Examining Macromolecular Orientation and Interaction at Crystal

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Examining Macromolecular Orientation and Interaction at Crystal Growth Steps Using an Energy-Based Dual Miller Plane Mapping Algorithm Yaohua Dai and John Spencer Evans* Laboratory for Chemical Physics, New York University, 345 East 24th Street, New York, New York, 10010 Received February 8, 2001. In Final Form: April 5, 2001

Introduction Conceptually, interfacial growth sites can be visualized as surfaces where stereospecific molecular recognition takes place.1-4 In nature, some aspects of biomineral and ice crystal growth such as dimensionality, aspect ratio, and morphology can be manipulated by proteins and polypeptides which stereospecifically interact with exposed crystal interfaces.5-11 It has been demonstrated5,6,12 or proposed13,14 that, in some cases, crystal growth regulation polypeptides bind at the boundaries of growing crystals (i.e., “growth steps”) where two interfacial surfaces intersect. As an example, it is known that protein binding at calcite growth steps leads to step edge roughening with subsequent cessation of crystal growth,5,6 similar to that observed for calcite screw dislocations in the presence of Mg(II),15 and can lead to the formation of a different crystal morphology at that site (i.e., polymorph switching).5 Thus, the interaction of macromolecules at crystal growth step sites represents an important phenomenon whose elucidation will not only impact our understanding of biomineral and ice crystal growth but also improve our current capabilities in template-controlled composite material design.16-20 * To whom correspondence should be addressed. E-mail: [email protected]. (1) Weissbuch, I.; Addadi, L.; Lahav, M.; Leiserowitz, L. Science 1991, 253, 637-645. (2) Weissbuch, I.; Frolow, F.; Addadi, L.; Lahav, M.; Leiserowitz, L. J. Am. Chem. Soc. 1990, 112, 7718-7724. (3) Shimon, L. J. W.; Vaida, M.; Addadi, L.; Lahav, M.; Leiserowitz, L. J. Am. Chem. Soc. 1990, 112, 6215-6220. (4) Vaida, M.; Weissbuch, I.; Lahav, M.; Leiserowitz, L. Isr. J. Chem. 1992, 32, 15-21. (5) Thompson, J. B.; Paloczi, G. T.; Kindt, J. H.; Michenfelder, M.; Smith, B. L.; Stucky, G.; Morse, D. E.; Hansma, P. K. Biophys. J. 2000, 79, 3307-3312. (6) Smith, B. L.; Paloczi, G. T.; Hansma, P. K.; Levine, R. P. J. Cryst. Growth 2000, 211, 116-121. (7) Addadi, L.; Weiner, S. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 4110-4114. (8) Jia, Z.; DeLuca, C. I.; Chao, H.; Davies, P. L. Nature 1996, 384, 285-288. (9) Worral, D.; Elias, L.; Ashford, D.; Smallwood, M.; Sidebottom, C.; Lillford, P.; Telford, J.; Holt, C.; Bowles, D. Science 2000, 282, 115117. (10) Albeck, S.; Aizenberg, J.; Addadi, L.; Weiner, S. J. Am. Chem. Soc. 1993, 115, 11691-11697. (11) Aizenberg, J.; Albeck, S.; Weiner, S.; Addadi, L. J. Cryst. Growth 1994, 142, 156-164. (12) Wierzbicki, A.; Sikes, C. S.; Madura, J. D.; Drake, B. Calcif. Tissue Int. 1994, 54, 133-141. (13) Yang, D. S. C.; Hon, W.-C.; Bubanko, S.; Xue, Y.; Seetharaman, J.; Hew, C. L.; Sicheri, F. Biophys. J. 1998, 74, 2142-2151. (14) DeLuca, C. I.; Davies, P. L.; Ye, Q.; Jia, Z. J. Mol. Biol. 1998, 275, 515-525. (15) Davis, K. J.; Dove, P. M.; De Yoreo, J. J. Science 2000, 290, 1134-1137. (16) Cha, J. N.; Stucky, G. D.; Morse, D. E.; Deming, T. J. Nature 2000, 403, 289-292. (17) Firouzi, A.; Schaefer, D. J.; Tolbert, S. H.; Stucky, G. D.; Chmelka, B. F. J. Am. Chem. Soc. 1997, 119, 9466-9477.

Figure 1. The POINTER-2 algorithm. In A, the general schematic for the phase space search at a dual Miller plane interface is presented. In B, a close-up of the polypeptide is given, indicating the rotation angles δ, δ′, χ, χ′ that are utilized during the search to reposition the polypeptide surfaces S1, S2 with respect to each interface at a value of σ, φ. (C) Perfect dual Miller plane growth step (no extra adsorbed water layer) showing only oxygen atoms. (D) As in (C) but featuring a single array of water molecules aligned along the x-axis. This extra ice layer is constructed by appropriate unit cell extension. System coordinates, surface definitions, and a full description of the algorithm are presented in Supporting Information.

Given the resolution limits in experimental surface techniques such as atomic force microscopy and scanning electron microscopy, the ability to visualize and probe macromolecule-growth step interactions at the atomic level is problematic. To address this issue, we have developed a novel algorithm, POINTER-2 (Figure 1), which represents an evolution from our earlier POINTER polypeptide-interfacial mapping algorithm.21 POINTER-2 maps the energy interactions and orientation(s) between the surface(s) of a protein (represented in CR, Cβ virtual bond format) and dual Miller interfaces (explicitly represented) which form a step on a crystal surface (Figure 1) (for a full description of the algorithm and initial conditions utilized in this study, please refer to Supporting Information). For benchmarking purposes, we map the interactions of an ice antifreeze polypeptide, eel pout AFP Type III, on a hexagonal ice (Ih) {100}/{001} growth step. This protein was chosen as a benchmark for the following reasons: (1) the three-dimensional structure has been established by NMR22,23 and X-ray crystallography8 studies; (2) mutagenesis studies have established critical residues for ice binding at the {100} interface;14 (3) it is believed that AFP Type III binds at or near the intersection (18) Ngankam, P. A.; Lavalle, Ph.; Voegel, J. C.; Szyk, L.; Decher, G.; Schaaf, P.; Cuisinier, F. J. G. J. Am. Chem. Soc. 2000, 122, 89989005. (19) Tannev, P. T.; Pinnavaia, T. J. Science 1996, 271, 1267-1269. (20) Xu, G.; Yao, N.; Askay, I. A.; Groves, J. T. J. Am. Chem. Soc. 1998, 120, 11977-11985. (21) Dai, Y.; Evans, J. S. J. Chem. Phys. 2000, 112, 5144-5157; errata 113, 2509. (22) Sonnichsen, F. D.; Sykes, B. D.; Chao, H.; Davies, P. L. Science 1993, 259, 1154-1157. (23) Sonnichsen, F. D.; DeLuca, C. I.; Davies, P. L.; Sykes, B. D. Structure 1996, 4, 1325-1337.

10.1021/la010216u CCC: $20.00 © 2001 American Chemical Society Published on Web 05/19/2001

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Figure 2. Representative section of contour plots (total system energy versus σ, φ angular orientation) for AFP Type III on the Ih {001}/{100} growth step. Each contour pixel represents the total energy (kcal/mol, z-axis) for the global x, y, z position for each value of σ, φ (given in degrees). Because of the large number of simulation points, only the relevant sector displaying the global minima is presented in this figure. (A) S1/{001}, S2/{100}, perfect; (B) S2/{001}, S1/{100}, perfect; (C) S1/{001}, S2/{100}, extra hexagonal ice layer; (D) S2/{001}, S1/{100}, extra hexagonal ice layer.

of the {001} and {100} planes of hexagonal ice;8,13,14,23,24 (4) for comparison, there exist published visual models of AFP Type III at the Ih growth step.8,14 Identifying Binding Site Complementarity For AFP Type III, we identified two contact surfaces on the polypeptide, surface 1 (S1), which encompasses the putative {100} ice binding residues Q9, N14, T15, and T18,8,13,14,21 and surface 2 (S2), which includes the putative {001} ice binding residues N14, Q44, and N46.8,13,14 To probe polypeptide binding site-interface complementarity, two parallel simulations were performed: “normal” (i.e., a phase space search involves the positioning of S1 and S2 above the I1 {100} and I2 {001} planes of the Ih growth step, respectively)8,14,21 and “mismatch” or juxtaposed (i.e., S1 positioned over I2 {001} and S2 over I1 {100}). If specific AFP Type III protein surfaces are complementary to specific Ih interfaces, then the two simulations should differ in terms of total energies and side chainsurface interactions. As shown in Table 1 and Figure 2, we find that the global minimum orientation obtained for the S1/{100}, S2/{001} simulation is lower in energy (≈200 kcal/mol) compared to the minimum obtained for the corresponding S2/{100}, S1/{001} simulation. Further(24) Chen, G.; Jia, Z. Biophys. J. 1999, 77, 1602-1608.

Table 1. Lowest Energy Orientations for AFP Type III on Ih {100}/{001} Growth Step simulation normal S1/{100}; S2/{001}

σ φ total energy water layera (deg) (deg) (kcal/mol)

no yes normal S1/{100}b no mismatch S2/{100}; S1/{001} no yes mismatch S1/{001}b no

2 0 N/A 126 126 N/A

238 240 N/A 276 277 N/A

-911.3 -986.5 -734.1 -717.8 -732.4 -692.4

a Simulations were performed with and without a single layer of water molecules aligned along the conjunction (x-axis) of the two Miller planes. b For comparison, we present results obtained from earlier POINTER simulations of AFP Type III binding on the individual {100} and {001} Ih planes (ref 21).

more, the global minimum σ, φ orientations obtained for both simulations are significantly different (Table 1, Figure 2). For both systems, we observe periodic minima along the σ and φ axes. The S1/{100}, S2/{001} simulation exhibits a unique minimum centered at σ, φ ) 2°, 238°. The S2/{100}, S1/{001} simulation contour plot exhibits multiple minima wells that run orthogonal to those obtained from the normal simulation, with the global minimum centered at the protein: {100}/{001} orientation of σ, φ ) 126°, 276° (Figure 2). For comparison, we provide the global minima obtained from earlier POINTER

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Figure 3. Sequence-specific first derivative (dEtotal/dr) force plot for the lowest energy σ, φ, x, y, z orientation, AFP Type III{001}/{100} Ih growth step. N ) normal; M ) mismatched. Units of force are given in kcal mol/Å. Note that AFP Type III residues that are not shown in this plot have values of dE/dr ) 0.

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simulations of AFP Type III on the individual {100} and {001} Ih planes (Table 1). Note the following: (1) the global minimum obtained for the POINTER-2 S1/{100}, S2/{001} simulation is ≈177 kcal/mol lower in energy compared to the original POINTER-determined global minimum orientation for the AFP Type III S2 region-{100} Ih plane simulation; (2) the global minimum obtained for the POINTER-2 S2/{100}, S1/{001} simulation is ≈25 kcal/ mol lower in energy compared to the POINTER-determined global minimum orientation for AFP Type III S2 region on the {001} Ih plane.21 From this, we conclude the following: (1) the POINTER-2-generated in-plane orientations are sensitive to protein ice binding motif-Miller plane pairings and (2) the combinatorial interactions offered by the Ih {100}/{001} growth step are more energetically favorable compared to the interactions offered by the individual {100} or {001} planes. Next, we calculated the first derivative of the total energy (dE/dr) as a function of each residue-specific Cβ atom at the lowest σ, φ orientation for each simulation run (Figure 3).21 These force plots permit comparisons of AFP Type III residue-specific Cβ nonbonding interactions (attractive, repulsive) with the Ih interfacial slab atoms.21 For the normal S1/{100}, S2/{001} scenario, attractive forces exist between the {100} Miller plane and S1-surfacespecific residues Q9, N14, T15, and T18 (Figure 3). These four residues have been identified as important constituents of the putative {100} ice binding motif of AFP Type III.8,13,14,21,22-24 We also observe attractive interactions between the {001} Miller plane and S2-specific residues R39, S42, Q44, N46, R47, D58, and K61 (Figure 3). If we examine the global minimum obtained from the mismatch S2/{100}, S1/{001} simulation, we now observe that the S1-, S2-specific residues exhibit attractive/repulsive interactions with their respective mismatched interfaces (Figure 3). However, note the reduction in mismatch force magnitudes (approximately 5-20%) for the majority of ice-interaction residues. Clearly, the juxtapositioning of AFP Type III ice binding motifs with Ih interfaces leads to less favorable CR, Cβ-interfacial interactions (Figures 2 and 3, Table 1). Hence, despite the high degree of surface atom symmetry on both the {100} and {001} Ih interfaces, each AFP Type III ice binding motif exhibits optimal complementarity to only one of the two Ih Miller planes. This confirms the S1/{100}, S2/{001} arrangement as the optimal AFP Type III-Ih growth step configuration as proposed elsewhere.8,14 Hydrophobic and “Cross-Interfacial” Interactions In the course of our benchmarking studies, we note two interesting phenomena. First, repulsive interactions are observed between AFP Type III hydrophobic or sterically “bulky” residues and the Ih growth step. Specifically, for the S1/{100}, S2/{001} scenario, L10, I11, P12, I13, L17, L19, and V20, which flank the hydrogen-bonding residues of the {100} ice binding motif, exhibit repulsive force interactions with the {100} interface (Figure 3). These findings are in agreement with our earlier POINTER simulations of AFP Type III on the {100} Ih interface.21 Similarly, L40, V41, M43, V45, L51, V60, and Y63 exhibit repulsive interactions with the {001} basal plane. Collectively, these observations are in qualitative agreement with experimental studies that implicate AFP Type III hydrophobic side chains in ice binding site stabilization and/or ice surface interaction.8-11,13,14,22-24 Second, minor attractive “cross-interface” interactions were observed for the {001} interface with S1-specific residues, L10, I11, P12, I13, N14, T15, L17, T18, L19, and V20 and the {100}

Figure 4. Ribbon/CPK representation of lowest energy polypeptide σ, φ orientation for AFP Type III on the Ih growth step, S1/{001}, S2/{100} configuration, featuring an extra hexagonal ice layer. The polypeptide backbone was regenerated from virtual chain representation and assigned a ribbon structure using the program MOLMOL. Each CR-Cβ vector is represented by a cylinder which branches off of the ribbon structure at each CR position. Ih water oxygen atoms are represented in CPK format. For clarity, the ice slab has been trimmed in all dimensions, and not all AFP Type III residues are labeled. For visual comparison, please see ref 8, Figure 2, and ref 14, Figure 2.

interface with S2-specifc residues R39, L40, V41, M43, Q44, V45, N46, R47, L51, V60, and Y63 (Figure 3). Corresponding cross-interfacial interactions were also observed in the mismatch S2/{100}, S1/{001} simulation. We attribute these minor interactions to the small size of the AFP Type III protein (33-35 Å diameter)8,22,23 which results in the placement of these residues within 9 Å of either interface (Figure 4). Because we are employing a 40 Å nonbonding cutoff (see Supporting Information), these weak pairwise cross-interactions are detected by our POINTER force field.21 It remains to be seen whether these cross-interfacial interactions actually exist in situ. Presence of a New Hexagonal Ice Layer We consider a hypothetical situation where one layer of water molecules has adsorbed at the junction of the two Miller planes prior to protein interaction (Figure 1D). This simulation mimics the “capturing” of guest molecules by advancing growth steps15 and patterns itself upon the Ih growth step representation utilized in earlier AFP Type III docking studies.8,14 However, in contrast with previous modeling studies,8,14 we performed parallel POINTER-2 simulations using a “perfect” growth step (Figure 1C) and a growth step featuring a single linear array of hexagonal ice positioned along the x-axis of the {100}/{001} growth step conjunction (Figure 1D). As shown in Table 1 and Figure 2, for both the normal S1/{100}, S2/{001} and mismatched S2/{100}, S1/{001} simulations, the presence of a “captured” water layer does not significantly alter the orientational result of the simulation. However, it can be observed from Table 1 that the presence of the captured water molecule array leads to a lowering of the total system energy for both the normal and mismatch simulation scenarios. These results can be attributed to the following: (1) an increase in attractive interactions between Q9, N14, T15, T18, and the {100} plane and between Q44, R47, D58, K61, and the {001} plane; (2) an increase in repulsive interactions for L10, P12, and L17 with the {100}

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plane and for V41, M43, and V45 with the {001} plane; (3) a decrease in repulsive interactions for L11, I13, and L19 with the {100} plane (Figure 3). Clearly, the most significant perturbations in attractive-repulsive interactions are noted for residues L10, N14, and Q44 (Figure 3), which lie in close proximity to the extra water array (Figure 4). These simulation results are consistent with published modeling studies which suggest that N14 and Q44 may interact with advancing water molecule(s) in the conjunction or hinge region of the growth step.8,14 From these results, we conclude that protein-growth step interactions are, in part, influenced by the degree of “host-guest” apposition1-4 at the growth step surface. Comparison with Published AFP Type III-Growth Step Modeling Studies Earlier AFP Type III-{100}/{001} Ih growth step modeling studies utilized a new hexagonal ice layer at the growth step conjunction and a S1/{100}, S2/{001} configuration.8,14 These studies presented their findings in a visual molecular modeling format. To correlate our findings with these earlier reports, we present a molecular “snapshot” of the POINTER-2 lowest energy σ, φ orientation for S1/{100}, S2/{001} featuring the extra hexagonal ice layer (Figure 4). As shown in this figure, (1) the planar putative ice binding motif (consisting of residues Q9, N14, T15, and T18) is positioned in close proximity to the {100} interface; (2) residues R39, S42, Q44, N46, R47, D58, and K61 are positioned near the {001} interface; (3) N14 and Q44 are positioned near the extra hexagonal ice layer. These findings are in agreement with previous docking studies.8,14 In summary, we find that the POINTER-2 algorithm can serve as a novel predictive tool for the selection of “initial guess” polypeptide-growth step starting geometries for other simulation methods. It is clear that several important insights regarding protein-growth step interactions can be gleaned from the POINTER-2 results. First,

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we find that two surface-accessible motifs of the AFP Type III protein possess several key residues, both polar and hydrophobic, that exhibit optimal interactions when the protein is positioned in a unique orientation at the growth step interface (Table 1, Figures 2-4). Second, compared to a single interface,21 a dual interface offers an attractive scenario to a protein: the presence of multi-interfacialprotein interactions (Table 1, Figures 3 and 4). These multi-interfacial interactions, in turn, would allow a protein to persist at a crystal growth site, blocking further apposition at the growth step6,7 or enabling polymorph switching.5 Third, there is evidence that specific AFP Type III residues (L10, N14, Q44) interact with newly ordered water molecules at the conjunction of the two Miller planes. In the absence of this newly captured water array, the energetics of protein-growth step interactions is significantly perturbed (Table 1, Figure 3). From this, we infer that the complementarity and binding energetics between the polypeptide and the growth step surface are dependent not only on the protein orientation but also upon the degree of host-guest apposition1-4 at the growth step surface. Obviously, further studies will be required to refute or verify these concepts. Acknowledgment. Support for this study has been made possible by grants from the National Science Foundation (DMR 99-01356 and MCB 98-16703) and the Army Research Office (Young Investigator Award, DAA1999-1-0225). This paper represents contribution number 14 from the Laboratory for Chemical Physics, New York University. Supporting Information Available: A full description of the POINTER-2 algorithm, initial simulation conditions, and phase space search protocol. This material is available free of charge via the Internet at http://pubs.acs.org. LA010216U