Proc. Natl. Acad. Sci. USA Vol. 90, pp. 6869-6873, July 1993 Biochemistry The linker of des-Glu84- is bent (calcium/mutagenesis/flexible tether/bent a-helix/crystallography) S. RAGHUNATHAN*, R. J. CHANDROSSt, B.-P. CHENG*t, A. PERSECHINI*§, S. E. SOBOTTKAt, AND R. H. KRETSINGER* Departments of *Biology and tPhysics, University of Virginia, Charlottesville, VA 22901 Communicated by William N. Lipscomb, April 1, 1993

ABSTRACT The crystal structure of a mutant calmodulin 111111111122 22222222 (CaM) lacking Glu-84 has been refined to R = 0.23 using data 123456789 012345678901 23456789 measured to 2.9-A resolution. In native CaM the central helix domain n nn n X Y Z(Y) X Z n nn n is fully extended, and the molecule is dumbbell shaped. In contrast, the deletion of Glu-84 causes a bend of 950 in the 1 ADQLTEEQIA EFKEAFSLF DKDGDGTITTKE LGTVMRSL39 linker region of the central helix at Ile-85. However, EF-hand 2 GQNPTEA ELQDMINEV DADGNGTIDrPz rLTMKARKP75 domains 1 and 2 (lobe 1,2) do not touch lobe 3,4. The length, by a-carbon separation, of des-Glu5"-CaM is 56 A; that of 3 76MKDTDSEE EIRZAFRVr DKDGNGYISAAE LRHVMTNL112 native CaM is 64 A. The shape of des-Glu"-CaM is similar to 4 GEKLTDE EVDEMIREA DIDGDGQVNYEE FVQMMTAK148 that ofnative CaM, as it is bound to the target peptide ofmyosin light-chain kinase. This result supports the proposal that the FIG. 1. The amino acid sequence ofvertebrate CaM is aligned by linker region of the central helix of CaM functions as a flexible its four EF-hand domains, each consisting of 29 residues. The side tether. chains whose oxygen atoms coordinate calcium are aligned under X, Y, Z, -X, -Z. The carbonyl oxygen of (-Y) coordinates calcium with its peptide oxygen. The insides ofa-helices E and F usually have Calmodulin (CaM) is the most extensively studied of the 30 hydrophobic side chains, indicated by n. The central helix consists subfamilies (1) that contain two to eight EF-hand domains. It of the second (F) helix of domain 2, the eight-residue linker 76-83, regulates numerous target enzymes and structural proteins and the first (E) helix ofdomain 3. Glu-84, the first residue ofdomain (2) and is inferred to be present in all cells of all eukaryotes. 3, is shown in italics as the residue deleted in des-Glu4-CaM. The We want to understand the mechanisms whereby CaM trans- region Lys-77-Thr-79 is predicted not to be helical. Native CaM with duces the information in a pulse of Ca2+ ions in the cytosol three glutamates has a slightly stronger helix-forming tendency than to a change of conformation of a target enzyme. To this end does des-Glu84-CaM with only two adjacent glutamates. There is a Persechini and Kretsinger (3) made three mutant CaMs in bend of 950 in the helix centered about Ile-85 and a bend of 40' at which the linker region of the central helix has one (des- Lys-75 in des-Glu"-CaM. Glu84-CaM), two (des-Glu83, Glu4-CaM), or four (des-Ser8l, range 3.1-2.9 A the redundancy is 2.1, the mean I/ar is 5.3, Glu82, Glu83, Glu84-CaM) residues deleted. Persechini et al. and the scaling residual is 0.115. (4) treated mutated CaM (Gln-3 -3 Cys, Thr-146 -) Cys) with bismaleimidohexane to cross-link this mutated CaM into a Given the difficulty of growing larger crystals and the bent form. On the basis ofthe abilities ofthese deletion CaMs assumed similarity in structure of native CaM and of des- to activate light-chain kinase Glu84-CaM, we computed self-rotation and molecular replace- (skMLCK), NAD kinase, and calcineurin (protein phospha- ment functions. We routinely describe the EF-hand domain in tase) and of the cross-linked CaM to activate skMLCK, they terms of 29 residues, as illustrated in Fig. 1 and explained in proposed "that the central helix of calmodulin functions as a its legend. The four canonical domains of CaM extend from flexible tether." residue 11 through 39, 47-75, 84-112, and 120-148. We anticipated that des-Glu84-CaM would have four EF-hand EXPERIMENTAL PROCEDURES domains with domains 1 and 2 related by an approximate 2-fold Des-Glu4-CaM was prepared, as described by Persechini rotation axis forming lobe 1,2, very similar to those observed and Kretsinger (3), and was crystallized by vapor distillation in native CaM and C (TnC). Lobe 3,4 should also from drops over reservoirs of 20%o saturated ammonium consist ofa pair of EF-hands related by an approximate 2-fold sulfate/10 mM CaCl2/50 mM 2-(N-morpholino)ethanesulfo- rotation axis. We hoped to determine the rotation operation nic acid, pH 6.1, at both 4°C and 20'C. Initial drops consisted relating lobe 1,2 to lobe 3,4. We found a single rotation axis in of 8.0 ,ul of des-Glu84-CaM at 10 mg/ml and 8.0 ,ul of the self-rotation function with the rotation value K 1800. We reservoir. Our largest crystals seldom exceeded 0.25 mm in could interpret this result only in terms of the subsequent any dimension; diffraction was weak beyond 2.9 A. Unit cell molecular replacement calculations. dimensions and intensity data were measured at the Multi- Molecular replacement searches were computed by using wire Area X-ray Diffractometer Facility (5). Systematic the package CCP4 (6). We used lobe 1,2 (residues 11-75) and absences indicate space group P212121 with unit cell dimen- lobe 3,4 (84-147) as probes (probe 1,2 and probe 3,4). The sions a = 45.3, b = 49.9, c = 62.4 A; one molecule per results were relatively insensitive to the exact definition of asymmetric unit. The 3396 unique reflections of 3483 avail- probe termini as well as to both radius of integration and able reciprocal lattice points were measured to a resolution of resolution of data used. On the basis of several preliminary 2.9 A. The redundancy ofmeasurements is 3.8. The mean I/oa trials we used 20-A radius, 10- to 4-A data, and all nonhy- is 6.6; 2371 reflections have I/cr > 3.0. The overall scaling residual - I,I/n is 0.09. In the resolution Abbreviations: CaM, calmodulin; TnC, ; skMLCK, skel- Y:.=j[7,jgi(Iji Ijj)/m etal muscle myosin light-chain kinase. *Present address: Institute of Solid State Physics, Academia Sinica, The publication costs of this article were defrayed in part by page charge Hefei, People's Republic of China. payment. This article must therefore be hereby marked "advertisement" §Present address: Department of Physiology, School of Medicine, in accordance with 18 U.S.C. §1734 solely to indicate this fact. University of Rochester, Rochester, NY 14642. 6869 Downloaded by guest on September 26, 2021 6870 Biochemistry: Raghunathan et al. Proc. Natl. Acad Sci. USA 90 (1993) drogen atoms. In cross-rotation functions probe 1,2 gave rotation peaks). As anticipated, the resulting maps were very three significant peaks (Fig. 2A), and probe 3,4 gave two of noisy because each search used only halfofthe molecule. We the three (Fig. 2B). Similar results were obtained when atoms then used each of several solutions to the translation function beyond the (3-carbon were omitted, the polyalanine model. for probe 1,2 as a fixed contribution, while we searched for Because the two lobes of CaM are so similar, it was expected the translation component of probe 3,4 and vice versa. We that both probes would give the same two peaks, one found only one rotation, translation solution for probe 1,2 corresponding to the 1,2 lobe of des-Glu84-CaM and one that gave a significant solution for probe 3,4 and conversely corresponding to lobe 3,4. We could not, however, at this only one rotation, translation solution for probe 3,4 that gave point determine which search peak corresponded to which a significant solution for probe 1,2 (Fig. 2 C and D). lobe of des-Glu4-CaM. At this point we had tentatively identified the correct We computed translation functions for both cross-rotation rotation and translation matrices for probe 1,2 and for probe function solutions (and as a control for several smaller 3,4 but did not know which corresponded to lobe 1,2 of A CROSS ROT PROBE 1,2 B CROSS ROT PROBE 3,4 -6C 1800 0 I~~so at 180 JR 7

360 360 a = 75, 80, 85 & 90 = 85&90 C D ci; tOSS TRANS FIX 3,4 PROBE 1,2 CROSS TRANS FIX 1,2 PROBE 3,4 ' Z 0.5 0 Z 0.5

v

X

--I= ocr

0.5 0.5

Y = 0.214, 0.228, 0.242, 0.256 & 0.270 Y = 0.343, 0.357, 0.371 & 0.385

FIG. 2. Molecular replacement calculations using both lobe 1,2 and lobe 3,4 ofnative CaM as probes revealed the orientations and translations of the two lobes of des-Glu84-CaM. The radius of integration is 20 A; data from 10-4 A are used. (A) Cross-rotation (CROSS ROT) function computed including all side chains of residues 11-75 (lobe 1,2) of native CaM as probe. The map is computed at 19 intervals of , from 00 through 900. Contours are drawn at 3.6 times the mean level, exclusive of the origin peak, and are displayed, in superposition, for levels i3 = 750, 800, 850, and 900. In ascending order of y the peak heights are 3.8, 4.1, and 4.2; the next highest peak in the entire map is 3.6. (B) Cross-rotation map using lobe 3,4 as probe. Contours are drawn 3.5 times the mean level and are displayed for levels (8 = 85° and 900. Peak heights are 3.9 and 4.0; the next highest peak in the entire map is 3.9. Both probes located the two lobes of des-Glu84-CaM at relative rotation angles a 440, 13830, and y 2120 and at 1370, 900, 380, as indicated by X. (C) Lobe 3,4 is held fixed in its correct position for the computation of structure factors and lobe 1,2 translated. The map is contoured at 3.7. The three highest peaks, all shown on these five sections (Y = 0.214-0.270), are 4.4, 4.1, and 5.0 in ascending values of X. (D) Lobe 1,2 is held fixed in its correct position, and lobe 3,4 is translated; the map is contoured at 3.3. The two highest peaks, 4.2 and 4.9, are shown on four sections, Y = 0.343 through 0.385. The third highest, 4.0, is not shown. (C and D) Correct peak is indicated by X. Downloaded by guest on September 26, 2021 Biochemistry: Raghunathan et al. Proc. Natl. Acad. Sci. USA 90 (1993) 6871

B F 65

G 25

D 64 82

I 63 a

N60 G 61

T 29

FIG. 3. The quality of the electron density maps is reasonable for 2.9-A resolution data as evaluated in OMIT maps in which the portion of the molecule under evaluation is omitted from phase calculations. (A) Computed electron density for Glu-82, Glu-83, and Ile-85, which were omitted from phase calculations. The insert shows the a-carbon trace ofresidues 66-92, in about the same orientation as in the OMIT map. There is a bend of 400 between the F2 helix, residues 66-76, and linker 77-83. The angle between the linker and the E3 helix, 85-92, is 85°. (B) (Fo - Fj) map, in which Ca2+ ions 1 and 2 were omitted from the phase calculation; their computed electron densities are quite strong with little other density appearing in the map. Traces of parts of the main chain are shown. The carbonyl oxygen atoms of the homologous Thr-26 and Thr-62 coordinate the Ca2+ ions at the -Y vertices (see legend to Fig. 1). des-Glu84-CaM and which to lobe 3,4. Packing calculations tether. The predicted structure of TnC (10), which was confirmed that neither lobe, when placed in one asymmetric applied to CaM, was based on three assumptions. (i) The two unit, overlapped with either its symmetry-related mates or domains of lobe 1,2 and the two of lobe 3,4 would be related with the other lobe in that or in any neighboring asymmetric by an approximate 2-fold axis, as are domains CD and EF in unit. We then asked whether the N terminus ofprobe 1,2 was parvalbumin. (ii) The 11-residue linker of TnC, or 8-residue within 15 A of the C terminus of probe 3,4 in any of the linker ofCaM, would be bent. (iii) The hydrophobic patch on reference or 26 surrounding asymmetric units. Then the lobe 1,2 would touch the homologous patch of lobe 3,4 (Fig. inverse question-C terminus ofprobe 1,2 and N terminus of 4A). The postulated structures oflobes 1,2 and lobes 3,4 were probe 3,4-was asked. Of the 54 (2 x 33) pairings only one confirmed in the crystal structures ofTnC (11, 12) and ofCaM was close enough that the seven-residue linker, Met76-GIu82, (13, 14) (Fig. 4 B and C). However, in both structures there would be long enough to link the two lobes. These two lobes is a long continuous a-helix consisting ofhelix F2, linker, and were then assigned to one molecule and provided the starting helix E3 (Fig. 1). Small-angle x-ray scattering experiments point for refinement of the structure. (15, 16) were interpreted in terms of an elongated structure. The structure was initially refined by using PROLSQ (7), in Persechini and Kretsinger (3) found that their deletion which the structure is adjusted to minimize the residual, R = mutants-des-Glu84-CaM, des-Glu83, Glu84-CaM, des-Ser81, 7[(IFoI - IFcI)/1IFoI subject to constraints of near canonical Glu82, Glu83, Glu4-CaM all activate skMLCK, NAD ki- bond lengths and angles. Initially only the two lobes were nase, and calcineurin with Km 5- to 7-fold higher than that of refined. Later the linker, residues 76-82, and residues 5-10, native CaM. Further, CaM with Gln-3 -- Cys and Thr-146 -* preceding the first domain were fitted into electron-density Cys mutations crosslinked into a bent form by reaction with maps computed by using observed amplitudes and phases bismaleimidohexane activates skMLCK and calcineurin but computed from the two lobes as positioned in the unit cell. activates NAD kinase only poorly. They proposed that the The entire molecule was refined by molecular dynamics, linker region of the central helix of CaM functions as a using the program X-PLOR (8). The quality and correctness of flexible tether permitting the two lobes to enfold an a-helical electron-density maps were evaluated by OMIT maps (9) in portion of the target enzyme (Fig. 4D). The flexibility was which the entire refined molecule, excepting the small region postulated in two senses: (i) to accommodate the function- under investigation, is used to compute phases (Fig. 3). ality of linkers of various lengths in mutant CaMs and in Details of the structure determination and refinement will be homologs of CaM and (ii) to enfold target helices of various published elsewhere.$ sequence and sense. Subsequently, small-angle x-ray scattering experiment RESULTS AND DISCUSSION measurements of CaM showed a shortening of CaM upon The significance of the crystal structure of des-Glu84-CaM is complexation with target helices (17-21). Des-Glu83, Glu84_ best understood in the context of the proposal that the linker CaM and des-Ser81, Glu82, Glu83, Glu84-CaM are elongated in region of the central helix of CaM functions as a flexible solution, as determined by experiments consistent with their having helical linkers two and four residues shorter than 1The atomic coordinates and structure factors have been deposited native CaM. Preliminary results indicate a similar elongated in the Protein Data Bank, Chemistry Department, Brookhaven shape for des-Glu84-CaM (M. Kataoka, personal communi- National Laboratory, Upton, NY 11973 (reference 1DEG). cation). Shortening of the molecule was observed for des- Downloaded by guest on September 26, 2021 6872 Biochemistry: Raghunathan et al. Proc. Natl. Acad. Sci. USA 90 (1993)

FIG. 4. Diagrams show the relative positions oflobe 1,2, linker, and lobe 3,4. The hemisphere ofeach cup, which symbolizes a lobe, has an axis ofrotational symmetry coincident with the approximate 2-fold rotation axis relating domains 1 and 2 or with the axis of 3 and 4. The hatched portion of the surface represents the hydro- phobic patch, which is on the opposite side and -20 A from the two calcium-binding loops on the other surface. The handle of cup 1,2 is almost perpendicular to the cup face and consists of the C terminus ofhelix F2 and the N part ofthe linker. The handle ofcup 3,4 is almost parallel to the cup face and consists of the C part of the linker and the N terminus of helix E3. In the crystal structures ofTnC and of CaM the two handles are continuous, and the central linker region of the central helix is represented by a rod. The target helix bound by CaM is also a rod. (A) Predicted structure of TnC. (B) Crystal structure of TnC. (C) Crystal structure of CaM. (D) Flexible tether model of CaM complexed with the target peptide M13. (E) NMR solution structure of CaM complexed with 577-602 of skMLCK. (F) Crystal structure of sarcoplasm calcium-binding protein. (G) Anticipated structure of des-Glu84-CaM. (H) Crystal structure of des-Glu84-CaM. The axis of approximate 2-fold rota- tion relating lobe 1,2 and lobe 3,4 is indicated in this and in E. Glu83, Glu84-CaM and for des-Ser8l, Glu82, Glu83, Glu84-CaM of CaM enfold the target helix with linker residues 75-81 in upon binding melittin (22), as had been observed for binding nonhelical conformation. Lobe 3,4 binds to the N-end of the target analogs by native CaM. Recently Ikura et al. (23) target helix with many hydrophobic interactions to the tryp- determined the structure in solution of CaM complexed with tophan of the target. Lobe 1,2 binds to the other side of the the 26-residue peptide that represents the CaM-binding re- helical target at its C end. Barbato et al. (25) concluded from gion of skMLCK by multidimensional NMR (Fig. 4E). The 15N relaxation using 2-dimensional NMR that the linker crystal structure of CaM complexed with the homologous residues Lys-77-Ser-81 have such high mobility that from the 20-residue peptide of smooth-muscle myosin light-chain ki- perspective of 15N relaxation the tumblings oflobe 1,2 and of nase has a very similar structure (24). In both, the two lobes lobe 3,4 are independent. Downloaded by guest on September 26, 2021 Biochemistry: Raghunathan et al. Proc. Natl. Acad. Sci. USA 90 (1993) 6873 The crystal structure of the sarcoplasm calcium-binding distribution of its structures. Whether Nature has evolved protein of the sandworm Nereis diversicolor (26) reveals a such a delicate balance of helical and bent structures in the bent linker with lobe 1,2 making numerous contacts with lobe score of other four domain EF-hand proteins remains to be 3,4 (Fig. 4F). The function of sarcoplasm calcium-binding seen. protein is not known; there is no evidence that it binds to a target protein. Further, it is not known whether the linker We thank T. N. Bhat for his assistance in the initial interpretation region is flexible. of molecular replacement calculations and R. B. MacDonald for We anticipated that the structure of des-Glu84-CaM would preparing figures. R.H.K. thanks the National Science Foundation be similar to that of CaM, except that lobe 3,4 would be 1.5 for Grant DMB-8917285 A closer to lobe 1,2 and rotated 1000 (Fig. 4G). The crystal 1. Nakayama, S., Moncrief, N. D. & Kretsinger, R. H. (1992) J. structure of des-Glu84-CaM (Fig. 4H), shows the linker to be Mol. Evol. 34, 416-448. bent in the absence of a target. 2. Manalan, A. S. & Klee, C. B. (1984) Adv. Cyclic Nucleotide The peak in the self-rotation function is 4 = 700, q, = 89°0 Res. 18, 227-277. K = 1800, significantly different from that anticipated from the 3. Persechini, A. & Kretsinger, R. H. (1988) J. Biol. Chem. 263, model (Fig. 4G). Lobe 1,2 ofdes-Glu84-CaM is related to lobe 12175-12178. 4. Persechini, A., Blumenthal, D. K., Jerret, H. W., Klee, C. B., 3,4 by an approximate 2-fold rotation axis, as for the CaM- Hardy, D. 0. & Kretsinger, R. H. (1989) J. Biol. Chem. 264, skMLCK peptide complex (23). The observed rotation axis in 8052-8058. the crystal structure is 4 = 890, q = 850 with the rotation K 5. Sobottka, S. E., Chandross, R. J., Cornick, G. G., Kretsinger, = 1760. R. H. & Rains, R. G. (1990) J. Appl. Crystallogr. 23, 199-208. The four calcium-binding sites are occupied at (near) full 6. CCP4 (1979) A Suite ofProgramsfor Protein Crystallography, occupancy (Fig. 3B). The structures ofboth lobes 1,2 and 3,4 The Science and Engineering Research Council Colloborative are quite similar to their homologs in native CaM. Small Computing Project 4 (Daresbury Laboratory, Warrington, U.K.). distortions in helices F2 and E3 will be described after 7. Hendrickson, W. A. & Konnert, J. H. (1981) in Biomolecular completion of refinement. The first four residues cannot be Structure, Function, Conformation and Evolution, ed. Srini- located in the electron density map. vasan, R. (Pergamon, Oxford), Vol. 1, pp. 43-57. In the crystal des-Glu84-CaM has numerous contacts to 8. Brunger, A. T. (1990) X-PLOR (Yale Univ., New Haven, CT), eight neighboring molecules. The residues that form the Version 2.1. hydrophobic patch on lobe 1,2 or the homologous patch on 9. Bhat, T. N. & Cohen, G. H. (1984) J. Appl. Crystallogr. 17, lobe 3,4 are not in contact with neighbors. Nor is it obvious 244-248. whether crystal packing would favor the observed bent form 10. Kretsinger, R. H. & Barry, C. D. (1975) Biochim. Biophys. Acta 405, 40-52. over the anticipated extended form of des-Glu84-CaM. 11. Herzberg, 0. & James, M. N. G. (1988) J. Mol. Biol. 203, The amino acid sequences of bovine (and all known 761-779. mammalian) CaMs, of CaM from Drosophila and from Par- 12. Satyshur, K. A., Rao, S. T., Pyzalska, D., Drendel, W., amecium are quite similar: mammalian CaM/Drosophila Greaser, M. & Sundaralingam, M. (1988) J. Biol. Chem. 263, CaM three differences, mammalian CaM/Paramecium CaM 1628-1647. 17 differences, Drosophila CaM/Paramecium CaM 18 dif- 13. Babu, Y. S., Bugg, C. E. & Cook, W. J. (1988) J. Mol. Biol. ferences. The linker sequences are identical. All three crys- 204, 191-204. tallize in space group P1 (13, 14, 27) with near-identical unit 14. Taylor, D. A., Sack, J. S., Maune, J. F., Beckingham, K. & cells and are nearly isomorphous. Yet Rao et al. (27) note Quiocho, F. A. (1991) J. Biol. Chem. 266, 21375-21380. 15. Heidorn, D. B. & Trewhella, J. (1988) Biochemistry 27, 909- very interesting differences. In Paramecium CaM (27) the 915. central helix has a radius of curvature of 75 A with no kinks. 16. Hubbard, S. R., Hodgson, K. 0. & Doniach, S. (1988) J. Biol. Mammalian CaM (13) has a kink at Asp-80; whereas Dro- Chem. 263, 4151-4158. sophila CaM (14) has two small bends at Lys-75 and Ile-85. 17. Heidorn, D. B., Seeger, P. A., Rokop, S. E., Blumenthal, Lys-75 and Ile-85 are the residues observed to be bent in D. K., Means, A. R., Crespi, H. & Trewhella, J. (1989) Bio- des-Glu84-CaM. chemistry 28, 6757-6764. We suggest that the linker of native CaM, which is strongly 18. Kataoka, M., Head, J. F., Seaton, B. A. & Engleman, D. M. conserved from fungi through animals, has a structure in (1989) Proc. Natl. Acad. Sci. USA 86, 6944-6948. solution distributed between multiple extended and bent 19. Kataoka, M., Head, J. F., Vorherr, T., Krebs, J. & Carafoli, E. measure- (1991) Biochemistry 30, 6247-6251. forms. Small-angle x-ray scattering experiment 20. Matsushima, N., Iaumi, Y., Masuo, T., Yoshino, Y., Ueki, T. ments detect ensemble averages and are especially sensitive & Miyake, Y. (1989) J. Biochem. (Tokyo) 105, 883-887. to the gross changes in structure associated with complexion 21. Trewhella, J., Blumenthal, D. K., Rokop, S. E. & Seeger, of targets by CaM or to the changes in overall length of P. A. (1990) Biochemistry 29, 9316-9324. des-Glu84-CaM, des-Glu83, Glu84-CaM, and des-Ser8', Glu82, 22. Kataoka, M., Head, J. F., Persechini, A., Kretsinger, R. H. & Glu83, Glu84-CaM relative to native CaM. Certainly in native Engleman, D. M. (1991) Biochemistry 30, 1188-1192. CaM and probably in these deletion mutants the linker is 23. Ikura, M., Clore, G. M., Gronenborn, A. M., Zhu, G., Klee, flexible, although usually extended, in solution. Binding to a C. B. & Bax, A. (1992) Science 256, 632-638. target, or in the case of des-Glu84-CaM sitting in a crystal 24. Meador, W. E., Means, A. R. & Quiocho, F. A. (1992) Science 257, 1251-1255. lattice, drives the distribution to the bent form. The deletion 25. Barbato, G., Ikura, M., Kay, L. E., Pastor, R. W. & Bax, A. mutants appear less helical in solution but still extended (1992) Biochemistry 31, 5269-5278. enough to appear elongated to small angle x-ray scattering 26. Vijay-Kumar, S. & Cook, W. J. (1992) J. Mol. Biol. 224, and to present binding sites accessible to targets. 413-426. We are left to marvel at the balance offorces that determine 27. Rao, S. T., Wu, S., Satyshur, K. A., Ling, K. Y., Kung, C. & the flexibility of this tether and at our inability to predict the Sundaralingam, M. (1993) Protein Sci. 2, 436-447. Downloaded by guest on September 26, 2021