that TmAFP maintains a preformed ice-binding conformation in solution, wherein the rigid array of threonines that form the AFP-ice interface matches the ice crystal lattice. A key factor in binding to the ice surface and inhibition of ice crystal growth appears to be the close surface-to-surface complementarity between the AFP and crystalline ice, and the lack of an entropic penalty associated with freezing out motions in a flexible ligand.

Keywords: Antifreeze protein; ß-helix; nuclear magnetic resonance; side chain dynamics

Abbreviations: AFP, antifreeze protein • DQF-COSY, double quantum filtered correlated spectroscopy • NMR, nuclear magnetic resonance • NOE, nuclear Overhauser enhancement • NOESY, nuclear Overhauser effect spectroscopy • 3Jß, 3-bond scalar coupling constant between spins H and Hß

	Introduction

TOP Abstract Introduction Results Discussion Materials and methods References

 
Antifreeze proteins (AFPs) present in various organisms, including fish (Fletcher et al. 2001), insects (Duman 2001), and plants (Worrall et al. 1998; Sidebottom et al. 2000), help them to survive the freezing conditions of the environments in which they live. The yellow mealworm beetle Tenebrio molitor, for example, produces AFP (abbreviated TmAFP) in its hemolymph while overwintering in the larval stage. AFPs bind to the ice surface, inhibiting ice crystal growth and depressing the freezing point of the solution below the melting point. The separation of the nonequilibrium freezing temperature and the melting temperature in a solution containing ice crystals is referred to as thermal hysteresis (for review, see Jia and Davies 2002). At millimolar concentrations, the TmAFP can account for 5.5°C of thermal hysteresis (Graham et al. 1997). This is much more active (10–100 times) than the well-studied fish AFPs (Davies and Sykes 1997).

Binding to the ice surface is believed to proceed via an adsorption-inhibition mechanism, although the details of this interaction at the molecular level are not understood (Raymond and DeVries 1977). AFP binding to ice is specific and occurs on defined planes. This is supported by observations that AFP solutions are able to shape the ice crystals to produce particular ice crystal morphologies (Houston Jr. et al. 1998). Crystal growth is slowed or stopped completely on the plane to which the AFP binds. For example, fish AFPs shape the ice crystal into a hexagonal bipyramid by binding to the pyramidal plane, whereas insect AFPs can bind to both prism and basal planes, shaping the ice crystal into a hexagonal plate (Graether et al. 2000). Ice etching studies can reveal the specific ice surface bound by an AFP and also support this hypothesis (Knight et al. 1991).

Early attempts to define the nature of the AFP–ice interaction focused on hydrogen bonding of polar side chains to the ice lattice, either through the inclusion of hydrogen bonding side chains into the ice lattice (lattice occupancy; Wen and Laursen 1992) or by a hydrogen bond match with ice surface oxygen atoms (lattice matching; Sicheri and Yang 1995). The models were based on the crystal structure of the -helical Type I AFP from winter flounder, the best characterized and simplest AFP to study. It is composed of 37 amino acids, with a tandemly repeated 11-amino acid unit with consensus sequence TX₂N/DX₇, where X is generally alanine (Davies and Hew 1990). The 16.5 Å spacing of the i, i+11 threonine residues matches the 16.7 Å distance of the water molecules on the pyramidal plane. However, further experimentation has led to diminished importance for the hydrogen bonding interaction, because mutation of threonine to serine, preserving the hydroxyl group and therefore the hydrogen bonding ability of the side chain, caused a severe loss in activity, whereas mutation to valine, preserving the methyl group and the shape of the side chain, resulted in a relatively minor loss of activity (Chao et al. 1997; Haymet et al. 1998, 1999; Zhang and Laursen 1998). Additionally, a redefinition of the ice-binding site of this AFP has occurred on the basis of mutation studies in which the alanine 17 to leucine mutant abolished antifreeze activity (Baardsnes et al. 1999). On the basis of these studies and a recent examination of Type III AFP ice-binding (Baardsnes and Davies 2002), a universal ice-binding mechanism relying on hydrophobic and van der Waals interactions is emerging.

The mechanism of ice-binding has also been complicated by questions concerning the relative rigidity or flexibility of the side chains. Upon refinement of the Type I crystal structure, the ice-binding threonine residues were observed to all adopt a rigid side chain conformation with a ₁ of -60° (Sicheri and Yang 1995). Those authors suggested that the rigidity and common orientation of these side chains are critical for the ice-binding mechanism. However, solution NMR studies at low temperatures indicated that the threonine residues of Type I AFP were in fact flexible, and can sample many possible rotameric states prior to ice binding (Gronwald et al. 1996).

The highly active TmAFP is an ideal candidate for this type of mechanistic study. TmAFP is a small (8.4-kD) highly disulfide-bonded, right-handed parallel ß-helix consisting of seven tandemly repeated 12 amino acid loops. Its crystal structure revealed an array of threonine residues on the ß-sheet side of the protein that all adopted the same ₁ = -60° rotameric conformation, and the spacing of the hydroxyl groups is a near-perfect match to the prism plane of ice and approximates the spacing on the basal plane (Liou et al. 2000b). In addition, the crystal structure also contained bound external water molecules that, along with the threonine hydroxyls, mimic a section of the ice lattice; this was the first time that this has been observed in an AFP structure. Furthermore, this threonine array has been defined as the ice-binding face by extensive mutagenesis (Marshall et al. 2002). The threonines not on the ice-binding face serve as internal controls. We previously solved the NMR solution structure of TmAFP and performed an analysis of the ¹⁵N backbone relaxation parameters which revealed it to be a well folded and rigid protein with restricted backbone internal mobility throughout, at both 30°C and 5°C (Daley et al. 2002). In the present study, we investigated the orientations of the threonine side chains in solution to examine the role of flexibility of these side chains in this binding interface, using high-resolution two-dimensional DQF-COSY experiments to measure the ³J_ß coupling constants. These coupling constants, in combination with NOE data, were used to determine the conformational states of the threonine side chains. These observations are important to the consideration of surface residues, for understanding both antifreeze protein activity and other systems involved in molecular recognition.

	Results

TOP Abstract Introduction Results Discussion Materials and methods References

 
The amino acid sequence and structure of TmAFP are shown in Figure 1, A and B, respectively. The threonine residues arrayed on the ß-sheet side of the protein, which were identified by mutagenesis as the ice-binding residues (Marshall et al. 2002), are highlighted in blue. Those threonine residues that are not part of the ice-binding face are colored red. Threonine 9, which is also not involved in ice binding, is colored green, because coupling constant data could not be obtained for this residue.

View larger version (60K): [in this window] [in a new window]   Figure 1. Sequence and structure of the T. molitor AFP isoform used in this study. (A) Amino acid sequence. The 17 threonine residues are colored to emphasize their positions in the protein. The threonines in blue are those that form the ice-binding face; the threonines in red and in green are not part of the ice-binding face. Coupling constant data could not be obtained for T9 (in green). (B) Structure of TmAFP (PDB 1EZG [PDB] ) with threonine side chains displayed in ball-and-stick representation and colored as for the sequence in (A). Figure generated using Molscript (Kraulis 1991) and Raster3D (Merritt and Bacon 1997).

View larger version (44K): [in this window] [in a new window]   Figure 2. 1H NMR spectra at 300 MHz of TmAFP in 100% D2O. Samples were cooled from 30°C to -20°C, as outlined in Materials and Methods.

ß

View larger version (25K): [in this window] [in a new window]   Figure 3. 2D DQF-COSY 1H NMR spectrum at 800 MHz of TmAFP at 30°C in 100% D2O. Part of the H-Hß region containing the threonine cross-peaks is displayed. The boxed threonines are those on the ice-binding face; the circled ones are not.

ß

ß

ß

ß

View larger version (20K): [in this window] [in a new window]   Figure 4. Expansions of the 2D DQF-COSY 1H NMR spectra at 800 MHz of TmAFP at (A) 30°C and (B) 15°C. The region containing the H-Hß cross-peak patterns of T40 (bottom) and T62 (top) are displayed to show the fine splitting of these cross-peaks. To distinguish between positive and negative peaks, the positive peaks are displayed as contoured peaks and the negative peaks are displayed as open circles.

View larger version (23K): [in this window] [in a new window]   Figure 5. Plot of 3Jß of each threonine residue as a function of temperature. The two observed populations correspond to those threonines on the ice-binding surface, in blue, and the threonines not on the ice-binding surface, in red.

View larger version (21K): [in this window] [in a new window]   Figure 6. Correlation of the 3Jß coupling constants and the corresponding NOE intensities with the 1 side chain torsion angles for the three most populated threonine rotamer conformations. The threonine side chain is shown in the Newman projection with the C in front and the Cß in the back.

ß

(1)

Solving this equation shows that the side chain of T52 populates the ₁ = -60° conformation 63% of the time at 30°C. At 5°C, this increases to 73% population of the ₁ = -60° rotamer. These calculations were performed for all of the observed coupling constants at all four temperatures, and these results are displayed in Table 1. The NOE analysis, as described in the Materials and Methods section, corroborates this observation of restricted rotation for the threonines on the ice-binding face.

View this table: [in this window] [in a new window]   Table 1. Percent population of the 1 = -60° rotamer conformation

	Discussion

TOP Abstract Introduction Results Discussion Materials and methods References

 
Although the ability of AFPs to bind ice and inhibit its growth is well known, it is not well understood. Study of the molecular mechanism by which these proteins adsorb to ice are hindered by two main factors: first, the wide structural variation of AFPs for which structures have been solved, and second, the unique characteristics of the AFP–ice interaction which make it difficult to examine directly. The structural diversity of AFPs and the inability to deduce any sequence similarity between different AFP types means that putative ice-binding faces have been proposed based on mutation studies. The only features these varied sites appear to share is the relative flatness of the interaction surface of the protein, and in the case of Type I and the insect AFPs, the evenly spaced ranks of threonine that match the ice lattice. As well, a significant proportion of the surface area of the protein is involved in the binding interface, and the ice-binding sites generally appear to contain more hydrophobic and less polar amino acid side chains than the rest of the protein. Mutation studies of Type I and Type III fish AFPs and the ß-helical insect AFPs have been very revealing, however. Mutations that introduce large side chains on the ice-binding surface sterically block the AFP–ice interaction, but are well tolerated elsewhere on the protein (Marshall et al. 2002). Mutations that decrease the size of the side chain are also deleterious; however, mutations that are isosteric, that is, those that do not change the shape or size of the side chain, are generally neutral (Baardsnes and Davies 2002). This suggests that loss of shape complementarity and with it, the hydrophobic and van der Waals contacts, is an important factor contributing to the ability of these proteins to bind to ice. Mechanistically, for the adsorption-inhibition model of crystal growth to explain the observed properties of AFPs, the proteins must bind irreversibly. Therefore the antifreeze must fit almost perfectly to the ice in order to prevent water molecules from entering the interface (Knight and Wierzbicki 2001). This can explain why any mutation that disrupts the "snugness" of the binding interface, whether larger or smaller, will have negative effects on ice-binding and thermal hysteresis.

In order to elucidate the molecular mechanism of ice binding, it is necessary to characterize the conformations of the critical side chains. The NMR analyses of TmAFP indicate that the ice-binding threonine residues have a clear preference for the ₁ = -60° rotamer conformation at all temperatures studied and that this preference becomes more pronounced at the lower temperatures, where the AFP is closer to physiologically relevant conditions. The threonine residues that are not on the ice-binding surface do not display this preferred conformation. This indicates that the TmAFP adopts a unique preformed ice-binding structure in solution prior to recognition of the ice surface. The rigidity of the side chains in the binding site reduces the entropic barrier to binding, as the side chains do not have to reposition themselves prior to ice binding. This experimental observation that the key binding side chains of TmAFP position themselves in the rotamer conformation they adopt in the interface supports the hypothesis proposed based on molecular dynamics simulations of proteins both alone and in complex that this allows biological recognition to proceed on a feasible time scale and yields an intermolecular affinity that reduces the entropic penalty of binding (Kimura et al. 2001). The flexibility and free rotation of the threonine residues not on the ice-binding face may help prevent the structuring of water molecules on the other side of the protein and prevent its engulfment.

The ß-helical structure of TmAFP provides the ideal scaffold to form this rigid ice-binding conformation. The repetitiveness of the primary amino acid sequence is reflected in the three-dimensional loop structure allowing a rigid array of threonine residues to perfectly match the ice crystal lattice. Notably, the ₁ = -60° rotamer conformation is a commonly preferred rotamer in ß-sheet secondary structure, due to favorable hydrogen bonding effects with the backbone (Dunbrack Jr. and Karplus 1994).

The inability to directly observe the molecular interaction of AFP and ice requires the use of various methods to indirectly probe the surface interaction. Computational docking studies and molecular dynamics simulations are commonly used ways to examine this interface; however, the potential flexibility of protein surface side chains has posed a challenge for correct modeling. In the present study, we show that the threonine side chains in the ice-binding face are not particularly flexible, and furthermore, we determined the rotamer conformation most preferred by these side chains. This knowledge will contribute to the ability to perform more accurate molecular dynamics simulations and docking studies. Finally, the indirect probe we employed, specifically solution-state NMR experiments to study protein dynamics, has proven very useful in characterizing the rigid nature of the AFP–ice interface and the behavior of the side chains in particular. Clearly the intimate surface-to-surface complementarity between TmAFP and crystalline ice is a key factor in binding.

	Materials and methods

TOP Abstract Introduction Results Discussion Materials and methods References

 
NMR spectroscopy
An unlabeled sample of TmAFP isoform 2–14 previously prepared was used for all experiments described. This sample was prepared by dissolving the lyophilized protein in 100% D₂O, with 0.1 mM DSS added for internal referencing. The final protein concentration was approximately 2 mM and the pH was adjusted to 5.6 with microliter aliquots of 100 mM NaOD or DCl as required. For the freezing experiment, a medium-walled tube (524-PP-8, Wilmad) was used to prevent tube breakage.

The freezing experiment was collected on a Varian Unity 300 MHz spectrometer using an indirect detection probe. The ¹H 1D NMR spectra were collected over a temperature range of 30°C to -20°C in 1°C decrements. After each temperature change, the sample was allowed to equilibrate for 30 min. For each temperature point, 16,000 complex data points were acquired with 256 transients using a spectral width of 4000 Hz. The 90° pulse width was calibrated to 7.5 µsec.

The 2D DQF-COSY (Rance et al. 1983) and NOESY (Jeener et al. 1979) spectra were acquired at 30°C on a Varian INOVA 800 MHz spectrometer equipped with a 5-mm triple resonance probe and x, y, and z-axis pulsed field gradients. For the DQF-COSY experiment, a spectral width of 7000 Hz was used in both dimensions. The acquired data consisted of 8192 F₂ x 1024 F₁ complex data points. The data were zero-filled to give a spectrum after transformation that contained 16,384 x 4096 data points. The 2D NOESY spectrum was acquired with a spectral width of 10,000 Hz in both dimensions and a mixing time of 100 msec. The data consisted of 8192 F₂ x 512 F₁ complex data points and were zero-filled to give a spectrum that contained 16,384 x 4096 data points after transformation.

All 2D DQF-COSY spectra collected for measurement of ³J_ß coupling constants were also measured on the Varian INOVA 800 MHz spectrometer. Spectra were acquired at 5°C, 15°C, 25°C, and 30°C with a spectral width of 2000 Hz in F₂ and 2400 Hz in F₁. Temperature calibration of the spectrometer indicates the reported temperatures are within 0.5°C from 5°C to 20°C and within 0.1°C from 25°C to 30°C. The data consisted of 8192 F₂ x 1024 F₁ complex data points. The spectra were processed with 16,384 x 4096 data points using an unshifted sine bell window function in combination with line broadening in both dimensions. All NMR data processing, including the integration of the NOEs, was performed using the Varian VNMR 6.1B processing software on a Sun Blade 100 workstation.

Determination of ³J_ß coupling constants
The ³J_ß coupling constants of the threonine residues were determined at four temperatures from the peak separation of the DQF-COSY H-Hß cross-peaks. Even with the high digital resolution of the spectra, it is difficult to obtain accurate values for coupling constants directly from the observed splitting, as the separation between antiphase components becomes increasingly different from the actual splitting with increasing linewidths (Wüthrich 1986). Slower molecular tumbling at decreased temperatures causes this effect to become more pronounced at the lower temperatures measured. For this reason we used the method of Kim and Prestegard (1989), which allows accurate calculation of scalar couplings by the measurement of peak-to-peak separations of the extrema in absorptive and dispersive plots of rows through the cross-peaks of interest. These peak-to-peak separations were measured along the higher resolution F₂ axis in the opposite trace orientation from that displayed in Figure 4A to reduce partial overlap in the multiplet.

Determination of ₁ side chain torsion angles
The ₁ side chain torsion angles are obtained by the analysis of the pattern of ³J_ß coupling constants and the relative intensities of the intraresidue NOEs involving the H and two Hß protons (Karplus 1959, 1963; Clore and Gronenborn 1989). Figure 6 displays the necessary information for determining these angles for the simpler case of threonine residues, which have only one Hß proton. From the observed coupling constant, it is possible to calculate the occupancy of a specific rotamer conformation on the basis of the expected coupling constants for the different staggered rotamers.

(2)

where P_i is the fractional occupancy of a conformation corresponding to the coupling constant J_i. For a side chain which is not fixed into one conformation, an average coupling constant will be obtained if the rotation is fast on the NMR time scale. In the case of a threonine side chain with no rotamer preference, a ³J_ß of 6.6 Hz would be obtained. In addition to the information about preferred rotameric states obtained from coupling constant analyses, the ratio of NOE intensities of the -, ß-, and -protons provides further evidence. The NOE ratio is defined by:

(3)

The H-H NOE is divided by 3 to account for the three threonine methyl protons as compared to the one ß-proton. As outlined in Figure 6, an NOE ratio significantly less than 1 corresponds to a ₁ of -60°, whereas an NOE ratio significantly greater than 1 corresponds to a ₁ of 180°. An NOE ratio of approximately 1 corresponds to either a ₁ of +60° or to a freely rotating side chain.

	Acknowledgments

 
We thank Dr. Peter L. Davies for the unlabeled Tm 2–14 protein we used in this study, Dr. Steffen Graether for assistance with the freezing experiments, and Gerry McQuaid for maintenance of the NMR spectrometers. We also thank the Canadian National High Field NMR Centre (NANUC) for their assistance and use of the facilities. The operation of NANUC is funded by the Canadian Institutes of Health Research (CIHR), the Natural Science and Engineering Research Council of Canada (NSERC), and the University of Alberta. This work was supported through grants from the CIHR and the Protein Engineering Network of Centres of Excellence (PENCE) to B.D.S. M.E.D. is supported by a CIHR Doctoral Research Award.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

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