Proc. Natl. Acad. Sci. USA Vol. 83, pp. 226-230, January 1986 Biochemistry Antigenic determinants in proteins coincide with surface regions accessible to large probes (antibody domains) (flexibility/contact surface/contour maps/crystal contacts/Debyc-Waller temperature factor) JIAI NOVOTN.', MARK HANDSCHUMACHER*, EDGAR HABER*, ROBERT E. BRUCCOLERI*, WILLIAM B. CARLSON*, DAVID W. FANNING*, JOHN A. SMITHt, AND GEORGE D. ROSE: *Molecular and Cellular Research Laboratory and tDepartments of Molecular Biology and Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114; and *Department of Biological Chemistry, Hershey Medical Center, Pennsylvania State University, Hershey, PA 17033 Communicated by Frederic M. Richards, August 30, 1985

ABSTRACT We evaluated surface areas on proteins that workers in the field. In principle, the antigenicity data is would be accessible to contacts with large (1-nm radius) never complete, being dependent on immunogenic potential spherical probes. Such spheres are comparable in size to of selected amino acid replacements in a set of homologous antibody domains that contain antigen-combining sites. We proteins (3) or on availability of apparently randomly ob- found that all the reported antigenic sites correspond to tained monoclonal antibodies. Moreover, identification of segments particularly accessible to a large sphere. The epitopes can only be based on indirect experimental proce- antigenic sites were also evident as the most prominently dures. In this situation, theoretical models of antigenicity are exposed regions (hills and ridges) in contour maps of the of particular value, but even here difficulties arise. For solvent-accessible (small-probe) surface. In myoglobin and example, the proposed correlation between antigenicity and cytochrome c, virtually all of the van der Waals surface is segmental flexibility is based on data that are influenced accessible to the large probe and therefore potentially significantly by conditions such as crystal packing (9) and antigenic; in myohemerythrin, distinct large-probe-inacces- static crystal disorder (10) and whose exact molecular mean- sible, and nonantigenic, surface regions are apparent. The ing is currently being debated (11-13). Hopp and Woods (14) correlation between large-sphere-accessibility and antigenicity suggested that antigenic epitopes can be located as those in myoglobin, lysozyme, and cytochrome c appears to be better segments ofprimary structure that are markedly hydrophilic. than that reported to exist between antigenicity and segmental However, a recent test (15) of their predictive algorithm flexibility; that is, sdrface regions that are rigid often constitute reports a success rate of only 56%. antigenic epitopes, whereas some of the flexible parts of the As protein antigenicity is clearly a surface property, we molecules do not appear antigenic. We propose that the decided to examine the static accessibility (16-19) of selected primary reason why certain polypeptide-chain segments are molecules to probes of various radii. In what follows, we antigenic is their exceptional surface exposure, making them discuss the possible relationship of the large-probe accessi- readily available for contacts with antigen-combing sites. bility and the location of antigenic epitopes. Exposure of these segments frequently results in high mobility and, in consequence, to the reported correlation between Crystallographic Data and Calculations antigenicity and segmental flexibility. Myoglobin, lysozyme, and cytochrome c coordinates and One ofthe issues currently discussed by immunologists is the B-factor values were obtained from the Brookhaven Data nature of protein antigenicity (1-4). Early experimental Bank (20), those for myohemerythrin were a gift from S. results (5, 6) were interpreted as suggesting that discrete Sheriff and W. Hendrickson (Columbia University, New antigenic epitopes exist, implying that certain regions of York). The van der Waals surface of the proteins was protein surface are more antigenic than others. A more recent constructed and its areas of contact to spheres of varying interpretation (3) of a larger experimental data base seems to radii were computed by use of the Lee and Richards (16) indicate, however, that a number of mutually overlapping algorithm, as implemented in the program CHARMM version epitopes exist on protein surfaces and that perhaps the whole 16 (21). Alternatively, the contact surfaces were computed surface can be antigenic. It has also been noted (1, 2, 4) that using a procedure (to be published elsewhere) that involves antigenic epitopes composed of contiguous polypeptide- calculations of the contact area on the van der Waals surface chain segments usually have higher-than-average backbone of an atom to a series of probes ordered smallest to largest. fac- A water-sized sphere with radius r = 0.14 nm and four other flexibility, as evidenced by Debye-Waller temperature spheres with radii 0.25 nm, 0.5 nm, 0.75 nm, and 1.0 nm were tors (7). The temperature factor, or the B value, is given by used in the computations. The last sphere, with diameter 2 B = -ir2(r2), where (r2)1/2 is the root-mean-square atomic nm, is comparable in size to an antibody-domain dimer (Fv 3 fragment) (22). displacement from the crystal equilibrium position. Thus, it The contact areas (17) used in this study should not be has been hypothesized that flexibility is an essential compo- confused with solvent accessibilities (16), defined as enve- nent of antigenicity (1, 2), perhaps because structural adjust- lopes ofprotein structures obtained by the path of the center ment is a prerequisite for antibody complementarity. [The of the probe as it rolls over the protein surface. The contact hypothesis has recently been reconsidered by one of these surface (17) is a set of disconnected patches representing authors (8), however.] those portions of the atomic surfaces that are in contact with These alternative, and often conflicting, interpretations of the surface of the probe. As the probe size increases to antigenicity bespeak conceptual difficulties confronted by infinity, the contact surface of a protein converges to a small value. On the other hand, protein accessibility (16) values The publication costs of this article were defrayed in part by page charge converge to infinity with increasing probe radii. payment. This article must therefore be hereby marked "advertisement" Residue sums were obtained from the contact-surface in accordance with 18 U.S.C. §1734 solely to indicate this fact. values of individual atoms. The sums were smoothed by the 226 Downloaded by guest on September 26, 2021 Biochemistry: Novotn' et al. Proc. Natl. Acad. Sci. USA 83 (1986) 227 seven-point moving-window procedure (23) and plotted (Fig. 1C), but evolutionary constancy of the primary struc- against residue numbers to obtain the smoothed contact ture of this molecule makes the delineation of antigenic sites profiles. particularly difficult. The two contact profiles displayed in Close intermolecular contacts in crystals were examined Fig. 1C, those oftuna and bonito cytochrome c, respectively, by use of the program CHARMM (21). Coordinates of suggest that contact-surface profiles of homologous proteins symmetry-related molecules were generated from the start- are likely to be very similar. ing sets of atomic coordinates by applying the rotational and Antigenic Epitopes and Average Backbone B-Factor Values. translational operations ofthe particular space groups P21 for The above-average maxima of B factors of backbone atoms hen-egg lysozyme (24) and sperm whale myoglobin (25), N, Ca, C, and 0 are plotted in Fig. 1 A-C by light lines. P21212 for cytochrome c (26), and P212121 for human Comparison of the two types of data-namely, the contact lysozyme (27). All possible pairs of symmetry-related mole- profiles and the backbone B factors-indicates that segments cules were then generated in the explicit hydrogen-atom of high flexibility constitute a proper subset of regions representation (21) and a list of atom pairs falling within the exceptionally accessible to the large-size probe. That is, of cutoff distance of 4 A was prepared. the 46 major peaks of the B factors, all but 1 (lysozyme Contour maps of myoglobin and lysozyme surfaces were residue 107) coincide with some of the 54 distinct or partially computed by a method described in greater detail elsewhere overlapping maxima of large-probe contact areas. It seems (41). Briefly, the molecular surfaces (17) have been deter- significant that there are several antigenic epitopes associat- mined using a 0.14-nm probe (19) (small sphere), and then ed with highly accessible regions of no exceptional flexibility contoured§ in increments of 1 A (i.e., 0.1 nm). The list of (residues 56-62 and 140 in myoglobin; 1,41,84 or 19,21 in points, whose coordinates define the molecular surface, is lysozyme; and 1-4 and 60,62 in cytochrome c), whereas there obtained using an algorithm by Connolly (19). The starting are no epitopes that are flexible but inaccessible to the large elevation is taken at the surface of a triaxial ellipsoid with probe. axes scaled to be 0.5 times the principal axes of the protein Next, the possibility was considered that the above- (28); points below the starting elevation are eliminated. The mentioned "rigid" epitopes are artificially constrained by maps are displayed as Mollweide projections, a representa- crystal contacts and might be more flexible in solution. An tion that preserves the relative areas of all features. exhaustive search for intermolecular crystal contacts showed no close appositions in the human (27) and hen-egg-white (24) Results and Discussion lysozyme crystals that would involve the epitope 1-41-84, although the side-chain terminal nitrogen of Arg-41 (human Correlation of Contact-Area Peaks with Locations of Anti- structure) is only 3.2 A from the CY atom ofPro-103. This site, genic Epitopes. Fig. 1 A-C shows contact-surface profiles then, can be said to be inherently "inflexible." The computed with the largest sphere size (r = 1 nm) for myoglobin epitopes 56-62 and 140, as well as the cytochrome myoglobin, lysozyme, and cytochrome c and smoothed over c epitopes 1-4 and 60,62 and the lysozyme site 19,21 were 7-residue segments. The smoothing procedure was chosen in involved in intermolecular interactions. The significance of order to account for the fact that the antibody binding site can this finding remains unclear, however, particularly because accommodate 6-8 amino acid residues (36). Also shown are our search also revealed multiple close intermolecular con- the 111 amino acid positions that were implicated in antigenic tacts involving residues from segments of above-average epitopes in all three proteins (3, 4). An overwhelming backbone B-factor values, as, for example, between Gly-153 majority of them coincide with prominent peaks shown on (backbone B value = 43.3) and Gln-91 (B = 10.0) in contact-surface profiles. Only 8 ofthe antigenic residues (140 myoglobin, or between the side chain of Lys-27 (B = 21.5) and 113-116 in myoglobin; 33, 34, and 93 in lysozyme) are and the backbone oxygen ofGly-77 (B = 15.0) in cytochrome located on minor local maxima, and 5 of the antigenic c. Clearly, crystal contacts are not incompatible with high positions (residues 12 and 144 in myoglobin; 94 in cyto- average backbone B factors (10) and, conversely, an exist- chrome c; and 5 and 25 in lysozyme) occur in regions not ence of close crystal contacts should not be taken to imply covered by any of the contact-profile peaks. However, these that the segment in question becomes more flexible in 5 positions are parts of larger epitopes that also contain other solution. Instead, independent evidence is needed to prove residues that are prominently exposed. Residue 12 in this point. The a-carbon B values computed from normal- myoglobin belongs to the epitope that also includes residues mode dynamics simulations on hen-egg-white lysozyme by 4 and 79, both located in contact-area peaks, whereas residue Levitt et al. (37) do not indicate an above-average backbone 144 forms an epitope with residue 83, which is located at a flexibility for the two epitopes 1-41-84 and 19-21, nor do maximum. Similarly, residues 89 (highly exposed) and 92 in they reproduce the B-value maximum at position 107 (not cytochrome c are parts of the same epitope; residue 5 in coinciding with any of the contact-profile peaks). Likewise, lysozyme belongs to the same epitope as the peak-associated dynamical simulations of Northrup et al. (38) do not suggest residues 7, 13, 14, and 125; and residue 25 of lysozyme is an above-average flexibility of cytochrome c residues 60-62, included in the epitope defined by the neighboring residues although NMR measurements (39) show the region around 20-23, all prominently exposed. The antigenic residues Ile-57 to be flexible. located on minor local maxima are likewise parts of larger Is the Whole Protein Surface Antigenic? Comparison of epitopes that also include major contact-profile peaks, with contact-area profiles computed with small- and large-probe the sole exception of residue 140 in myoglobin. Consequent- radii (e.g., 0.25 nm and 1 nm) revealed an interesting fact: the ly, the reported antigenic regions can be said to correspond, magnitudes of the contact areas obtained with the two probe with a single exception, to surface regions particularly well radii differed, yet the positions of the maxima of the small- accessible to the large probe. probe and large-probe profiles were virtually identical in all The reported antigenic epitopes cover virtually all the the three cases (cf. Fig. 1D for myohemerythrin). Thus, prominent maxima of the contact-area profiles in lysozyme residues that are accessible to contacts with large, protein- and most of those in myoglobin. Only a small part of the sized spheres correspond to surface regions with an excep- accessible surface of cytochrome c is known to be antigenic tional exposure to small, water-sized spheres. Fig. 1D shows that amino acid sequences of the two "cold" myohemeryth- WFortran Subroutine SRFACE, General Surface Projection and rin peptides, whose antibodies fail to crossreact with native Contour Mapping (National Center for Atmospheric Research, myohemerythrin (2), are located in regions that are only Boulder, CO), July 1980. marginally accessible to a small-size probe and are inacces- Downloaded by guest on September 26, 2021 228 Biochemistry: Novotny et al. Proc. Natl. Acad. Sci. USA 83 (1986) 40 sible to contacts with large spheres. It is easy to understand, then, why antibodies elicited by these peptides are not able to react with the parent molecule (2). The presence of a small local maximum around position 28, in Fig. 1D, might be .l30 interpreted to mean that this region has a very weak antigenic potential. Indeed, minute amounts of antibodies elicited against peptide 22-35 were found to precipitate with myohemerythrin, but the peptide itself failed to inhibit the 21 antibody-myohemerythrin reaction (2). It thus seems that relative antigenicity of different parts of the molecule corre- lates with their relative exposures to the large probe and that the large-probe contact profiles can be interpreted as prob- 11.3 abilities, for a surface region, to be antigenic. Fig. 1D predicts that antibodies elicited by a peptide corresponding to myohemerythrin residues 53-60 will react 54 poorly, or not at all, with the myohemerythrin molecule. Such antibodies were not described by Tainer et al. (2), who "-4 studied the antigenicity of myohemerythrin. Although anti- x bodies against the peptide 57-66 have been tested (2), the end 42 E ofthis peptide overlaps the strong predicted epitope centered on residue 65 and therefore would be expected to elicit o crossreacting antibodies. Note that the average B value ofthe 30 # backbone segment 53-60 is comparable to those of some 8 ¢ other, fully antigenic peptides (e.g., residues 7-16). x Contour Maps ofProtein Surfaces. Figs. 2 and 3 contour the 17.2 entire surface of myoglobin and of lysozyme (41), showing that antigenic epitopes generally coincide with major convex cir.. protrusions on the surface. Contouring is a common method of graphically representing topographical features of sur- c)cds faces. Each contour line in Figs. 2A and 3A is a trace of co 0 constant elevation. When the interval between contour lines uc) is constant, the patterns formed by the lines quantitate 28 surface features such as peaks, valleys, and steepness of slope. A particular advantage of a contour map is that global features, such as the highest portions of the surface, can be seen at a glance. In Figs. 2B and 3B, only contours 8 A or 21.5 more above the reference ellipsoid are shown, corresponding to the globally exposed regions of the protein surface. Superimposed on the maps are outlines ofreported antigenic 15.3 regions for these two proteins. Sequence numbers ofresidues in highly exposed regions are also shown. In Fig. 2B, virtually every globally exposed portion of the myoglobin molecular surface is implicated in protein antigenicity. A similar situ- ation exists for lysozyme (Fig. 3B); there are two major globally exposed regions. The peripheral region below antigenic site III is virtually covered by reported antigenic residues, but the central region is less well covered. It should

addition to two discontinuous epitopes [4, 12, and 79; 83, 144, and 145 (29)]. The average backbone B factor of deoxygenated myoglobin (25) is 10.2 (B range from 5.0 to 46.0). (B) Hen-egg-white lysozyme. One contiguous epitope [64-80 (30)] and seven discontinuous ones [5, 7, 13, 14, and 125 (6); 62, 87, 89, 93, 96, and 97 (6); 33, 34, 113, 114, and 116 (6); 45-48 and 68 (31); 19 and 21 (32); 102 and 103 (32); Sequence number 1, 41, and 84 (32)] were identified experimentally. The above-average backbone B factor of human lysozyme (27) is plotted. The average FIG. 1. Profile of contact areas computed with a spherical probe backbone B-factor value is 17.2 (B range from 9.5 to 55.3). (C) Bonito ofradius 1.0 nm. Contact areas (1 A2 equals 0.01 nm2) represent those and tuna cytochromes c. The experimentally defined antigenic parts of the van der Waals surface of a protein that come in direct epitopes are 1-4 (33), 42-46 (34, 35), 60 and 62 (34), 89 and 92 (34), contact with the sphere, when the sphere is rolled over the surface and the carboxyl terminus (3). The average backbone B-factor values (16). The larger the spherical probe used, the smaller the contact of the tuna structure (26) is 15.3 (B range from 10.0 to 23.4). (D) area. Residue-contact areas shown were obtained as sums of com- Profiles of contact areas of myohemerythrin computed with a puted atomic-contact areas. The values were smoothed by a 7-point spherical probe ofradius 0.25 nm. As discussed in the text, the profile moving-window algorithm (23) and plotted against the residue of contact areas to a large-sized probe (such as 1.0 nm in radius) can numbers. The above-average backbone B factors, as computed from be obtained from the one displayed in the figure by considering only the atomic B factors contained in the Brookhaven Protein Data Bank those parts of the profile that show an exceptional exposure (heavy (20), are drawn in light lines. Residues identified as parts ofantigenic lines). Bars drawn in dashed lines denote positions ofpeptides known epitopes are indicated by symbols, with symbols belonging to the as "cold" (2); antibodies elicited against these peptides fail to react same epitope drawn on the same ordinate. (A) Sperm whale with the intact myohemerythrin molecule. It can be seen that myoglobin. Six contiguous epitopes were identified experimentally polypeptide-chain segments corresponding to "cold" regions are [residues 15-22, 56-62, 94-99, 113-119, 146-151 (5) and 140 (29)], in poorly accessible to large-size probes. Downloaded by guest on September 26, 2021 Biochemistry: Novotn' et al. Proc. Natl. Acad. Sci. USA 83 (1986) 229

FIG. 2. (A) Mollweide projection of the molecular surface of FIG. 3 (A) Mollweide projection of the molecular surface of myoglobin, with contour interval 1 A (0.1 nm). The heme pocket is lysozyme with contour interval 1 A (0.1 nm). The active-site cleft can seen as a hole below antigenic site III. (B) Contour map ofmyoglobin be seen as a hole adjacent to antigenic site IV on the left side of the limited to contours .8 A. Superimposed in heavy lines are outlines of map. (B) Contour map of lysozyme limited to contours .8 A. the molecular surface of antigenic regions. Site I, residues 15-22; site Superimposed in heavy lines are outlines of the molecular surface of II, residues 56-62; site III, residues 94-99; site IV, residues 113-119; antigenic regions; broken line delimits the "loop" antigenic deter- site V, residues 146-151; site VI, residues 4, 12, and 79; site VII, minant (30). Site I, residues 5, 7, 13, 14, and 125; site II, residues 62, residues 83, 144, and 145; site VIII, residue 140. Also superimposed are 87, 89, 93,96, and 97; site III, residues 33, 34, 113, 114, and 116; site sequence numbers of residues that contribute points to the molecular IV, residues 45-48 and 68; site V, residues 19 and 21; site VI, surface. residues 102 and 103; site VII, residues 1, 41, and 84; site VIII, residues 64-80. Also superimposed are sequence numbers of resi- be noted, however, that additional sites determined by dues that contribute points to the molecular surface. The exposed monoclonal antibodies to lysozyme (32) implicate residues 1, surface on the periphery of the map is almost entirely covered by 19, and 21, together with other residues already included antigenic regions. The exposed surface in the center ofthe map is less here, as antigenic. These three residues cover additional well covered, but an additional antigenic determinant, described by portions of the exposed surface. Amit et al. (40), includes residues 117, 119, 121, and 125. Amit et al. (40) recently reported the crystallographic structure of a protein-antigen-antibody complex: lysozyme molecule is more compact, having more secondary structure with a monoclonal anti-lysozyme Fab fragment. The anti- (76%) than lysozyme (56%) and acquiring an additional body binds to two regions of the sequence that are highly stabilization from its heme ligand; however, myoglobin, exposed (contact-area peaks around residues 10-20 and although apparently more rigid than lysozyme, is an equally 115-125). good antigen. The only highly flexible segments of myoglo- bin, yet to be implicated by experimental studies as major Concluding Remarks independent epitopes, encompass residues 1-4 and 151-153. We have found that the availability of particular regions of The same is true of the flexible region in lysozyme, residues protein surface to contacts with large, spherical probes 105-110. correlates well with the antigenicity of myoglobin, lysozyme, This static-accessibility model of antigenicity incorporates cytochrome c, and myohemerythrin. Although most of the both the contiguous and discontiguous epitopes. Profiles in highly accessible regions of the backbone are also more Fig. 1 A-C suggest that virtually all the molecular surface is flexible than the average, there are distinct antigenic epitopes potentially antigenic, in accordance with the conjecture of that seem to be rigid, particularly the residues 1, 41, and 84 Benjamin et al. (3), whereas Fig. 1D seems to indicate that in lysozyme. protein molecules exist for which a significant part of the A comparison of individual B-factor values found in surface is not accessible to contacts with antibody and myoglobin and lysozyme crystals strengthens this interpre- therefore is not antigenic (2). Because large-probe accessi- bility and above-average B factors in any given structure are tation. Most ofthe above-average B-value maxima associated strongly correlated, it is difficult to assign antigenicity with with antigenic regions in myoglobin (average backbone B complete confidence to one factor or the other. It would value of 10.2) are significantly smaller than those found in appear that somewhat greater consistency can be found for lysozyme and rarely exceed the value B = 17 ((r) = 0.6 A). the accessibility correlation, but more experimental work is Conversely, the average backbone B-factor values in hen- needed to clarify this issue. egg-white and human lysozymes are 13.9 and 17.2, respec- tively, whereas the resolution of the human structure (1.5 A) We thank Professors Martin Karplus (Harvard University, Cam- is comparable to that of myoglobin (1.4 A). The B-factor bridge, MA) and Frederic M. Richards (Yale University, New Haven, differences may be attributable to the fact that the myoglobin CT) for comments. We also thank Dr. Wayne Hendrickson (Columbia Downloaded by guest on September 26, 2021 230 Biochemistry: Novotnf et al. Proc. Natl. Acad. Sci. USA 83 (1986) University, New York) for providing us with myohemerythrin coordi- 15. Tanaka, T., Slamon, D. J. & Cline, M. J. (1985) Proc. Natl. nates and for communicating his results to us prior to publication. The Acad. Sci. USA 82, 3400-3404. idea to explore large-probe contact surfaces was also conceived by Dr. 16. Lee, B. K. & Richards, F. M. (1971) J. Mol. Biol. 55, 379-400. Don Wiley (Harvard University), who suggested it independently to 17. Richards, F. M. (1977) Annu. Rev. Biophys. Bioeng. 6, M.H. We note that Dr. Mitchell Lewis (Smith, Kline & Beckman, 151-176. Philadelphia) has arrived at a conclusion similar to that presented here, 18. Richmond, T. & Richards, F. M. (1978) J. Mol. Biol. 119, by different methods; similarly, Drs. Greg Petsko, Robert Campbell, 537-555. James Mottonen, and Robert Tilton (Massachusetts Institute ofTech- 19. Conolly, M. L. (1983) Science 221, 709-713. nology, Cambridge) have found that a large fractional hydrophobic 20. Bernstein, F. C., Koetzle, T. F., Williams, G. J. B., Meyer, accessibility is observed for antigenic regions, thus supporting the view E. 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(1986) Biopoly- USA 78, 3824-3828. mers, in press. Downloaded by guest on September 26, 2021